In memoriam This site is dedicated to the memory of Dr. Erwin Van den Enden, who passed away unexpectedly on May 5, 2013. The Illustrated Lecture Notes are Dr. Van den Enden's magnum opus. They form an invaluable body of reference for both students and practitioners of tropical medicine. Dr. Van den Enden spent decades on this project, expanding on the knowledge of his predecessors at the Institute of Tropical Medicine. His colleague and mentor, Dr. Jef Van den Ende, explains how Erwin enriched his life: On May 5, 2013, our colleague Erwin Van den Enden was unexpectedly taken from us. Dr. Van den Enden was a corner stone and buttress of the Medical Services of the Institute of Tropical Medicine, Antwerp. First and foremost, he was a homo doctus. He could engage in more than knowledgeable conversation about the Higgs particle, butterflies, crystals or even the Hubble telescope and distant galaxies. He pointed out to me one day that a medical doctor should read the 'Scientific American' too, to be truly deserving of the title “Doctor”. I subscribed the same day. As homo universalis he was a bicyclist and invested an enormous amount of time in raising his two sons and being a caring husband. He went on missions to Africa under dreadful conditions and took me by surprise once by - out of the blue - playing two magnificent pieces of jazz on my piano. Above all we will remember him as a clinician and professor. He could thoroughly analyze clinical cases, study the publications and lead the team on a journey through biochemistry, physiopathology, pathology and the history of medicine, all based on one case. As professor (he unfortunately never obtained the title because of a shift in focus from education to research at the end of the 20th century in most universities), he was unparalleled. He was a guest teacher in Amsterdam, at John Hopkins, in Lima and elsewhere, and could completely enthrall students with his lectures about tropical spiders and snakes - even those suffering from a serious hangover from the previous night. "They don't make these kinds of people anymore," a student once said about him. Duly noted. Jef Van den Ende, ITM. Dr. Van den Enden preparing vaccine Marymount Mission Hospital, Zimbabwe, 1981

 

Electronic exercises For electronic exercises on tropical clinical cases, click on the following link http://e.itg.be/erwin

A tropical disease tour We are taking a panoramic tour through a number of medical subjects. An attempt has been made to provide a picture of a number of problems. Hopefully this has whetted the reader’s appetite and he/she will be motivated to continue to learn and contribute to the ever-increasing knowledge and use this for commendable purposes. We hope this course will be highly interactive, and not lead to "death by power-point" in the class room. Many rural areas in the tropics seem to be frozen in time (although cell phone use spreads fast), while the flow of medical and scientific information has become a deluge. Getting information resembles trying to drink from the water of a fire hose. We are living in the middle of several revolutions, from explosive expansion of genetic and biomedical data, to fundamental changes in information / communication strategies. Rapid advances in robotic surgery, rapid diagnostic tests, the promise of stem cell therapy, the approval of the first gene therapy in 2012 (Glyvera), brainbow cell staining, fast internet access, attosecond lasers, quick gene cloning, optogenetics, metagenomics and transgenic organisms suggest that we enter a world with surprising new possibilities and risks. There is so much new information available that we are in danger of becoming overwhelmed by it, so choosing what to study becomes more and more important. Maybe a part of the style of learning is changing right under our noses as well. With lots of information coming in small "Mc"-bite size portions, there is a danger of loosing an overview or a proper reference frame. With electronic data overload, certain students might have a diminishing ability to stay focused on a single item for a prolonged and reasonable time. We have to get a firm broad and constant education, based on factual knowledge and mastering applications, together with empathy as well as fluency in divergent and convergent thinking. I think it is a good idea that a physician can talk with a marine biologist, a geochemist and an entomologist and of course the patient. Drawing hard dividing lines between academic disciplines makes it more difficult for researchers to communicate and cooperate. Other items on a very different level -to name just a few- are the looming freshwater crisis in certain geographical areas, the dramatic increase of the world population, the "population greying" in several countries and loss of habitat, biodiversity and the near-exhaustion of unique natural non-renewable resources such as geological phosphate deposits. The unrelenting spread of multiresistant pathogens is becoming a global emergency and will become one of the most serious infectious disease problems facing the world in the near future. The spectrum ranges from multidrug-resistant malaria,  Staphylococcus aureus , tuberculosis, ceftriaxone-resistant gonorrhea, carbapenem-resistant Enterobacteriaceae to triclabendazole-resistant Fasciola hepatica . Treatment of bacterial infections with bacteriophages is still experimental at this moment. Another threat is the possibility of a new pandemic of a highly contagious and lethal disease, be it a new influenza, a SARS-like pathogen or something nobody expected. For a good review of the SARS epidemic of 2003, see Science 2013;339:1264-1273. In this course, the emphasis is on tropical medicine. Let's try to get the outlines right, getting a good grip on basic principles as currently understood, before diving deep. It is true that a jack-of-all-trades is usually a master of none. So be it, but it eases communication between disciplines. A study of the details comes afterwards (since we'll all be life-long learners) and often shows how rich nature is if we only want to see. Admiration of nature goes best together with understanding of nature. Understanding the structure of a flower does not diminish the beauty of a rose. Hence the following by William Blake. Van den Enden Erwin, MD Antwerp, Belgium April 2013   Photo courtesy of Mr Jan Van den Enden

General 

" To wrest from nature the secrets which have perplexed philosophers in all ages, to track to their sources the causes of disease, to correlate the vast stores of knowledge, that they may be quickly available for the prevention and cure of disease - these are our ambitions". Sir William Osler.

In this age of fast and spectacular new developments in science, including biology, affluence, attention to  environmental problems and the current financial crisis, it is all too easy to see that an important part of humanity lives below the poverty line. The problems facing the population in the Third World are numerous. Improvements in medical care will only solve a few of these, less than improvements in socio-economic conditions. A large proportion of the local population lives in unhealthy conditions, mostly as a direct consequence of poverty. Lack of income rather than the tropical climate is the leading factor in the occurrence of many diseases. When money is available, access to clean water, healthy food etc becomes much easier. There is a strong correlation between health parameters (life expectancy, child mortality) and economic parameters (GNP, per capita income, income distribution). The fact that health and development are closely related leads to the concept of diseases of poverty. Many of the problems can only get worse if the population continues to increase. Demographers expect that global population will increase to about 9 billion people in the next 30 years. This is two extra Chinas well within the lifetime of most readers. It is unclear if one or more global breaking points are going to be reached in this period (energy, food, pollution, climate change, disturbances of the global nitrogen cycle, global phosphate stocks depletion, increasing rate of biodiversity loss, limits of global fresh water and land use, plagues or others). Large swaths of food plant monocultures are standing invitations for rapid multiplying pathogens. One can expect major and probably dramatic changes in at least one, but more likely many important areas.

Poverty in Somalia

Why are current cultures so different from each other? Why is it that until recently Papuans were using neolithic stone tools, while Americans were walking on the Moon? Human history is affected by chance events and as such may seem unpredictable. Over a broader timescale however, there may be repeatable patterns. A fascinating hypothesis is that differences between cultures can arise from very early environmental differences. Some scholars (e.g. Jared Diamond) think that those cultures whose environments contained animals which could be domesticated and wild plants that could usefully be cultivated, developed farming. These cultures grew rapidly. This growth can lead to the establishment of political systems and written language, both aiding general knowledge and the development of new technologies, including weapons. This allowed these cultures to dominate others, as did the germs they brought with them. Some of these germs came from the animals they lived with. They spread easily due to high population densities and absent immunity due to lack of previous exposure. Large sections of the indigenous population of Latin America and the Pacific islands were wiped out when they first came in contact with measles and smallpox brought to them by the Europeans who contacted them.

Normal children and sheep, Andes Mountains  

Medical disorders, diseases and infections are not isolated phenomena. Public health depends to a large extent on living conditions, social infrastructure, economic development and engineering programmes and to a lesser extent on medical services and medication. Naturally, vaccines, surgery and curative medicines have their place, but sanitary facilities (toilets, sewers, waste water treatment), safe drinking water, a safe and balanced diet, a knowledge of basic medical principles through education, good housing, lifestyle, etc., also have a role that should not be underestimated. As an illustration, reference may be made to the decline of cholera, tuberculosis and leprosy in Europe around the turn of the 19th-20th century, even before the time of antibiotics.

The great majority of diseases in tropical regions are cosmopolitan which means they are found throughout the world: pneumonia, burns, fractures, diarrhoea, asthma, diabetes, hypertension and schizophrenia. Some disorders were also previously found in Europe, but here they have largely disappeared: leprosy, vivax malaria, plague. Only a few diseases occur exclusively in tropical regions, e.g. African sleeping sickness. A number of diseases have disappeared in the West as a result of the improvement of living conditions. The classic, predominantly parasitic tropical diseases are for the most part not the main cause of disease in developing regions, except in certain localised areas where there is a high prevalence. The main medical problems in Third World countries at present continue to be respiratory tract infections, diarrhea, tuberculosis, malaria, AIDS, measles, accidents, anemia and pregnancy-related problems. Hepatitis B and C, salpingitis (P.I.D.) and epidemic meningococcal meningitis are also frequent problems.

It is important to realise that the distribution and incidence of diseases are constantly evolving. In the past few years there has been a marked reduction in poliomyelitis, river blindness and leprosy. Conversely, there has been a spectacular increase in West African sleeping sickness, dengue, multiresistant P. falciparum malaria and AIDS. Now and again new diseases appear. The risk of epidemics is real. When we think for example of what the so-called "Spanish Flu" caused at the end of the First World War, we must remain cautious and alert. [Spanish Flu was first observed in March 1918 and in the following year 20-40 million people across the world died from the disease.] The appearance of SARS in 2003, avian influenza type H5N1 with transmission to humans in 2004 and the "Mexican" swine flu H1N1 in 2009, reminds us of the dangers.

As economies develop, other diseases previously first seen in Western countries will become more common, such as cancer, caries, cardiovascular diseases and multiresistant micro-organisms. Problems typical of large cities will become more important in the near future as urbanisation increases in Third World countries. The poor neighbourhoods and slums of conurbations such as Cairo, Lagos and Kinshasa in Africa, Sao Paulo, Rio, Lima and Bogota in South America, Dhaka, Calcutta, Bombay, Delhi, Karachi and Manila in Asia pose their own problems, but also offer opportunities for improvement.

The outskirts of Lima, Peru. Poverty is a main determinant of illness. Copyright Alexander von Humboldt Institute, Peru.  

In the West, the fear of attacks with biological weapons is increasing. Anthrax, plague, botulism and certainly variola major (smallpox) are high on the agenda. Although smallpox has been eliminated for decades and officially there are only two places where the virulent pox virus is stored (CDC in Atlanta, USA and the State Research Centre of Virology and Biotechnology, Novosibirsk, Russia), the possibility of the deliberate spread of the virus is considered to be real. An attack of this kind could be an unparalleled catastrophe (30% mortality in a non-immune population). For this reason, production of a large stock of vaccine was started in 2001 (Acambis, Cambridge, MA, USA). Vaccination of certain target groups in the USA was resumed.

Some diseases are due to infections, such as tuberculosis, while others are caused by inadequate diet, e.g. beri beri, kwashiorkor, pellagra. Others in turn are genetically determined, such as sickle cell anaemia. There still remain, however, numerous uncertainties and gaps in our knowledge of and insight into many diseases. An understanding of how current concepts have arisen (how we know what we know) is important. We will provide an overview of individual diseases currently associated with tropical regions and areas of extensive poverty.

Some concepts recur constantly and are explained below.

Parasite :A parasite is an organism that lives in or on another organism and draws its nourishment from it (from the Greek "para-sitos": beside food). Strictly speaking, it has no connotation of harmfulness or otherwise. Usually, however, the meaning is taken in a narrower sense and the term is used to refer to various worms, protozoa and arthropods which have another organism as their habitat. Parasites often have a complicated life-cycle with well-defined hosts and a specific mode of transmission.

Paratenic host : a host in which a parasite lives and survives, but does not develop further.

Vector : an intermediate host, which transports a parasite from the previous host to the subsequent one. E.g.: the tsetse fly is the vector of African sleeping sickness.

Arthropod : Invertebrate animal with articulated legs. In medical practice the main arthropods belong to the group of insects and arachnids (including ticks and mites). Copepods are also arthropods and are vectors for a number of organisms.

Epidemic : infection which fairly suddenly affects a large number of people at the same time. E.g. the plague epidemics in the Middle Ages in Europe, the meningitis epidemics in the Sahel.

Pandemic : epidemic which spreads around the whole world. E.g.: Flu (influenza), AIDS pandemic

Endemic : a disease is endemic if it is chronically present in a particular region. E.g.: in Africa there are foci of endemic malaria.

Transmission : Transport of an organism can occur in various ways.

Pathogenic organisms

After the German physician Koch had formulated his postulates about pathogenic organisms in 1891, numerous practical problems arose in meeting these for each presumptive causative agent. For many parasitic diseases these postulates have still not been met today. In particular, in vitro cultivation raises problems (fastidious organisms, complex life-cycles in various hosts). This has resulted over the course of history in the separation of bacteriology (+ virology at a later date) on the one hand and parasitology on the other.

These postulates are:

The disease organism must be associated with the disease.

The organism must be isolated and then grown in pure culture.

The cultured organism must cause the disease after inoculation into a sensitive host.

The organism isolated from the second host must be identical to the first.

 "Common" bacteria : e.g. those causing plague, cholera, typhoid fever, bacterial meningitis. Antibiotics often are first choice in treatment. On the basis of the structure of the outermost layer of the bacteria, organisms can be classified into Gram-positive and Gram-negative. These can be distinguished by means of a particular staining method (Gram stain). Bacteria can be rod-shaped or round (bacilli and cocci, respectively). Some are spiral (e.g. spirochaetes) or comma-shaped (e.g. vibrios). Mycoplasma are the smallest free-living bacteria. They are rather difficult to detect by Gram stain. Chlamydiae and rickettsiae are also difficult to detect under the light microscope because of their small dimensions (often smaller than the optical resolution of the instrument).

The Danish physician Hans Christian Gram in 1884 developed a staining method which still bears his name today. Bacteria in suspension are fixed on a glass slide by heating for a short time. Two substances (crystal violet and lugol [containing iodine]) are added successively and then react to form a coloured complex in each bacterium. When the carrier glass is subsequently rinsed with an alcohol or acetone, bacteria with a thick cell wall retain the stain, while those with a thin cell wall lose it. A second counterstain is then applied with a weaker red stain (safranin or dilute carbol fuchsin). [The latter should not be confused with strong carbol fuchsin used in Ziehl's stain]. Gram staining thus reveals differences in cell wall structure.

Gram-positive bacteria have a cell wall that consists of a very thick peptidoglycan layer. It forms a thick latticework around the bacterium. The peptidoglycan layer in Gram-negative bacteria is only 1 to 2 molecules thick. Peptidoglycan is a polymer of a disaccharide (2 different sugars, N-acetylglucosamine and D-acetyl muramic acid). The sugar chains are bound to one another by oligopeptide bridges. Peptidoglycan synthesis is impaired for example by beta-lactam antibiotics. In Gram-negative bacteria, the thin peptidoglycan layer is surrounded by an outer membrane that consists of lipopolysaccharides (containing endotoxin). These are of great importance in pathogenicity.

Comparison of molecular structure of Gram positive and Gram negative bacterial cell walls. Copyright ITM

Structure of bacterial Gram positive cell wall. Chains consisting of linked disaccharide-units are cross-linked by short oligopeptide bridges. Copyright ITM

Gram-staining technique, overview of the procedure. Copyright ITM

Mycobacteria : e.g. Tuberculosis, leprosy, Buruli ulcer. Treatment of these diseases requires a different set of antibiotics. Special staining of the wax-like capsule is necessary to detect the organisms (Ziehl-Neelsen stain).

Chlamydiae : e.g. Trachoma, psittacosis, lymphogranuloma venereum. Some cause pulmonary inflammation, others congenital infections and eye inflammations. They possess an incomplete metabolism and are obliged to live intracellularly. They are susceptible to some specific antibiotics.

Rickettsiae : e.g. typhus. Very small, obligate intracellular bacteria named after their discoverer, Howard Ricketts. Tetracyclines constitute the basic treatment.

Fungi : Moulds (mycelium formation) and yeasts (no mycelium, sometimes pseudohyphae). A mycelium is a cluster of branched filaments formed by the organisms. Some organisms live preferentially on the skin (dermatophytes) and some on mucous membranes ( Candida ). Others cause deep infections e.g. cryptococcal meningitis. Specific therapy is indicated. Some fungi produce toxic substances. When these find their way into food, health problems can follow, e.g. ergotism ( Claviceps purpurea ) and aflatoxicosis ( Aspergillus niger ). Allergic problems are common.

Viruses : e.g. yellow fever, dengue, rabies, polio, herpes, HIV. These organisms cannot reproduce independently and do not possess their own metabolism. They are obliged to reproduce intracellularly. Sometimes treatment with virostatics is indicated, but often only symptomatic therapy is possible.

Prions : e.g. kuru, Creutzfeldt-Jacob disease, bovine spongiform encephalopathy (BSE, "mad cow disease"). In the first half of the 20th century kuru was a major neurological problem with a fatal outcome. This disease only occurred in cannibals of the Fore tribe in Papua New Guinea. Our knowledge of this and related diseases is clearly inadequate. The current hypothesis is that the disease is caused by a modified form of a normal protein. This modified protein possesses a well-defined three-dimensional configuration (tertiary structure). This form of the protein catalyses the conversion of other proteins from the one steric, non-pathogenic form of the protein to the other pathogenic form. As a result, the protein acts like an infectious agent. The change of form, however, can also occur as a result of a genetic mutation in the DNA coding for the protein.

Protozoa : unicellular organisms that contain a cell nucleus surrounded by a nuclear membrane: eukaryotes (as opposed to prokaryotes - bacteria). There are specific treatments for each disease.

e.g. Sleeping sickness, malaria, amoebiasis, leishmaniasis, giardiasis, toxoplasmosis.

Metazoa : Multicellular eukaryotic organisms, diverging considerably in size and taxonomic relationship. E.g. whip worms, bilharziasis, scabies, lung flukes.

Transmission

A number of major diseases such as malaria, leishmaniasis, trypanosomiasis, the various forms of filariasis, bacteria (recurrent fever, typhus, plague, tularaemia, bartonellae, etc...) as well as arboviruses are transmitted by bloodsucking arthropods: mosquitoes, biting flies, sandflies, bugs, lice, fleas, ticks, mites. Sometimes this involves purely human diseases (e.g. malaria), but often there is a zoonotic cycle in nature between arthropods and vertebrate animals. Propagation of self is the driving force of evolution. This is reflected in efficient pathogen transmission within a host population. Survival of the host is driven similarly. Adaptive immune responses have evolved in mammals to protect against infectieous disease. There are two transmission patterns: (a) pathogens replicate quickly and are transmitted early, followed by the development of an effective immune respons which clears the pathogens. Above a minimum susceptible host population size, there are abundant opportunities for transmission during the early stages of infection, (b) persistent infection in which the pathogen evades host adaptive immunity. Opportunity for transmission might not occur until long after the development of a mature immune respons. Besides masking, mimicry and hiding in immunoprivileged hard-to-reach sites, several pathogens generate antigenic variation while in the host, thereby defeating the adaptive immune response. This differs from the concept of "quasi-species" of the RNA-based HIV retrovirus. Mutation alone seems insufficient in generating the diversity of DNA-based eukaryotic parasites. Non-reciprocal homogous recombination (gene conversion) whereby the donor sequence remains unchanged, is one mechanism to generate antigenic diversity. It is used by certain pathogens which are taxonomically very far apart, an example of convergent evolution.

Transmission of pathogens from insect to man can occur in various ways:

mechanical transmission , comparable to sharing a dirty needle. This can occur in rapid repetitive blood meals of mobile insects on different hosts, e.g. the host reacts to the pain caused by the bite and interrupts the insect's feeding. The hungry insect will soon try to bite a second host and infect him via the blood of the first host which is still sticking to its mouthparts. This sort of transmission, however, is rare, e.g. tularemia spread by tabanid flies.

biological transmission , in which the pathogenic organism either (1) reproduces in the vector (e.g. plague, arboviruses), (2) undergoes maturation before it becomes infectious (e.g. river blindness), (3) both reproduces and undergoes maturation (e.g. malaria, sleeping sickness).

 How precisely does the vector transport the organism? Haematophagous arthropods have fine mouthparts with which they attempt to puncture a narrow blood vessel. Similar vessel-feeders or solenophages include mosquitoes, lice, bedbugs and many fleas. Other arthropods tear the skin and its capillaries and drink from the pool of blood that then forms. These are the pool-feeders or telmophages (simulids, sandflies, Culicoides sp.). Pathogens can be introduced into the wound via the saliva or by regurgitation ("vomiting") of intestinal contents via the mouthparts. South American assassin bugs defaecate during bloodsucking and the liquid excreta can contain pathogenic organisms. A similar system is found in lice (epidemic typhus and trench fever). Some ticks secrete a liquid between their legs ("coxal fluid") which can contain organisms. Borrelia recurrentis is only transmitted when the vector, the body louse, is crushed.

The portals of entry may vary : Examples

sexual : e.g. gonorrhoea, portal of entry : mucous membranes

aerogenic : via the inhalation of droplets that are coughed up or sneezed, e.g. tuberculosis: portal of entry : lungs.

skin-skin contact : e.g. impetigo, portal of entry : skin

food : e.g. trichinosis, portal of entry : intestine

drinking water : e.g. dracunculosis, portal of entry : intestine

water-skin contact : e.g. schistosomiasis, portal of entry : skin

soil-skin contact : e.g. hook worms, portal of entry : skin

faeco-oral : hands, drinking water or food contaminated with faeces, e.g. oxyuriasis, giardiasis, shigellosis. Portal of entry : intestine

blood : contaminated blood transfusion or dirty injection needles, e.g. Hepatitis B, portal of entry : blood

vector: A bloodsucking insect infects a human, e.g. through its bite, e.g. malaria, portal of entry the skin (transcutaneous), or by its faeces e.g. Chagas

Transplacental : the pathogenic organism can reach the foetus via the placenta, e.g. Treponema pallidum (congenital syphilis).

Classification items

 Phytophotodermatitis after exposure to Ruta graveolens ("wijnruit"), a mediterranean plant growing in many European gardens. Photo Dr Van den Enden, copyright ITM

Yellow Oleander, Thevetia peruviana . Used as suicide poison. Photo Dr Van den Enden, copyright ITM

It is useful to be acquainted with some of the basic concepts from botany and zoology. It is regrettable that in a number of medical curricula these subjects are missing or became less important. As a general training, an introduction to botany and zoology is highly recommended. Were this to be restricted to taxonomy and to overwhelming students with various life cycles, the benefit of such a foundation course might be questionable. The importance lies not so much in the details, but in the recognition of a framework of reference within which details may be organised. Not only do plants form the basis of most food chains and are thus the basis of all higher life but they also form an important part of the world economy. In addition to the discussion of the direct medical problems which some plants can cause, a number of subjects may be discussed during an introduction to evolutionary theory such as fertile hybrids, tetra- or polyploidy in most food plants, the consideration of chloroplasts and mitochondria as descendants of endosymbionts, etc. The same types of argument can be advanced for zoology. In order to be able to better understand some subjects it is useful to have some notions of comparative anatomy and basic concepts of (disease) ecology. Likewise a basic knowledge of zoology is useful for a good understanding of zoonoses, vectors (mosquitoes, fleas, flies, ticks, mites, snails, copepods, etc, ...), scabies, various worm infections, venomous animals (snakes, scorpions, jellyfish, etc.) or traumatogenic animals (from processionary caterpillars to cat scratches). The discovery of specific fly larvae in a corpse can be important in forensic medicine. Medical problems related to xenotransplantations we shall simply leave out of consideration. Further removed is the possibility that in the future a plant disease might be transmitted to human hosts (one of the reasons why plant diseases are included in ProMed). Generally therefore, there is a very strong argument to keep botany and zoology in a medical curriculum. Regrettably enough, this falls outside the scope of tropical diseases, although occasionally reference will be made to it. The full impact of very rapid developments in the area of genetic engineering is still not clear. The work on transgenic vectors is still in its infancy. However, various arthropods can already be genetically manipulated in the laboratory to become resistant to infection by certain pathogenic organisms. Thus, it has been possible recently to make Aedes aegypti resistant to the dengue 2 virus. Studies of transgenic mosquitoes and yellow fever, transgenic tsetse flies and sleeping sickness, and transgenic bugs and South American trypanosomes are in full development.

Once a hybrid has been found and its parentage established, it is named. The name can be either a formula or binomial. Formulas consist of the two parents' names with a multiplication sign x placed between them, e.g. Adiantum latifolium x A. petiolatum . Hybrid binomials resemble the usual scientific names but with the x placed before the specific epithet, e.g. Asplenium x ebonoides (= A. platyneuron x A. rhizophyllum ). It is best to name hybrids with binomials instead of formulas.  A formula can change if one or both of the parents' names change. This can happen when an older name is found (the rules of nomenclature require that the oldest legitimate name is used), when a name is later considered a synonym of another (when two species are "lumped" together), or when parentage is proved different from the combination originally proposed. These changes do not affect a binomial, which stays the same no matter what happens to the names of the parents.

Relationships between species and hybrids can be depicted with so-called reticulograms. Reticulograms show which species have come together to form hybrids. Usually, they also indicate whether the hybrids are sterile or fertile. Nearly all hybrids are sterile when first formed, but if they double their chromosome number, becoming tetraploid or polyploid, they usually become automatically fertile (not with their diploid parents, but with similar tetraploids of the other sex, a kind of "instant species formation"). Polyploid formation typically starts with an abnormality in meiosis. Polyploidy is often associated with hybridization. Hybrids are formed when the sperm from one species fertilizes the egg of another. By doubling a hybrid's chromosome number, each chromosome will have a partner during meiosis, its duplicate or homologue, to pair during meiosis. Normal pairing and reduction division can follow. Examples are wheat (for pasta) which is tetraploid, and wheat for bread, which is hexaploid, with important differences in gliadin-expression. Aneuploidie can occur, for example in sugar cane which is a very vigorous plant, or triploidy in banana plants ( Musa paradisiaca ), ensuring that they are sterile and will form no seed (and can only be multiplied by cuttings). This might become a problem in the long run, however, especially for pathogen defence and genetic degeneration.

There are several taxonomic methods:

Classical taxonomy (eclectic, traditional or evolutionary taxonomy), in which a value is attributed to specific characteristics. This can range from a morphometry to geographical distribution to stratigraphic distribution of fossils. Specialists can differ in their opinions about the weighting of characteristics and the method is sometimes inconsistent. For this reason new methods have been sought since the 1950s. Each method has its own advantages and disadvantages.

Numerical taxonomy (phenetics), in which clusters of similar characteristics are sought. Parallel or convergent evolution in organisms sometimes causes problems.

Cladistics . The emergence of species is viewed as a dichotomous process in which the species branch off at divergence points. An attempt is made to develop a genealogical tree with as few junctions as possible. Relationships are based on the occurrence of inherited, evolutionarily adapted characteristics. Different calculations can yield different dendrograms.

Note concerning taxonomy, nomenclature and the concept of "species"

The only true entity is a specimen, be it an individual plant, mould, protist, bacterium or animal. Since many individuals resemble one another (a pheasant resembles another pheasant more than a quail), individuals are grouped into species. Much ink has flowed in the discussion of species as "true entities" (cf. the discussion about asexual organisms, races and fertile hybrids). The subject suddenly becomes less esoteric if one sees practical implications. For example: some species and subspecies are listed as endangered. If the taxonomic classification changes, it is possible that a population of previously protected animals would suddenly be allowed to be wiped out, because the rules have changed. Another example is certain island animals, which initially were considered as unique to the island (and therefore ought to be protected), were considered invasive pest species after taxonomic reclassification. For a medical example, see the difference between limes and lemons in the chapter on vitamin C. There are more than a million different species of living organisms on our planet. Some of the most well-known or common have been given a popular name. These names, however, can be somewhat confusing, particularly in the exchange of information between people from different countries. For this reason formal Greek or Latin names are used. The reason for this is that Latin was previously the language of science. Sometimes strange linguistic hybrids are formed, e.g. " Australopithecus ": "Auster": south (Latin) and pithecos = monkey (Greek). The Swedish professor, Carl Linnaeus (1707-1778) proposed a simplification in the existing system of nomenclature. He attempted to classify all living organisms to uncover any underlying pattern in their creation (later identified with evolution). In 1753 Linnaeus published a two-volume work, " Species Plantarum " or "The Kinds of Plants"). In this work, Linnaeus used multi-word (polynomial) designations for all species of plants as a means of describing and naming them. He regarded these polynomials as the proper names for the species included in these volumes. In adding an important innovation, he laid the foundation for the binomial system of nomenclature - the one we still use today. In the margin of the " Species Plantarum ", next to the polynomial name of each species, Linnaeus wrote a single word that, when combined with the generic name, formed a convenient shorthand designation for the species. For example, catnip, which was formally named Nepeta floribus interrupte spicatus pecundularis (meaning "Nepete with flowers in an interrupted pedunculate spike"), Linnaeus wrote the word "cataria" (meaning cat-associated) in the margin of the text, thus calling attention to a familiar attribute of the plant (the plant contains the chemical nepetalactone, a  feline "aphrodisiac" which drives cats into weird affective behaviour). He and his contempories soon began calling this species Nepeta cataria , which is still its official designation today. Initially, only proper Latin of Greek words could be used, but these days, it is sufficient that the word "looks OK". There is for example the small fly Dicrotendipes thanatogratus (thanatos: dead) and gratos: gratefull, referring to the favorite rockband on the entomologist who described the little insect. In 1758 Linnaeus published the tenth edition of " Systema naturae ", in which he used the binomial system consistently, also for animals. This work represented a turning point in zoological terminology. It is due to his work that have a naming system with two parts: first the genus and then the species. E.g. Schistosoma mansoni , Escherichia coli , Aedes aegypti . If there are subspecies (races), a third word is added, e.g. Trypanosoma brucei gambiense . Thus, living organisms are divided into hierarchical groups according to the similarities in their structure. The successive groups are: Kingdom, Phylum, Class, Order, Family, Genus and Species. A mnemonic sentence to help to remember the sequence is " K ing P hillip C ame O ver F or G ood S paghetti".

Sometimes additional levels are needed. Thus, the order of Hymenoptera (membrane-winged) is classified into two large suborders: the Apocrita (wasps, bees and ants) and the Symphyta (saw-flies, wood wasps). A suborder can then be further divided into a number of superfamilies. Thus, the suborder Apocrita are split into the superfamilies Apoidea and Vespoidea.

Sometimes a suborder is subdivided into individual infra-orders (these then end with the suffix -morpha). Superfamilies end in -oidea. Families of the animal kingdom always end in -idae (in the plant kingdom the family names always end in -aceae). [Note the difference between "-ea-" and "-ae-"]. Subfamilies always end in -inae. A subfamily can be subdivided into tribes which end in -ini. Sometimes a subgenus is given and is written between brackets, e.g. Aedes (Stegomyia) aegypti . When there are species complexes, as in Simulium damnosum , reference is often made to S. damnosum s.l. (sensu lato - in the broad sense, i.e. the species complex) or S. damnosum s.s . (sensu stricto - in the narrow sense). Different groups within a complex may exhibit very different patterns of behaviour. Thus, Anopheles gambiae sensu strictu is highly anthrophilic, while the sister species Anopheles quadriannulatus is totally zoophilic and has no medical significance. The presence of the latter in an environment, however, can cause confusion in a control programme.

Example: Order: Diptera

Suborder: Nematocera

Infra-order: Culicomorpha

Superfamily: Chironomoidea

Family: Simuliidae

Subfamily: Simuliinae

Tribe: Simuliini

Genus: Simulium

Subgenus: Erwardsellum

Species: Simulium (Edwardsellum) damnosum

Initially, this can appear to be too much of a good thing, but what is most important is to have a basic idea of how medically important arthropods are classified. According to the "International Code of Zoological Nomenclature", the genus name is always written with a capital letter and the species name always with a lower case letter (e.g. Glossina tachynoides ). This applies even if the name is derived from a proper name, e.g. Culicoides grahamii . In scientific publications, genus and species name are italicised or underlined. Names also never contain an accent, apostrophe or umlaut (thus no Aëdes aegypti or Tipula o'neili ). Two words are sufficient, e.g. Mycena luxaeterna for a luminescent mushroom species (instead of Mycena lux aeterna ). The name of the genus can be abbreviated, e.g. Anopheles funestus becomes A. funestus if this does not lead to confusion or potential mistakes in the text. Sometimes the generic name is abbreviated to two letters to prevent confusion. Suppose a text contains the mention of Culiseta and Culex. If both are abbreviated to C . then it is no longer possible to know to which this refers. If Culex is identified by Cx . then clarity is restored. Sometimes the name or the initials of the discoverer of the species are included (not italicised), possibly with the year of description: e.g. Enterobius vermicularis (Linnaeus, 1758). This mention of the name, however, is optional and does not form any part of the actual scientific name. Because Linnaeus described so many species, his name is sometimes only indicated with an L. The same applies to Fabricius, whose name is abbreviated to F. The principle of this notation system is internationally accepted. In view of the fact that knowledge and opinion are constantly changing, taxonomic classifications (certainly the "middle groups") sometimes differ from author to author and according to the time of publication. There is no such thing as "The One Final Correct Classification".

There are a number of difficulties, particularly associated with the concept of "species". The species is not a constant unit. It develops and often splits up into smaller units, which are known as subspecies or geographical races. Initially a species was defined on purely morphological grounds. However, as a result situations occurred where, for example, the male and female wild duck (which differ externally) were assigned to different species. Subsequently, reproduction became the focal point in terms of taxonomy. Conventionally, the species is defined as a population which can reproduce among itself and which is reproductively isolated from other populations. This appears clear when we talk for example of humans, horses, wild ducks or rattlesnakes, but with other organisms it is much less obvious. What is the situation with the taxonomy of extinct species? What about symbiotic organisms, from lichen to protozoa, which cannot live without their symbiont? Some organisms have no sexual reproduction (for example amoebae). If there are sterile hybrids (e.g. horse x donkey-> mule), then this is an answer. Sometimes however there are fertile hybrids (some animals, many plants). Sometimes only the number of sets of chromosomes in each cell differs. A number of plants reproduce by apomixis (= parthenogenesis), i.e. formation of seeds without prior fertilisation. Small variations in the offspring are immediately fixed and from then on transmitted to the following generations. Thus, extensive series of highly similar plants differing only in minor characteristics can occur. Parthenogenesis also occurs in animals, and likewise hermaphroditism. It all means that the concept of "species" starts to become somewhat blurred in certain cases. It becomes even more complex when we include the fact of the lateral transfer of genes in the discussion. Thus, it might be that the previous history of a particular gene in an individual organism differs totally from that of another gene in the same organism (cf. transposons, see e.g. mariner element). The problem of species definition is central in biology at present. This has practical implications for example for the better understanding of the variability of diseases such as amoebiasis, leishmaniasis or Chagas' disease. Better insights into vector populations also depend on good definitions (some morphologically identical mosquitoes appear genetically to consist of various complexes with, for example, differing biting or reproductive behaviour).

Identification and classification are two related, but differing concepts. Thus, we know for example of pentastomiasis (porocephalosis) caused by infection with Armillifer armillatus . The parasite is easy to identify, but there is considerable uncertainty in terms of its taxonomic status. Blastocystis hominis is another example. The organism is "incertae sedis" (Lat. "of uncertain location"). Bacteria and the "species" concept. Bacteria tend to have small genomes compared with eukaryotes. They also tend to be very promiscuous. It seems easy for them to exchange pieces of DNA, even between unrelated species. Many bacterial species live in environments with abundant diversity of donor DNA. It is not immediately clear if the species concept can be used to categorize bacteria. Everybody will agree to the astonishing variety and enormous diversity of bacteria in nature. How do species keep their specific identity if their DNA gets swapped around so easily? Bacteria do not display a continuum mediated by promiscuous gene exchange, but seem to form clusters of genetic related strains (species). Genetic recombination results in the transfer of DNA, even between relatively distantly related bacteria, although there is a consistent decline in the recombination rate as a function of genetic distance. Barriers to these exchange processes are not immediately apparent. Are there constraints on recombination at the genomic level which potentially allow species distinctness to emerge? How distinct must clusters be for the genetic barrier to be effective enough to maintain separation? Do most clonal lines finally get extinct? Does this extinction rate somehow compensate for the continuous creation of new species? What about the role of population bottlenecks in "bloom-bust" cycles? It is likely that certain combinations of genes will be favored in certain environments, as compared to other combinations. Competition in a certain stressful environment would drive the emergence of optimal genetic combinations. Still, the amount and frequency of lateral gene transfer between bacteria is staggering. Their genomes often look like quilts. Instead of a classic Darwinian "tree of descent", they will form something more akin to a net, with branching and reconnection. When Craig Venter (of Celera genomic fame) returned from his ocean voyage in 2007, after sampling sea water in different locations for DNA analysis, he found an astonishing variety of bacterial DNA. It was expected that in open sea, where certain conditions of salinity, temperature, concentration of nutrients etc… are present, at most a few microorganisms would dominate. The opposite was found. There is still plenty to learn on this subject. Most eukaryotes can reproduce via sex. However, bacteria are not able to reproduce sexually. In bacterial reproduction, one individual divides itself into two identical descendants. Therefore, one would expect bacterial populations to have a clonal structure. However, there are three possibilities for transferring genetic information (horizontal gene transfer) between bacteria: conjugation, transduction and transformation. During conjugation, a piece of DNA  is copied in one bacterium and transferred to another via a temporary connection, a conjugative pilus. This piece is not random, but a specific mobile plasmid or transposon containing the genes which code for the transportsystem, as well as some other maintenance functions.  It typically concerns non-essential genes which are not important for the basic metabolism, but which can become important in certain circumstances. A gene or plasmid coding for antibiotic resistance for example can be transmitted in this way to another bacterium. Conjugation is widespread, but does not damage the concept of species too much: there is no random mixing of all hereditary characteristics.  In transduction, the tranfer of DNA takes place with the aid of bacteriophages. The gene that codes for cholera toxin is spread by transduction. Also small random pieces of DNA can be transported this way. In transformation, DNA which is located outside the cell (e.g. from a dead bacterium) will be imported via a specialised mechanism, after which -via recombination- the DNA will replace a piece of the original DNA in the chromosome of the host. A lot of mucosal pathogens are capable of transformation: Neisseria, Haemophilus, Helicobacter pylori, Streptococcus pneumoniae. Pneumococci can change their penicillin-binding proteins (target of beta lactam antibiotics) via transformation, diminishing their sensitivity to this class of antibiotics. Antigen variation of the pneumococcal capsule is based on transformation. This example is historically very important because it led to the identification of DNA as the carrier of genetic information. Griffith studied transformation and the antigenic variation of S. pneumoniae , but it were Avery, McLeod and McCarthy who identified DNA as the carrier of the information. Subsequently, Watson and Crick determined the double helical structure of DNA. Soon afterwards, the genetic code was cracked, and the rest is history in the making. Or at least this was thought for a while. Afterwards, it was shown that methylation patterns of the nuclear bases were important, mutations in non-coding areas can have major consequences and that so-called neutral mutations in DNA coding areas can results in very different mRNA secondary structure and dynamics, leading to strong downstream effects. Taxonomy of Bacteria Taxonomy can be divided in three major areas: nomenclature (giving names), classification (ordening of bacteria into groups based on common properties) and identification. To date approximately 5000 bacterial and archeal species are recognized. For nomenclature, bacteriologists have agreed on a set of rules for naming Bacteria. These rules are known as the “International Code for the Nomenclature of Bacteria”. The name of a bacterial species is only valid after it has been published in the International Journal of Systematic and Evolutionary Microbiology. A complete list can be found in Bergey’s Manual of Determinative Bacteriology. Classification can be artificial or natural. Artificial systems of classification are based on expressed characteristics, the phenotype (e.g. Gram staining properties, shape). Natural or phylogenetic systems are based on the purported evolution of the organism. Until the biological DNA revolution at the end of the 20th Century, all bacterial classifications were artificial, because there simply was no way to determine the evolutionary history. The Gram stain happens to be still useful phylogenetically because two of the phylogenetic groups of bacteria are gram-positive (i.e. Firmicutes and Actinobacteria). The other 20 or more are gram-negative. The systems can give different results. For example Mycoplasma sp stain gram negative, but have been found to be members of the Firmicutes through 16SrRNA analyses. They apparently have evolved from gram-positive bacteria that lost their peptidoglycan cell wall. Cell shape is still important for some groups. For example the spirochetes phylum contains all of the helical shaped bacteria with periplasmic flagella. Phylogenetic data have confirmed this grouping. Also chemical data are useful. So far, all methane-producing bacteria have been found to be members of the Archea. The definition of a bacterial species is different from that of plants and animals. A bacterial species is defined as follows: two strains of the same species must have a similar mole percent guanine plus cytosine content (mol% G+C) and must exhibit 70% or greater DNA-DNA reassociation. A common procedure to determine GC ratios is by thermal denaturation (“melting” the double stranded DNA). The hydrogen bonding of a GC basepair is stronger than an AT basepair, and therefore, higher temperature is needed for DNA with a higher GC content. The strain separation is tracked with a spectrophotometer set at 260 nm, a wavelength that DNA absorbs strongly. Bacteria multiply by fission and a lineage of bacteria is determined in large part through this vertical inheritance. However, bacteria can also acquire genetic material via lateral or horizontal gene via conjugation, transformation of transduction. For conjugation, think of bacterial sex via pili, e.g. plasmid exchange. Transformation refers to the uptake of naked DNA. Think of Avery’s experiment with Streptococcus in which DNA was proven to be the carrier of genetic information. Transduction occurs via bacteriophages. As a result of lateral gene transfer, prokaryotic organisms may undergo dramatic changes, such as multidrug resistance. If horizontal gene transfer would happen too often, it would pose a major problem for phylogenetic classification. Luckily, it does not pose a major problem for 16S rRNA gene trees. These days, numerical taxonomy is commonly used at species and strain level, when phylogenetic relatedness has already been established by ribosomal RNA sequencing and DNA-DNA reassociation. In this system, all characteristics are given equal weight. Each strain is compared with every other strain. A similarity coefficient S between two strains A and B is defined as S AB = a/(a + b + c), where a represents the number of properties shared in common by strains A and B; b represents the number of properties positive for A and negative for B, and C represents the number of properties positive for B and negative for A. It is best to have as many tests of phenotypic characteristics as possible, typically at least 50 independent characters. Typically S AB values are greater or equal than 70% within a species and greater than 50% within a genus.

Parasite genome initiatives

The revolution in molecular biology, which began in the second half of the 20th century has also had an impact on the study of various organisms that cause problems in tropical countries. Various initiatives have been launched to map the genetic material of eukaryotic parasites such as Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Trypanosoma brucei, Leishmania major and Brugia malayi. Various prokaryotes have also been sequenced and are intensively studied and annotated, from Rickettsia prowazekii to Salmonella typhi. The first completed genomes from viruses, phages and organelles were deposited into the EMBL Database in the early 1980's. It was in 1995 that the first bacterial genome was sequenced ( Haemophilus influenzae).   In 1996 Saccharomyces cerevisiae (baker's yeast) was the first eukaryote genome sequence to be released. This was followed in 1998 by the first genome sequence for a multicellular eukaryote, Caenorhabditis elegans . In 2001 a draft of the human genome was published, followed in 2006 with the complete sequence. By mid-2007, genomes from more than 350 bacterial species and strains had been sequenced, including most human bacterial pathogens. At present, it seems like nearly every week, another genome is sequenced. The above number of sequenced genomes was outdated from the moment it was written. To give an example, in the first half of November 2009, an additional 40 genomes were deposited in the international EMBL databank.  Massive parallel signature sequencing will allow the identification of the transcriptional activity of many genes. Sequencing is only a first step. Annotating the genomes is next, a daunting task. Acquiring information has been likened to trying to take a sip of water from a firehose. It will be a considerable challenge to make effective use of the mass of information that has become available. Analysis of genome sequences from organisms (veterinary pathogens as well as human pathogens) as diverse as Anaplasma marginale, Borrelia hermsii and Trypanosoma brucei suggests convergent evolution to common mechanisms of antigenic variation and immune evasion. Gene conversion is a convergent strategy for pathogen antigenic variation. Common pathogen features in tick-born Rickettssiae, such as decreased metabolic capacity and deficiencies in certain biosysthetic pathways (e.g. ability to synthesize amino acids), have been identified. For vector borne pathogens, the presence or absence of transovarial transmission and adaptation to a hematophagous vector are important. In the host, the pathogens encounter the same or similar hostile environment, for which similar solutions have to be present (e.g. host hemostasis when a vector bites, complement inactivation, avoidance of digestion in a phagolysosome, ...). A recurrent theme is the expansion of families of immunodominant outer membrane proteins, facilitating antigenic variation. Hypervariable gene families adjacent to the telomeres are common. To give an example: Lateral DNA transfer from T. cruzi to the human genome, including the human germ line, has been demonstrated. In such interspecies DNA transfer, mitochondrial minicircle DNA was integrated into human chromosomes, leading to subsequent inheritance by the children. Much more information will become available in the near future. Synthetic biology and designer organisms seem not to be so far off, even when clean water for all is still a pipe dream.  Let's hope that all this new information will lead to practical solutions for existing problems.

Diagnosis

There are various ways of reaching a diagnosis. The saying: "One recognises only what one knows" is of great importance. Knowledge of diseases and pattern recognition are the basis. Recognition of clinical presentations and reaching a diagnosis is the outcome. In the case of infectious diseases, an attempt can be made to detect the pathogenic organisms directly by microscopy (for example thick smear for malaria, Ziehl-Neelsen staining of sputum for pulmonary tuberculosis, faecal specimen for amoebae, bone marrow aspirate for visceral leishmaniasis, etc). Cultures and serological tests are usually difficult or impossible in rural areas. Radiology and ultrasound are mostly of limited availability.

A patient will have certain complaints: symptoms, examples of which are neck pain, cough or loss of strength in the legs. There will also usually be objective signs identified by the physician treating the patient. Examples are neck stiffness, crepitations and hyperreflexia. One and the same disease may take different forms in different people. There is a spectrum of manifestations: there is individual variability (for example immunological resistance) and furthermore the symptoms and signs depend on the stage of the disease. Sometimes the degree of infection (for example worm load) is important. Whether a particular symptom, for example blood in the urine (haematuria), is highly indicative of a specific diagnosis depends on the local frequency (prevalence) of the disorder (for example, bilharziasis is frequent in Africa but not in India). A symptom may be specific to a greater or lesser extent, for example fever can be caused by numerous diseases. Fever is thus fairly aspecific for a certain diagnosis. Muscle spasms triggered by sudden noise are strongly indicative of tetanus. This sign rarely occurs in other diseases and is thus relatively specific for tetanus. Often, a definite diagnosis is not possible and a probable diagnosis must be established: the disease that is most likely in the differential diagnosis. The differential diagnosis is the list of those diseases that might explain the patient's clinical picture. It is not advisable to make a long list by including all sorts of rare possibilities. By definition, rare diseases are always rare. It is, however, important to think of a rare disease if it is severe and treatable in the given circumstances.

Treatment

Priorities play a major role. For example: with a limited budget, a renal dialysis unit will not be built at the expense of everything else. "What is the importance for this patient?" must be asked, but: "What is the importance for public health?" should also be considered. In the choice of medications, cost price and availability are also of importance. The WHO has compiled a list of essential medicines. Since the the discovery of penicillin in 1928 and it's production in 1938 and Prontosil and related sulfa drugs after 1932, antibiotic resistance has been on the rise. This is currently threatening many gains in infectious diseases which have been made over all those decades. The first penicillin-resistant bacteria where already identified before penicillin came on the market in the 1940s, methicillin-resistance was documented in the 1980s and vancomycin-resistance in the 1990s. It is estimated that more than 50% of the world production of antibiotics is for use in the agricultural sector, not so much to cure sick animals by veterinarians but for food additives. New threats include multiresistance in a multitude of pathogens including Plasmodium falciparum, Mycobacterium tuberculosis , methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci and the appearance of Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM-1) in several Gram-negatives in the first decade of the 21st Century. Since (1) Gram-negative bacteria easily share resistance genes across species, (2) less new antibiotics are discovered due to several reasons, (3) and the increase in world travel -both numbers of people and speed of travel-, the conditions for a perfect storm ("total resistance") are in the making, threatening to bring us back to conditions similar to where we were during World War One. The bottom line in pharmaceutical industries is financial gain. The development of new classes of drugs to treat multiresistant bacteria is rather challenging. Even if succesfull, the drugs will probably be used for a short period of time before resistance arrives, and therefore are not considered to be worth the great expense of research and development time. Better incentives (e.g. longer patent protection) are needed. Maybe formal agreements such as were used during poliovaccine development are needed to protect companies against financial disaster. Basic as well as applied research needs to be boosted. Let's hope that we won't need a Manhattan-type of project if things seriously get out of hand. In our direct workplaces as doctors and nurses, meticulous desinfection of hands and surfaces will need to be instituted to limit spread and outbreaks.

Prevention

Prevention is better than cure. Sometimes prevention is the only feasible measure (for example AIDS).

Prevention is based on:

Vaccination: for example measles, polio, diphtheria, tetanus, whooping cough, yellow fever

Chemoprophylaxis: for example the regular intake of antimalaria tablets

Interruption of transmission. A good knowledge of the biological cycle of the pathogen is necessary for this. For example, control of the tsetse fly for sleeping sickness. Interruption of epidemic typhus transmission by delousing.

Information, health education and encouragement of personal hygiene e.g. via school.

Genetic counselling has its place in a number of hereditary diseases.

Clean drinking water and food, use of good sanitary facilities. Quality control of food and drinking water is essential if it can be coupled to action to improve the situation.

Food supplements: e.g. vitamin A, iodine deficiency

Rapid isolation and treatment of infectious diseases (e.g. Ebola fever, open pulmonary tuberculosis, plague).

Epidemiological surveillance (regional, national, international) is important.

Combating poverty is the best prevention

Useful manuals: Tropical medicine

Medical practice in developing countries by Krawinkel (ISBN 3-8243-1276-X).

Médecine tropicale by Gentilini & Duflo.

Manson's Tropical Diseases by Cook and Zumla (published by Saunders, latest edition Dec 2008).

Tropical Medicine and Parasitology by Goldsmith (published by Prentice-Hall International).

Care of the critically ill patient in the tropics and subtropics by Watters (published by McMillan).

100 Clinical Problems in Tropical Medicine by Harries (published by Baillière Tindall). Common Medical Problems in the Tropics. Ed: Schull. MacMillan Publishers. ISBN 0.333.67.999.7

Lecture notes on Tropical medicine by Bell (published by Blackwell Science). On line exercises (McGill University): http://www.medicine.mcgill.ca/tropmed/cantropmed/Default.htm

On line clinical cases Gorgas course (Peru): see http://gorgas.dom.uab.edu/ On line: http://www.merck.com/pubs/

On line tropical radiology: http://tmcr.usuhs.mil/

On line mycology: http://www.mycology.adelaide.edu.au/Mycoses/ On line medical algorithms (requires free log-in): http://www.medal.org/Visitor/login.aspx

Reflection and Desiderata

The current lecture notes have grown out of a modest number of pages in the last decade of the 20th Century and the first of the 21st, aimed at medical students following courses at the Institute of Tropical medicine, Antwerp, Belgium. It took a fair amount of work to bring the texts to their current state. The resulting compilation is rather large, sometimes to the despair of students, when I go of on a tangent.  My excuse is that it is a work of love, and very much, it is a work in progress. Medicine is far from static. The work facing clinicians in the tropics mainly consists of cosmopolitan problems. The classic tropical diseases will probably become less important, but stay relevant for the individual who is affected by one. This while obesity, hypertension, heart disease are increasing in the "third word" and a massive diabetes epidemic is unfolding. A tsunami of new data threatens to overwhelm scientists, although creative use of networked silicon chips helps. Computer power still seems to follow Moore's law at present.  Many important changes are expected in the near future. In society, the population in the West is aging with all its expected problems. Overpopulation, habitat destruction, loss of biodiversity, climate change, pollution, exhaustion of some natural resources are some of the problems facing us and our children. Access to clean water will become worse for a large part of the world population and might lead to new wars. All in all, wisdom is the one thing that still eludes us. In medicine, we would like not only to form highly qualified professionals, but also balanced caring people with a gentle touch and a spark in their eyes. The illustrations and case studies discussed during the actual teaching course will help to identify the important points. Sometimes, after all the studying, it is nice to let your mind drift and reflect on the big picture and dream your dream(s).  The following has nothing to do with tropical medicine, but I would like to include here one of my favorite poems, just because I think it is beautiful. It was written by Max Ehrmann in 1927.

DESIDERATA Go placidly amid the noise and the haste, and remember what peace there may be in silence. As far as possible, without surrender, be on good terms with all persons. Speak your truth quietly and clearly; and listen to the dull and ignorant; they too have their story. Avoid loud and aggressive persons; they are vexations to the spirit. If you compare yourself with others, you may become vain or bitter, for always there will be greater and lesser persons than yourself. Enjoy your achievements as well as your plans. Keep interested in your career, however humble; it is a real possession in the changing fortunes of time. Exercise caution in your business affairs, for the world is full of trickery. But let this not blind you to what virtue there is; many persons strive for high ideals and everywhere life is full of heroism. Be yourself. Especially do not feign affection. Neither be cynical about love; for in the face of all aridity and disenchantment, it is as perennial as the grass. Take kindly the counsel of the years, gracefully surrendering the things of youth. Nurture strength of spirit to shield you in sudden misfortune. But do not distress yourself with imaginings. Many fears are born of fatigue and loneliness. Beyond a wholesome discipline be gentle to yourself. You are a child of the universe, no less than the trees and the stars and you have a right to be here. And whether or not it is clear to you, no doubt the universe is unfolding as it should. Therefore, be at peace with God, whatever you conceive Him to be. And whatever your labours and aspirations, in the noisy confusion of life, keep peace with your soul. With all its sham, drudgery and broken dreams, it is still a beautiful world. Be cheerful. Strive to be happy.  

Notice

The editors of this work have checked with reliable sources in their efforts to provide information that is complete and generally in accord with the standards accepted at the time of publication. In the view of possible human error or changes in the medical sciences, neither the editor nor the publisher nor any another party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete. They are not responsible for any errors or omissions or the results obtained from such information. In particular, readers are advised to check the product information sheet included in the package of each drug they plan to administer to be certain that the information contained in this CD-ROM is accurate and that changes have not been made in the recommended dose or in the contraindications for administration.

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights to translation, reprinting, reuse of illustrations, recitation, reproduction on microfilms or in other ways, and storage in data banks. The use of registered names, trademarks, etc. in the publication, does not imply even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Acknowledgements

This edition of this CD-ROM (anno 2012) was conceived to become the Illustrated Lecture Notes on Tropical Medicine, in the first place for the students at the Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium, but also for interested people working or studying elsewhere. Writing a work with a scope as broad as this one required the assistance of many people. We are grateful to the following persons for their help, contributions, their generous comments and suggestions : Alex Van den Daele, nursing staff and management Ann Stevens, MD Colonel Raymond Wouters, MD Emmanuel Bottieau, MD, PhD Soentjens Patrick, MD Jan Clerinx, MD Correwyn, San-Ho (Graphics/Technics) Jean Pierre Wenseleers (Graphics) Lut Lynen, MD, PhD Prof Alfons Van Gompel, MD Prof Bob Colebunders, MD PhD Prof Bruno Gryseels, MD PhD Prof Herwig Leirs, PhD Prof Jef Van den Ende, MD PhD Vekemans Marc, MD Vlieghe Erica, MD Zolfo Maria, MD The Welcome Foundation, London (certain pictures) Special thanks to the golden fingers of San-Ho Correwyn, without who this project would not have been possible, and the patience and support of my wife Yuki Hori. Enjoy the study!   Erwin Van den Enden, MD Institute of Tropical Medicine Nationalestraat 155 Antwerp, Belgium

Summary

Malaria is very common; a very important cause of mortality and morbidity Four main parasites: Plasmodium falciparum, P. vivax, P. ovale, P. malariae Occasionally infection with zoonotic malaria, such as P. knowlesi, regionally important (Southeast Asia) Transmission via female Anopheles mosquitoes which bite at night Symptoms: atypical, fever, anaemia, kidney failure, splenomegaly, cerebral malaria  Infections often asymptomatic in semi-immune people (generally low parasitaemia) Clinical diagnosis not reliable. Often clinical over-diagnosis of malaria and under-diagnosis of other disorders in endemic areas Problem of chronic malaria carriers with another disorder Diagnosis via thick smear, thin smear, rapid antigen-detection, DNA-based methods Treatment of P. malariae : chloroquine Treatment of P. vivax and P. ovale: chloroquine if possible with primaquine (hypnozoites, G6PD). Resistance of P. vivax to chloroquine is rising in several areas Increasing multidrug resistance of P. falciparum, including resistance to artemisinin derivatives Combination treatment of  P. falciparum infection is strongly advised. ACT: artemisinin combination treatment (e.g. artemether + lumefantrine; = Co-Artem, Riamet). Avoid monotherapy with artemisinin derivatives) Quinine + (doxycycline or clindamycin) Atovaquone with proguanil (Malarone) Individual prevention via pyrethroid-impregnated bednet ± chimioprophylaxis Stand-by self-medication for certain travellers Population protection via vector control Resistance of mosquitoes to various insecticides Vaccination is still in the experimental stage Fighting counterfeit medication via authentication codes relayed by instant text messaging

History

History, discovery of the parasite

Malaria has been with humanity since millenia. The most famous historical case of falciparum malaria is probably King Tutankhamen, the boy pharaoh from Old Egypt, in whose 3,000-year-old mummy the parasite was demonstrated. Although usually associated with the tropics, malaria was endemic in North America and large parts of Europe until the middle of the 20 th century. Malaria transmission occured in Belgium, the Netherlands, Sweden, Finland and the United Kingdom. It was a significant impediment for the European nations during the colonial period. In Northern Europe, only P. vivax and P. malariae occured. In Southern Europe, malaria was due to infection with P. falciparum, P. vivax and P. malariae . It is not clear how much the Mediterranean strain of P. falciparum was different from tropical P. falciparum .  In the Middle Ages, the word "ague" (from French "aigue") might be used for any febrile illness. It's use became more restricted in the 18th Century in order to describe illness with recurrent fever. The effects of kina-bark were not clear (role of dose, origin of the bark, adulturation, concentration of active ingredients, duration of treatment, timing, etc). There is a lot of doubt that the Walcheren malaria epidemic in 1809 was actually due to malaria (idem for the 1826 epidemic in Groningen, North Holland).  Malaria played an important part in the wars of the 19 th and 20 th centuries. For many years it had been known that people who died of malaria had large amounts of black pigment in their liver, kidneys, spleen and bone marrow. Yet the cause of this disorder long remained a riddle. In 1880 the French army doctor Charles Louis Alphonse Laveran discovered malaria parasites in fresh blood from malaria patients in the coastal town of Bone (Annaba), Algeria. He was also the first to observe exflagellation (see below), which normally only takes place in the stomach of the mosquito. The names which were given to the microscopic organisms were " Laverania falcipara " and " Oscillaria malariae ". At that time there were as yet no staining techniques (Romanowsky stains were introduced in 1891), and microscopes were monocular, quite primitive and with a limited magnification. In 1884 the German Ernst Karl Abbe, together with Carl Zeiss, developed the oil immersion lens and a few years later the optical condenser, which allowed a greater magnification and clearer, sharper views. In 1891, the Russian Dimitri Leonidovich Romanowsky (1861-1921) and Ernst Malachowski both developed a staining method based on water-soluble eosin (anionic), methylene blue (cationic) and absolute methanol. Methanol was used because aquous dye solutions were unstable. This staining techniques made microscopic identification easier. Variants of these stains include Giemsa, Leishman and Field stains. The findings of Laveran could now be verified by others. Laveran received the Nobel prize in 1907.

'Laveran medal of the ''Societe de Pathologie Exotique''. Laveran was the first to describe the malaria parasite in 1880. Copyright Wellcome'

History, staining methods

The study of dyes led to the development of various techniques for staining bacterial preparations and histological sections. Romanovsky stains are based on a mixture of basic dyes (positively charged) such as methylene blue, and anionic dyes (negatively charged), such as eosin. The positively charged dye will bind to negative structures (nucleic acids, ribosomes), and the negative dye will attach to positively charged structures (many cytoplasmic proteins). The specific mixtures, techniques and variants were named after those who developed them, e.g. Giemsa, Wright, May-Grünwald.

History, discovery of transmission

The transmission of malaria had for long been a mystery. One of the researchers was the Briton Sir Ronald Ross. He left for India with a personal mission to prove transmission via insects. In 1897, after three years of hard work, he demonstrated parasites in mosquitoes which had bitten patients. Later he also demonstrated transmission of avian malaria via mosquitoes ( Plasmodium relictum transmitted from one sparrow to another via Culex fatigans ). He was able to describe the complete development of the parasite in the mosquito and also demonstrated that transmission took place via the bite of the mosquito (and not via the presence of dead mosquitoes in drinking water, as his mentor Patrick Manson had initially thought). For this he received the Nobel prize in 1902. Subsequently the Italian Giovanni Battista Grassi and the Briton Patrick Manson confirmed that human malaria could be transmitted by Anopheles mosquitoes by carrying out extensive experiments in Italy and by allowing Manson's own son to be bitten by Anopheles mosquitoes which had fed on a patient with P. vivax malaria. Plasmodium ovale was discovered much later, in 1922. The realisation that P. knowlesi is responsable for frequent human misery in Southeast Asia is much more recent, in the last decades.

General

General, genus Plasmodium

Plasmodium falciparum trophozoites in thin blood smear. Copyright ITM

Plasmodium malariae in thin blood smear. Copyright ITM

Plasmodium vivax in thin blood smear. Schizonts have numerous nuclei. Copyright ITM

Plasmodium ovale in thin blood smear. Notice the elongated shape of the erythrocyt. Copyright ITM

Malaria is the common name for diseases caused by infection with single-celled parasites of the genus Plasmodium . They belong to the Apicomplexa. Among the parasites of the genus Plasmodium five species have been identified which regularly cause disease in humans:

Plasmodium falciparum Plasmodium vivax Plasmodium ovale (subdivided in P. ovale wallikeri and P. ovale curtesii ) Plasmodium malariae Plasmodium knowlesi Human malaria parasites have a restricted host-specificity. They don't develop disease in rabbits, rats or mice, but need to be maintained either in human volunteers or in primates. Human parasites infects only a few primate species (including the chimpanzee, the owl monkey and the squirrel monkey), but the course of infection and the pathology resulting from this infection are different from that in man. A common used less-than-optimal substitute is to perform experiments on primate, rodent or avian malaria parasites in their natural host. Most animal models are inadequate and, while they can help the researcher in answering specific questions, any extrapolation to human disease has to be considered with extreme care. For example, dexamethasone was considered to be useful in severe malaria caused by P. knowlesi in rhesus monkeys, but was found to be harmful in humans.

Simian zoonotic malaria

In 1908 Daniels found malaria parasites in Macaca fascicularis monkeys. Via inoculation studies of volunteers, he was able to proof the parasites were infectious for humans. Monkeys infected with malaria parasites usually show few if any symptoms, with the exception of P. knowlesi infection in Rhesus monkeys. The natural hosts of P. knowlesi are Macaca fascicularis  (long-tailed macaque) and Macaca nemetrina (pig-tailed macaque). In 1927 Julius Wagner-Jauregg won the Nobel prize for his discovery of malaria therapy for treatment of late stage neurosyphilis. However, this iatrogenic-induced malaria infection could rapidly become uncontrollable. After several fatalities, its use was discontinued in favor of the less virulent Plasmodium vivax . In 1932 Knowles and Das Gupta studied experimental transmission of monkey malaria to man. Simian malaria gained prominence again in 1960 when P. cynomolgi accidentally infected a researcher via a stray mosquito bite. The first natural infection by P. knowlesi of an American man was recognised in 1965 in Pahang, Malaysia. Another similar infection was diagnosed in Johore (peninsular Malaysian province close to Singapore). Further study showed that their are at least 17 different simian malaria species, of which 7 can be transmitted to man. These include four Asian species ( P. cynomolgi, P. knowlesi, P. inui, P. eylesi ), two species of the New World ( P. brasilianum, P. simium ) and one African species ( P. schwetzi ). In 2009, P. gaboni was identified in chimpanzees. This parasite is closely related to P. falciparum and P. reichenowi . In the past it was thought that because of their rare occurrence they posed no problem in daily clinical practice in most places. It is also possible that due to their rarity and /or their morphological similarity to human parasites, these zoonotic infections are missed in the normal clinical setting. As more PCR and cloning / sequencing is done, we hope to get a better understanding of their role in malaria pathology. An example of the value of molecular techniques in malaria research was the discovery of a cluster of human P. knowlesi infections in Sarawak. Afterwards, infections were identified in the rest of Borneo (including Kalimantan), Thailand, Myanmar, Yunnan (China), Singapore and the Philippines. P. knowlesi  has a shorter life cycle (24h) than P. malariae (several days), and leads to higher parasitemia, more aggressive pathology, including occasionally mortality. Young P. knowlesi trophozoites resemble P. falciparum. Older stages resemble P. malariae very closely. Ref: A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet. 2004 Mar 27;363:1017-24. White N (editorial)  Plasmodium knowlesi : The Fifth Human Malaria Parasite. Clin Infect Dis 2008:46;172-173. Clin Infect Dis 2008;46:165-171.

General, subgenera and subspecies

Sometimes subgenera are used: Plasmodium (Plasmodium) vivax , P. (P.) ovale, P. (P.) malariae and P. (Laverania) falciparum. Only P. (L.) falciparum and P. (L.) reichenowi belong to the subgenus Laverania . Lemurs and lower mammals are sometimes infected with Plasmodium sp. which are classified under the subgenus Vinckeia . P. vivax has some subtypes: P. vivax hibernans , a strain adjusted to moderate and cold climates, P. vivax chesson   and P. vivax multinucleatum . There may be other subtypes and P. vivax -like parasites. The names of the different strains of P. falciparum are not generally accepted ( P. f. immaculata, quitidiamum, tenue, perniciosa, aethiopicum . We shall not discuss subgenera any further.

General, malaria in animals

There are several Plasmodium species which cause infections in certain animals. Several mammals, birds and reptiles have their own form of malaria. Thus for example P. berghei and P. chabaudi are known in rodents. P. gallinaceum, P. relictum, P. elongatum and P. lophurae cause infections in birds. Chickens and even penguins (zoos!) may be infected, with serious consequences for these animals. Avian malaria has severly affected native (non-immune) bird populations in geographically isolated islands, such as Hawaii, after the introduction of the vector and the parasite. Even lizards have their own form of malaria. Some of these animal malaria parasites are used in experimental research of the malaria problem. P. cynomolgi is often used as a model for P. vivax infections. Another much-used model is P. falciparum infection in Aotu s monkeys, but the physiopathology in these animals does not correspond with the course of illness in humans. P. fragile and P. coatneyi can cause cerebral lesions in Rhesus monkeys ( Macaca mulatta ). These parasites are often used as an animal model for severe P. falciparum infections. The chimpanzee parasite P. reichenowi has about 20 times more genetic diversity than its very close relative P. falciparum , suggesting that P. falciparum started to diverge from it since about the end of the last Ice age. Since some great apes can be infected with certain human malaria parasites, theoretically they could form a reservoir. The life-cycle of avian malaria parasites is similar to that of mammalian parasites, but with some differences: avian malaria transmission is mostly by Culicine mosquitoes instead of Anophelines. more than one cycle of exo-erythrocytic schizogony takes place in reticuloendothelial cells, rather than hepatocytes. Exo-erythrocytic merozoites can reinvade reticuloendothelial cells and these exo-erythrocytic stages may be found in circulation in peripheral blood. There is a small number of pre-erythrocytic merozoites (less than 100, compared to 2,000-20,000 in mammalian parasites) Since avian erythrocytes contain a nucleus, erythrocytic schizogony of avian malaria  takes place in a nucleated cell Avian parasites are less host specific than mammalian parasites. When used in experimental models, the parasites are used in a convenient domestic bird rather than their natural host. Some avian malaria parasites have been extensively used in the development of experimental models and identification of antimalarials drugs.  - P. relictum and P. elongatum in passerines and canaries - P. lophurae and P. cathemerium in ducklings - P. fallax and P . durae in turkeys - P. gallinaceum in chickens P. relictum was the first standard drug screen in the search for new synthetic antimalarials in the 1920's, which identified pamaquine and mepacrine. P. cathemerium and the P. gallinaceum were used in screening drugs for their antimalarial activity. This led to the identification of proguanil, pyrimethamine, primaquine and chloroquine as antimalarials.

The malaria genome sequencing project

Three genomes per parasite: nucleus, mitochondrium and apicoplast At present, the genome of P. falciparum , as well as the vector Anopheles gambiae , and the human genome have been sequenced. As genetic analytical techniques improve, better understanding of the diversity of malaria parasites becomes available, witness the recent discovery that P. ovale subdivides in P. ovale wallikeri and P. ovale curtesii. During the life cycle, the malaria parasite is haploid with the exception of a brief phase after fertilisation (ookinete). In 1996 an international project was started to sequence the complete genome (23 Megabasepair) of P. falciparum. It is estimated that P. falciparum has about 5200 genes. The number depends upon the type of software used to search the databank. The nuclear genes are located on 14 chromosomes. Besides the nuclear genome, there is DNA in the mitochondria and in the apicoplast. The discovery of a nonphotosynthetic plastid in malaria was unexpected. Multiple lines of evidence indicate that the extant plastids of apicomplexans and dinoflagellates were inherited by linear descent from a common red algal endosymbiont. The apicoplast resembles a plastid (cf. photosynthesis in the chloroplasts of plants). Apicomplexa however are not photosynthetic. The cell organelle is surrounded by four membranes, which suggest an endosymbiotic origin. The apicoplast is transmitted via the macrogamete.  The apicomplexan plastid is involved in several biosynthetic pathways such as production of fatty acids for the cell. Some of these essential pathways use enzymes that are only found in other plastids or prokaryotes. It is therefore possible that drugs or compounds that inhibit these plastid enzymes specifically would harm the parasite without affecting the host cell (human cells don't have plastids).  

The Sanger Centre (UK), The Institute for Genomic Research (TIGR) in Rockville, Maryland (USA) and Stanford University in Palo Alto, California (USA) co-operated in this project. A large proportion of the costs was borne by the Wellcome Trust Foundation and the NIH (National Institutes of Health, USA). A rough genetic map was produced by the end of 2000. A nearly complete genome sequence of the 3D7 reference strain of P. falciparum was published in the October 2002 issue of Nature. Genomic DNA from both males and females of the PEST strain of Anopheles gambiae was also sequenced. The PEST strain was derived from a cross between a laboratory strain and a field-collected isolate of A. gambiae . This strain was choosen for several practical reasons. 

Life Cycle of parasites

After the cause and transmission of malaria became known, it was logic to assume that the parasites inoculated via a mosquito bite would directly penetrate red blood cells. This wrong idea was proposed in 1903 by Fritz Schaudinn, a distinguished German microscopist. It was based on faulty observation and due to his authority, it entered some textbooks. It was known that when blood from a patient with active malaria was inoculated into a healthy volunteer, the volunteer would develop malaria and would become infectious nearly instantaneously. However, when a volunteer was inoculated via a mosquito bite, the blood was not infective for 6 days (in case of P. falciparum ) to 9 days ( P. vivax ). Why? This was a vexing problem which took decades to answer. It was by very careful animal experiments with P. cynomolgi , a primate malaria species, that the puzzle was solved. Shortt and Garnham collected a large number of infected mosquitoes, mashed them to pulp and injected the lot (including sporozoites) into monkeys. After waiting a period, they killed the animals and searched the various organs and tissues. The parasites (with a different shape) were found in the liver. They had to support their hypothesis of the existence of a pre-erythrocyt stage with a species of human malaria. They used P. vivax and a human volunteer. This man was inoculated IV with sporozoites isolated from 200 mosquito salivary glands. A week later, the volunteer was operated on and a piece of liver tissue was obtained. The parasites were present in the liver. A year later they obtained a strain of P. falciparum , infected 770 mosquitoes and inoculated another human volunteer. About 6 days later, a liver biopsy was taken and again the parasite was found. The following is the life cycle as it is known today: When a mosquito alights on the skin, it attempts to pierce a small blood vessel with its proboscis in order to suck blood. To prevent the blood from coagulating and thromocytes to activate and clump, the mosquito first injects some saliva. Besides vasodilating agents this saliva contains anticoagulantia and enzymes such as apyrase which inhibit blood platelet aggregation (apyrase hydrolyses ADP, a substance which promotes the aggregation of thrombocytes). However, the saliva may also contain micro-organisms. When a human is bitten by an Anopheles infected with malaria, parasites (sporozoites) [Gr. sporos = seed, spore; zoon = animal] are introduced into the human body. On average 10-20 sporozoites are injected per bite, although this number can reach higher, e.g. 100. Initially, the sporozoites follow an erratic pathway through the dermis with a far greater velocity than sporozoites in artificial media or in the mosquito proboscis. When they encounter a capillary or lymph vessel, the sporozoites probe their way into the vascular lumen. A minority of the inoculated sporozoites arrive in regional lymphnodes. There is no further development in the local dendritic cells, but in the endothelial cells, some forms develop which resemble early liver schizonts. It is doubtful that these parasites mature fully or produce progeny, although they may affect immunity. Clearly much more needs to be learned. Although in theory, a single sporozoite might be sufficient to induce an infection, there is a difference between theory and practice (especially in practice). The real-world probability of malaria infection after an infective anopheline bite is 30 to 50%. The salivary glands of a lab-bred infected mosquito may contain 10,000 to 200,000 sporozoites. A naturally-infected mosquito usually carries much fewer, at most a few thousand sporozoites. Each droplet of saliva produced during probing during a blood meal will transfer a few sporozoites to the host, on average about 10, with less than 10% of the droplets containing more than 100 sporozoites. Some sporozoites will get stuck in the skin during probing or will be carried by the lymphatic fluid to the local lymph nodes. Some sporozoites will enter a blood vessel where a percentage will be destroyed by local immune cells. Of those which reach the liver (generally within 30'), some will be destroyed by Kuppfer cells, and the remaining will penetrate one or several hepatocytes before coming to rest. A certain protein of the parasite, (the circumsporozoite protein, CSP), plays an important role in the penetration of the sporozoite into a liver cell (cf. Mosquirix vaccine). Sporozoites reproduce asexually in liver cells, by schizogony [Gr. schizo = split, divided; cf. etymology of schizo phrenia, a "split" mind]. This is called exo-erythrocytic or pre-erythrocytic reproduction. This part of the life cycle was first described by Shortt and Garnham in 1948. The parasite grows and undergoes several nuclear divisions without the cytoplasm dividing. It reaches a diameter of 30-70 µm (for P. falciparum ). The form of the parasite produced in this way is called a liver schizont. No malaria pigment is present because no hemoglobin is consumed by the liver stage, because liver cells do not contain hemoglobin. Division of the cytoplasm follows nuclear division and the multinuclear schizont splits into many thousands of small offspring (merozoites) [Gr. meros = part; cf. etymology poly mer ]. Every successful sporozoite can produce some 20,000 merozoites. These initially measure only 0.7 to 1.5 µm. After some time the infected liver cells burst and the merozoites enter the blood stream. While the parasites are still reproducing in the liver, there are no symptoms. Neither the sporozoites, nor the liver forms are sensitive to most of the drugs used in malaria prophylaxis (Malarone is an exception). The minimally required time from infection to the appearance of the first merozoites, is the prepatent period. The incubation period is somewhat longer because signs and symptoms do not appear until the parasitaemia is sufficiently advanced.

In the case of P. vivax and P. ovale only some of the infected liver cells burst. The parasites in the liver cells which do not burst (hypnozoites) [Gr. hypnos = sleep; cf. hypnosis] may remain viable for years and are responsible for new attacks of the disease if reactivated. The trigger which reactivates the hypnozoites is not known. The existence of hypnozoites in P. vivax was only formally demonstrated in 1985, via fluorescence microscopy. Reactication of these "sleeping" forms explains delayed exacerbations of the disease after treatment with chloroquine. Chloroquine kills the blood forms, but not the liver forms. Hypnozoites are not present in P. falciparum and probably not in P. malariae (although this is controversial). This is important for treatment, because hypnozoites are not sensitive to chloroquine, quinine, mefloquine or artemisinin. Accidental inoculation with infected blood (blood containing trophozoites) may lead to infection, e.g. transfusion malaria or malaria via shared contaminated syringes by drug users. Since the infection in these cases is not transmitted by sporozoites, there are no liver forms. Liver forms are also absent in congenital malaria. This is important for treatment (no primaquine for congenital malaria with P. vivax or P. ovale ). The chronic nature of infections with P. malariae is traditionally explained by assuming that the parasite can induce a very low parasitaemia for many years, which is below the detection threshold of normal diagnostic methods. More study is needed on this subject.

The merozoites carry certain proteins on their membranes (MSP-1 or merozoite surface protein-1). This MSP-1 binds to the surface of the erythrocyte. The merozoites subsequently penetrate the red blood cells. Precisely how the parasites penetrate the red blood cells without haemolysing them is not yet completely understood. They apparently use an unusual actin-myosin molecular motor. P. vivax uses the Duffy blood group antigen to enter the erythrocyt. P. falciparum uses glycophorin to enter the red blood cell, but the details are rather complex and only partially understood. The Duffy blood group antigen is a receptor for a family of proinflammatory molecules ("Duffy Antigen Receptor for Chemokines" (DARC) or the Duffy chemokine receptor. DARC might play a role as a scavenger to eliminate excess of toxic chemokines produced in some pathologic situations. Duffy protein has functions in the brain, spleen and kidneys as well. A separate DNA enhancer sequence is responsable for the expression of Duffy antigen on different cell types. In most West Africans, a T-to-C mutation in the one of the Duffy enhancers prevents the expression of the Duffy antigen on red blood cells, but the antigen is still present on other cell types. Once in the red blood cell, the parasites remain in a vacuole in the erythrocyte. Some researchers think that there is a pathway between the extracellular space and the vacuole containing the intracellular parasite. This system could explain how macromolecules get into the parasite (erythrocytes have no capacity for endocytosis). This concept is, however, controversial and could possibly be due to an in-vitro artefact. The MSP-1 protein is highly variable and the parasite may change structural details even within the course of a single infection. The parasite uses this to escape the host's immunological defence mechanisms.

In the red blood cell the parasite feeds on haemoglobin. The form of the parasite is now known as a trophozoite (Gr. trophe = nutrition). The young parasite possesses a digestive vacuole with lysosomal enzymes. This vacuole contains proteinases (plasmepsin and falcipain). The vacuole can be clearly seen in a blood smear and explains the ring shape of the young parasite. The breakdown of haemoglobin results in an iron-containing pigment: haemozoin. This can be seen after 12-24 hours as malaria pigment (see below: diagnosis). The vacuole disappears as the parasite becomes older. The trophozoites will once more reproduce asexually and lead to the formation of a multinuclear parasite (schizont). The latter divides to form merozoites. Each schizont produces 8 to 24 merozoites, depending on the species, within a time span of 48 hours ( P. falciparum , P. vivax , P. ovale ) or 72 hours ( P. malariae ). The infected red blood cells burst after a while so that once more merozoites appear in the blood from where they will penetrate new erythrocytes within a few seconds. This bursting (lysis) of the red blood cells is accompanied by a bout of fever. If the development is synchronous (all parasites being at the same stage of development) the fever will follow a typical pattern (see below). This is, however, unusual: asynchrony is more common than synchrony, especially early in infections. The development from merozoite to schizont takes place in the peripheral blood and all stages can be observed. In P. falciparum usually only very young forms (ring forms) can be observed in the peripheral blood because older parasites adhere to the endothelium of blood vessels in deep organs (e.g. the brain).

After a few days some of the merozoites transform into male or female gametocytes. These are necessary for sexual reproduction of the parasite. Generally at least two schizogonous cycles must be completed before gametocytes appear. The trigger for the production of gametocytes is not known, and it is also still unclear what determines the sex of the gametocytes. It has been shown by capturing the progeny of P. falciparum merozoites of single asexuals in clusters on a erythrocyt monolayer, that all develop in the same way. P. falciparum gametocytes have a maturation period of around 8-10 days. Immature gametocytes sequester from the peripheral blood circulation, which means that the ratio of gametocytes to asexual blood forms at a certain moment in time is not a good indicator of how much the parasite invests in gametocyt production.  Gametocytes are responsible for transmitting the disease but do not themselves cause symptoms. Adult P. falciparum gametocytes are not sensitive to chloroquine and quinine, while those of P. vivax , P. ovale and P. malariae are sensitive. This means that following adequate treatment of P. falciparum there may still be gametocytes in the blood, and this may continue for several weeks. This does not mean that the treatment has failed. One interesting hypothesis is that chloroquine might significantly increase the gametocytaemia of chloroquine-resistant P. falciparum, resulting in an increased infectivity for Anopheles . This could, therefore, contribute to the rapid spread of chloroquine resistance.

'Xanthurenic acid, also known as the ''Gametocyte Activating Factor''. This is a simple molecule which is necessary for the further development of the malaria parasite in the mosquito. Copyright ITM'

Malaria, Plasmodium life cycle. Copyright Wellcome

" Plasmodium falciparum , penetration of mosquito salivary glands. Adapted from ""Trends in Parasitology"""

Plasmodium sp. Ookinete in the intestine of a mosquito. Photo prof Wéry. Copyright ITM

If the gametocytes are ingested during a bite from an Anopheles , the male gametocyte will quickly divide mitotically 3 times and develop several flagellae (exflagellation) [L. flagellum = whip]. After ten minutes eight microgametes [Gr. gamos =marriage] are formed from one male gametocyte. A microgamete is approximately 20 µm long and actively motile. Exflagellation is triggered among other things by a fall in temperature and by rising pH (between 8 and 8.3). The latter, however, only applies in vitro. The acidity in the insect's stomach is approximately the same as that of the blood ingested. A certain substance in the insect, the gametocyte-activating factor, plays a role in the activation which makes alkalisation unnecessary. This factor is a small molecule (xanthurenic acid). This heterocyclic double ring originates from the catabolism of tryptophan and is a by-product of the synthesis of ocular pigment in the insect. The substance is present in higher concentrations in malaria vectors than in human blood. That is one of the reasons for the absence of gametocyte activation in humans. On really rare occasions, exflagellation and microgametes can be seen in a fresh blood smear from a patient.

The peak of exflagellation occurs within 25 minutes after the blood meal. The female gametocyte will undergo a slight change in shape and is then called a macrogamete. The gametes are anisogamous (they differ in size). Within 3 hours after the blood meal the microgamete and macrogamete will fuse in the mosquito's stomach to form a diploid zygote (fertilised ovum) [Gr. "zygotos": yoked together]. Thus fertilisation occurs in the insect!

In the following 5 hours the zygote will undergo meiosis, resulting in 4 haploid parasites. During meiosis cross-over may occur between homologous chromosomes, which results in genetic recombination. Since in natural infections parasites are often of different genotypes, this is a mechanism for maintaining genetic diversity within one species. Later the zygote will become motile. This form of the parasite is now called an ookinete (Gr. oon = egg; kinetos = movement). The blood in the mosquito's intestine is separated from the midgut epithelium by a semi-permeable membrane (like a dialysis membrane). Because this membrane envelops the food, it is called the peritrophic membrane (Gr. peri = around; trophe = nutrition). The ookinete penetrates this chitinous membrane which lines the inner side of the mosquito's intestine. To do this the parasite secretes a prochitinase, an enzyme which will be converted to an active form by the mosquito's digestive enzymes (trypsins). The ookinete subsequently penetrates the mosquito's intestinal wall, mainly via certain epithelial cells (Ross' cells) which contain fewer microvilli than other nearby intestinal cells. The ookinete does not penetrate the basal membrane which encloses the intestinal cells on the haemocoel side. All the steps described above are essential for the maturation of the parasite and the midgut is therefore an important barrier. Only a few gametocytes in the blood meal will become successfully penetrating ookinetes. Once the ookinete has penetrated the intestine, it attaches to the outer side of the intestine, under the basal membrane. There the ookinete changes into an immobile oocyst. This is initially quite small (6-8 µm). It then grows to a diameter of 40-60 µm. After approximately a week (depending on the temperature) and after repeated mitotic nuclear divisions in the oocyst, countless 10-15 µm long fusiform parasites are produced. Thousands of sporozoites are formed, which after rupture of the oocyst will migrate to the mosquito's thoracic three-lobed salivary glands. However, this process seems inefficient. They seem to have a preference for the distal-lateral and medial lobes of the salivary glands, but seem unable to enter the proximal lobes. The sporozoites mature in the salivary glands and are then ready to be injected into a human during the insect's next blood meal. A certain number of salivary glands cells are destroyed by the penetration of the parasites in Plasmodium -infected mosquitoes. The processes whereby the ookinete penetrates the midgut and sporozoites penetrate the salivary glands, depend upon receptor-ligand interactions. If these can be blocked, transmission would become impossible (see malaria prevention, genetic altered vector). Note : malaria pigment, hemozoin Malaria pigment is not melanin-related. Inside erythrocytes, malaria parasites consume hemoglobin. Hereby they liberate free hematin (ferriprotoporphyrin IX), which is toxic for the parasite. For a long time, it was thought that the parasite protects itseld by polymerising the heme groups, but the concept of a polymer structure is not accepted by everyone. Synthetic beta-hematin seems to be identical (dimers forming chains linked by hydrogen bonds). Chloroquine interferes with this detoxification process. Hemozoin is formed by a kind of biocrystallisation at the interface between the aqueous medium of the food vacuole and very small lipid drops (nanospheres). The resulting crystals measure about 1 µm x 0.4 µm x 0.2 µm, but exact dimensions vary depend upon the parasite species and growth stage. After red cell lysis, the malaria pigment is phagocytosed. In repeated or chronic malaria infections, pigment will accumulate in liver, bone marrow and spleen, coloring them black. In history, the absence of black pigmentation of large spleens in kala azar patients helped in the suspicion that this illness was not malaria-related. Hemozoin is birefringent and paramagnetic (becomes temporary magnetic after application of an external magnetic field). The birefringence allows hemozoin detection by polarisation microscopy. Darkfield microscopy can also be used. Application of a magnetic field to a blood sample has been shown to accumulate intraerythrocytic parasites, allowing concentrations much higher than in thin films. This is being studied for clinical applications. Hemozoin has immunomodulatory effects and may contribute to immune pathology of malaria. Note: Ratio gametocytes/trophozoites In an infected person, countless parasites can often be seen in the peripheral blood. Since gametocytes are the only forms which are responsible for transmission in nature, it is remarkable how few gametocytes are generally found. Often the number of gametocytes is only a small percentage of the number of trophozoites. Yet higher gametocyte densities must lead to higher transmission. Why gametocytes occur in such low proportions compared to the number of trophozoites is still not clear. Ratio male /female gametocytes Generally there are more female than male gametocytes, certainly early on in an infection. There is thus a female-biased sex ratio. This is explained by the fact that one male gametocyte can form (as maximum) 8 viable male gametes, unlike the female gametocyte. Because each male can fertilize more than one female, an equal sex ratio would result in a wasteful surfeit of male gametes. This is in fact the case when infections are monoclonal, yet in mixed infections (i.e. at least two different strains without any inbreeding) the ratio would have to be 1/1 according to population genetics (Hamilton's theory of "local mate competition"). Later in the infection agglutinating antibodies are produced which immobilise male gametes and thus inhibit their function. Agglutination of female gametes has no effect on their function. To compensate for this the parasite produces more male gametocytes later in infection, possibly under the influence of increasing concentrations of erythropoietin. The latter hormone increases as anaemia increases.The details of sex ratio adjustment and kin discrimination in malaria parasites (in mixed infections) is still being studied.  A single haploid clone of P. falciparum can produce both male and female gametocytes. Precisely how this achieved is not clear. Plasmodium falciparum gametocytes need 7-10 days for maturation. The sex ratio is determined by the erythropoietin content 7-10 days before they mature. It is interesting to note that P. falciparum seems to exhibit increased infectibility in humans with sickle cell anaemia, regardless of the gametocyte density. This is explained by the chronically increased erythropoietin in this disorder, which leads to a higher percentage of male gametocytes. Nevertheless much study is still needed before the details will be fully understood.

Note on glucose metabolism, folate and ribosomes The trophozoite has no carbohydrate reserves and needs to consume glucose continually. The glucose metabolism in infected red blood cells is 50-100 times higher than that in non-infected cells. This probably contributes to the hypoglycaemia which is often seen in severe infections. The parasite does have mitochondria, but these play a minor role in the provision of energy (the last word on this has not yet been said). Glucose is converted by anaerobic glycolysis to pyruvate and then to lactate. This latter step, as in humans, is catalysed by the enzyme lactate dehydrogenase (LDH). The parasite's LDH is clearly different from that of humans and forms the basis of a diagnostic test (see below). Some glucose is processed in the hexose monophosphate shunt, which serves to produce NADPH. NADPH is necessary for the protective antioxidant glutathione, for reductive biosynthesis and for the de novo production of purines (see also G6PD-deficiency). Purines are necessary to produce parasitic DNA and glutathione is needed as a protection against oxidative stress. This may explain why G6PD-deficiency should offer relative protection against malaria and why this deficiency occurs frequently in malaria regions. However, opinions are divided on this subject. Plasmodium sp. synthesize folate. Malaria parasites increase red blood cell folate levels without providing a significant folate source to their human host. It makes it difficult to assess folate status during active infection. Bone marrow will respond to recurrent hemolysis, increasing the demand for folate. Some antimalarial drugs inhibit the production of folate by acting on dihydrofolate reductase and thymidylate synthetase, two enzymes which in parasites are part of a single bifunctional protein (DHFR-TS). The fact that the parasite relies more on de novo folate synthesis pathway than folate salvage, explains the antimalarial synergy of sulpha drugs (which inhibit de novo synthesis) and pyrimethamine (which inhibits DHFR). [Compare this with the action of sulfonamides and trimethoprim in cotrimoxazole]. In 1999 it was shown that Plasmodium berghei   uses two different kinds of ribosome, one which is active in the mosquito and one in its normal rodent host. Whether this is also the case with other parasites is as yet unclear. It is also not clear how this came about during evolution of the parasite.

Malaria. Plasmodium falciparum oocysts on the mosquito gut. The black part is the partial digested blood in the insect's intestine. Copyright ITM

Plasmodium falciparum gametocyt in a thin blood smear. Copyright ITM

Geographical distribution

Map of Africa showing the most important malaria vectors ( Anopheles mosquitoes). Copyright ITM

Plasmodium falciparum malaria anno 2007-8.

Many lay people regard malaria as a purely tropical disease. However, the distribution of malaria used to be world-wide. Today, it still occurs in some 100 countries. The situation varies from region to region. Until 1938 there was still P. vivax malaria ("polderkoorts") in Belgium, and in the Netherlands as late as 1958, although there was an unexplained (possibly autochtonous) case of P. malariae infection in a child in Zealand in 1969. The WHO declared the Netherlands officially malaria-free only in 1970. It is chiefly the pollution of surface waters which makes reproduction of Anopheles mosquitoes difficult. Yet Anopheles plumbeus , a vector which preferably breeds in water in hollow trees (and car tyres), Anopheles atroparvus , a brackish water mosquito, and its close relative A. messeae , still occur in the Low Countries. [ A. atroparvus prefers water with a chloride concentration of 800-2,500 mg/L. If the chloride concentration rises above 8,000 mg/L, the larvae die]. Anopheles atroparvus is able to transmit Plasmodium vivax malaria, but cannot transmit Plasmodium falciparum . Anopheles plumbeus was previously an exclusively zoophilic vector. In recent years a change in behaviour occurred and the mosquito started to feed more and more on humans. This mosquito can transmit tropical falciparum malaria. In the last century there were important changes in the lifestyle of humans, resulting in less human/mosquito contact. Effective therapy was available. All these factors mean that malaria has disappeared in Northwest Europe. Cases in Western countries are generally dealt with swiftly and satisfactorily, and one person with malaria very rarely leads to the infection of others. Chronic large scale reintroduction of the disease in Europe is thus improbable, although with the combination of the current economic crisis with its plummeting health budgets, the massive influx of tropical migrants refugees and global climate change, makes this possibility more real at present than in the last decades of the 20th Century. To maintain an infectious disease it is necessary for one infectious case to lead to one other infectious case, otherwise the disease will die out in the area. One would need sufficient gametocyte carriers and vectors to ensure the continuation of the disease. We have to remain vigilant. This was proven in 2012, when  by November of that year, a total of 12 autochthonous Plasmodium vivax cases were reported in Greece, together with a large outbreak of dengue in Madeira (also mosquito-borne disease).

  Malaria is a very important public health problem in most endemic countries. The number of clinical cases is estimated at 150 to 300 million per year. Of these, approximately 1 million, mainly young children die every year in Africa alone. Most lethal infections are due to Plasmodium falciparum . The disease causes symptoms such as fever, shivers, headache and muscular pain, anaemia and splenomegaly. Involvement of the brain often leads to death. For some years P. falciparum has been developing increasing resistance to chloroquine and other anti-malaria products. This, of course, makes treatment difficult. Many mosquitoes which are responsible for transmission of the disease are becoming resistant to a number of insecticides and this makes vector control difficult. P. falciparum is the most common form in sub-Saharan Africa. It occurs chiefly in Africa, tropical South America and Southeast Asia. The parasite occurred previously in the Mediterranean basin. P. vivax has the widest distribution area (previously as far as London, Norway, Denmark, New York, southern Canada and even Siberia). In 1922 the number of cases in Texas was estimated at 500,000. It is the most common form in certain regions (e.g. Maghreb countries). P. vivax preferentially penetrates young red blood cells (reticulocytes). In 1976 Miller discovered that P. vivax uses the Duffy blood group antigens (Fya and Fyb) as receptors to penetrate red blood cells. In order to invade a red blood cell, P. vivax displays a specific merozoit ligand, the Plasmodium vivax Duffy Binding Protein (PvDBP). This protein binds to a receptor on the erythrocyte's surface (the Duffy antigen/receptor for chemokines, abbreviated "DARC"). People who do not have this protein on their red blood cells cannot be infected with P. vivax . These antigens do not occur in the majority of humans in West Africa [phenotype Fy (a-b-)]. As a result P. vivax does not occur in West Africa, or occurs in low numbers (and could be systematiicalt missed). Duffy blood group negative erythrocytes are, in vitro, also resistant to infection with P. knowlesi (monkey malaria). In 2011, it was reported that  P. vivax could be found in Duffy negative individuals. This finding suggests the idea that this parasite is able to use receptors other than Duffy to invade erythrocytes. P. ovale is found chiefly West Africa, less elsewhere in Africa and sporadically in the Far East. P. ovale wallikeri and P. ovale curtesii seem to be sympatric, but more study is needed. P. malariae  is not very common anywhere. Often confused with P. knowlesi . P. knowlesi is known from Malaysia (including Borneo), The Philippines, Singapore and Thailand. The vector is present in India (Kerala) and Sri Lanka, but in these areas there is no known zoonotic reservoir. Often confused with P. malariae .      Could malaria reappear in Europe? Since the beginning of the 21st Century, a lot of information about the possible future climate change became available. One of the many questions is : "Will malaria reappear in Europe?". Each year many European travellers to tropical countries return with malaria (> 3000/year). Many immigrants bring also certain parasites with them. This means that gametocytes sources are available from time to time. What about mosquitoes? Sometimes tropical mosquitoes arrive via airports (see note on airport malaria), but they tend to die out when ecological conditions are not suited for them. Several indigenous European anophelines have vectorial capacity, such as Anopheles sergentii (Sicily), A. sacharovi (Italy, Corsica, Balkan, Greece, Turkey), A. labranchiae (Italy, Dalmatian coast), A. atroparvus (temperate regions of Europe, including Iberia), A. plumbeus, A. claviger  and others. The vector status of several anophelines is still unclear. With the exception of A. plumbeus which is a tree-hole breeder, all European anophelines use ditches and natural pools as larval habitat. Larval development rate is temperature-dependent. Members of the A. maculipennis complex ( A. atroparvus, A. messae, A. maculopennis ss ) overwinter as adults, where there is gonotrophic dissociation. This quiescence is triggered by decreasing day length and broken by rising spring temperature. A. atroparvus  continues to feed in blood during the winter, but A. messeae undergoes complete diapause as adults, not feeding from September to March. Malaria parasites which might be present slowly degenerate at low temperature. Many European anophelines are associated with livestock, only some of these mosquitoes bite humans. Lack of suitable winter resting sites for A. atroparvus caused by change in house design and modernisation of livestock farms, have led to a decrease in A. atroparvus populations. A. plumbeus might become a more efficient vector under changing climatological circumstances and this species remains a bit of a wild card. Malaria can only persist naturally when climatic conditions are suitable for the vector(s) and for the development of the parasites in the vector. Increased rainfall and higher temperatures may make larger areas favourable for malaria transmission in the future. Tropical P. falciparum requires a minimum temperature of 18°C, while tropical P. vivax strains require a minimum of 16°C. The European strain of P. vivax which persisted in the high North was uniquely adapted, with summer temperatures being sufficiently high for P. vivax development in the mosquito. Infection of future patients occured in autumn (September / October), especially when mosquitoes started to enter homes looking for shelter. The P. vivax parasites in humans had a very long incubation period of 6 to 9 months. A patient infected with these northern strain of P. vivax would remain asymptomatic during winter untill the following spring. This clearly differs from tropical P. vivax dynamics. In Southern Europe P. falciparum used to be common in Portugal, Spain, Italy and Greece. It is likely that the strain of this parasite was genetically different from tropical strains. Certain experiments showed that specific European mosquitoes were refractory to tropical P. falciparum , but research with A. atroparvus from Portugal demonstrated susceptibility of these mosquitoes to African P. falciparum. It is possible that specific environmental conditions need to be present for vector competence to be achieved. It is clear that more research is needed on this subject. However, for malaria to become reestablised, a sizable parasite reservoir (gametocyt carriers) must be present. This did not happen in other circumstances, such as after World War II, when great numbers of people (patients and gametocyt carriers) returned from tropical areas. In the current health system and socioeconomic situation in Europe, it is likely that patients will be treated early, diminishing the reservoir and lessening the threat of new epidemics. Small outbreaks and some local transmission might occur from time to time, but large reinvasion of the North European landmass is unlikely. South Europe would have a somewhat larger risk. Transmission of autochthonous P. vivax has been detected in East Turkey, Aragon in Spain (2010) and in the wetlands of Evrotas in the district of Lakonia, Peloponnese region, southern Greece (in 2012 there were 16 cases of autochtonous P. vivax malaria).   Origin of P. falciparum malaria in the New World. Malaria caused by Plasmodium falciparum originated in Africa, but now can be found on all continents – with the exception of Antarctica, where no people live to harbor the parasite, and no mosquitoes to transmit it. The parasite obviously travelled along with its human hosts when they conquered the world. But how and when did it arrive in the Americas? With the first Asian travelers across the Bering Strait, some thirteen thousand and maybe even sixty thousand years ago? With Leif Ericson and his Vikings, AD 1000? With Columbus and the conquistadores, as did measles? DNA analysis of blood samples from 17 countries in Africa, the Middle East, Latin America, Oceania and Asia showed that the American parasites were genetically distant from their Asian cousins, but close to the African ones. Moreover, the trade routes showed up: the American parasites come in two groups: one in the countries that received their slaves through the Spanish trade, and another group in Brazil, furnished by the Portuguese slave trade. Both groups are more closely related to the African strains in their countries of origin than to each other.

Epidemiological classification - stable versus unstable malaria

There is no completely satisfactory epidemiological classification of malaria. Stable malaria means that the clinical disease is characterised by preferentially affecting children and achieving a protective "immunity" in adults. Stability does not mean that there can be no variation in transmission. In some regions seasonal malaria occurs. In other areas there is unstable malaria: transmission differs greatly from year to year and sometimes epidemics occur. The disease then also occurs in older persons. This is important in many respects, including the fact that irregular control of malaria may lead to changes in the immune status of the population. Sometimes malaria may appear again in a region after a long abscence. For example: in 1972 the disease was eradicated in South Korea following an intensive eradication campaign with case detection and vector control. In 1993 one case of P. vivax was observed. There then followed 22 cases in 1994, in 1995 there were 107 cases, 356 in 1996 and more than 1600 in 1997. In 1995 all cases were still limited to the border area with North Korea, but in 1996 there was also transmission outside the demilitarised zone. After entomological surveys had shown that Anopheles   sinensis was the chief vector, measures were taken to control the disease.

Vector 

Vector, Anopheles mosquitoes

Malaria is transmitted via the bite of infected female Anopheles mosquitoes.

Malaria is transmitted by Anopheles mosquitoes. This applies to the malaria of all mammals. Avian malaria on the other hand is chiefly transmitted by Culicinae. There are some 400 Anopheles species, 40 of which are good vectors while 28 are poor vectors. Anopheles mosquitoes are relatively small (8 mm), two-winged insects (Diptera; Gr. di = two and pteryx = wing). They often assume a typical posture while feeding: head down and the lower body upwards. [There are exceptions to this such as Anopheles culicifacies , which, as its name suggests, resembles Culex .] As with many mosquitoes there are countless scales on the body and wings. In Anopheles there are darker and lighter coloured scales arranged in groups, which produces a distinctive marking on the wing (a speckled pattern). Culex mosquitoes on the other hand are of an even colour. Anopheles mosquitoes undergo induced color change based on perception of the background against which they are cultured. When larvae are reared on either a black or white background, they become pigmented dark or pale. The degree of darkening depends in part on the length of time the larvae have been cultured on a black background and the degree of fat body development. This color change phenomenon is called homochromy. Anopheles mosquitoes are active at night. They do not buzz much and are not easily noticed. Every species of mosquito has its own characteristics as to behaviour, reproduction, biting habits, etc. This is of course important for mosquito control.

Comparison of Anopheles and Culex mosquitoes. Copyright Wellcome.

Many invertebrates have an open circulation, in which blood pumped by the heart empties via an artery into an open, fluid-filled space, the haemocoel, which lies between the ectoderm and the endoderm. The fluid contained within the haemocoel is referred to as haemolymph or blood. The blood is not circulated through capillaries but bathes the tissues directly. This blood circulation is of minor importance for oxygen and CO 2 transport. Instead they have a tracheal system in which respiratory gases are transported directly to tissues through air-filled tubes. Most insects have a large capacity for aerobic metabolism.

At tropical temperatures the first oviposition occurs 2 to 3 days after the first blood meal. Each time the females lay about 100 eggs in water. The eggs are not attached to one another, unlike those of Culex mosquitoes. The eggs cannot survive drying out, unlike those of Aedes . After they have laid their eggs the females will once more search for a blood meal. The interval between sucking blood and laying eggs is called the gonotrophic cycle. The period of this cycle is important in determining vector capacity. The more often the mosquito feeds on blood, the more risk there is of becoming infected with parasites. At lower temperatures the gonotrophic cycle is longer, some 4 or 5 days. This reduces the risk of disease transmission because: (1) the number of times that the mosquito can take up or inject parasites is smaller, (2) the speed of development of the parasite is dependent on temperature and is slower in a cold climate, and (3) the mosquitoes have a greater chance of dying before they become infectious. If the lifetime of the mosquito is shorter than the development time of the parasite (extrinsic incubation period) there will be no transmission of the disease. In vector control, one tries to make the average life span of the mosquitoes shorter than the extrinsic incubation period of the parasite. The aim is to kill the majority of female mosquitoes before they become infectious. How can one measure the average life span? One way to ascertain the age structure of an insect population is to determine the proportion of females who have laid eggs at least once. This can be done by examining the ovaries. Specific attention is paid to the shape of the trachea (breathing tubes). In nulliparous insects these have a different shape to those of insects which have already laid eggs. They exhibit a scar, caused by the swelling of the eggs.

Vector, Anopheles larvae and pupae

Anopheles eggs. Notice the lateral floats. Copyright ITM

Anopheles stephensi pupa (mosquito, malaria vector). Copyright ITM

Anopheles stephensi larva. This mosquito is an important malaria vector in India. Copyright ITM

Anopheles larva. Copyright ITM

After 2-3 days legless larvae emerge from the eggs. There are four larval stages. The larvae of Anopheles lie parallel with the surface of the water, unlike Culex larvae. They breath air via small caudal openings (spiracles) [the larvae also have dorsal "gills", but these are actually osmoregulatory organs]. This surface position makes them susceptible to chemicals which float on water, e.g. oil (with special dispersion detergents to use less product per hectare). Larvae can also to a limited extent take up oxygen dissolved in the water. An oil film on water results in mechanical obstruction protection of the respiratory apparatus of the immature insects, is an ancient but useful method of vector control. The larvae are filter feeders and have oral tufts of hair ("mouth brushes"). They feed on all kinds of microscopic organisms. For example they can take up dead Bacillus thuringiensis var. israelensis. The spore of this bacterium contains toxins which kills the larva and this is used for vector control (see also onchocerciasis). The entomopathogenic Bacillus sphaericus may also be taken up.

After 7-20 days a larva will form a pupa. This pupa is quite active (motile) but does not feed. The rate of development of the larva and pupa is highly dependent on temperature. Malaria transmission quickly increases if the average temperature rises. A feature of the pupa is the two trumpets with which the animal breathes. Anopheles sexes can be distinguished easily in the pupal stage. This is often desirable in the laboratory, so that sexes can be separated before adulthood to avoid mating of adults intended for genetic crosses and otherwise to determine sex before adulthood. Claspers are present only in the male (visible on the rear of the pupa). After eclosion, the adult insect appears. Metamorphosis can be disturbed by various pesticides with a hormonal action. The metamorphosis is affected to an important extent by the corpora allata, small lobes near the insect brain. These corpora release what is called the juvenile hormone, a sesquiterpene. Methoprene (Altosid®) is a juvenile hormone analogue which prevents metamorphosis (the actual hormone is too unstable to be used). The shedding of the old cuticula is also under hormonal control. The brain excretes an activation hormone, called the prothoracicotropic hormone. This acts upon the prothoracic gland, which in turn excretes ecdysone, a steroid hormone. Due to the effect of ecdysone the old cuticula is separates enzymatically from the new cuticula which is forming underneath (apolysis). After the old cuticula has been discarded (ecdysis) the new one, which is still soft, quickly hardens under influence of bursicon, a hormone.

Vector, ecological habitat of the Anopheles mosquito

The ecological habitat which larvae need, varies greatly from one species to another, from permanent water surfaces to temporary puddles, fresh or brackish water, edges of streams or still water, in the open sun or in deep shadow, from pure water to polluted water, marshes or small water collections in plants, trees, rocks, hoof-prints or rubbish. Larvae are generally rare on large water surfaces, lakes or fast rivers (except at the edges). No larvae will be found in fast flowing water. The characteristics of the breeding grounds are precisely defined for each Anopheles species, so that if the local vector is known, the breeding grounds can be targeted for selective control. After the Southeast Asian boxing day tsunami in 2004, there was a fear that local population of Anopheles sundaicus would proliferate, compounding the problem. The world's most important vector is Anopheles gambiae . It typically breeds in exposed sunlit and often transient aquatic habitats such as pools, puddles, and irrigation channels.  An. funestus breeds in more permanent shaded waters often with aquatic vegetation.

Vector, mosquito diet

Like all two-winged insects mosquitoes can only feed on liquid food. Both sexes suck plant juices and nectar, but females need blood to make eggs. Without blood no or few eggs can be laid. Only the females suck blood. The life of a male mosquito consists of a constant search for sex and nectar.

Mouthparts

Structure of the head of male and female Anopheles mosquito. Copyright ITM

The mouthparts must not be confused with the palpi and antennae. The long palpi lie on either side of the proboscis. The antennae have a distinctive structure (plume shaped and hairy in the male). The structure and action of the mouthparts of a female are important for the transmission of malaria. The mouth (proboscis) consists of 7 parts. Above is the upper lip (labrum) and below is the larger U-shaped lower lip (labium) which ends in a blunt thickening (labellum). The labrum and labium enclose a space containing 2 awl-shaped serrated mandibulae, 2 similar maxillae and 1 hollow hypopharynx. The cavity in the hypopharynx is the extended outlet from the salivary glands. When a female bites, the blunt labellum is placed on the skin. This cannot penetrate the skin and the labium bends backwards. This allows the other 6 mouthparts to penetrate the skin. Saliva is injected to prevent the blood from coagulating and blocking the mouthparts. It is in this saliva that the sporozoites are to be found. Mosquito bite tends to be painless (before becoming itching). In 2010, Seiji Aoyagi from Kansai University, Osaka tried to develop a better hypodermic needle. By etching silicon he created a device which imitated three of the seven mosquito mouthparts: two serrarated maxillae and a tubular labrum. Each part is driven by piezoelectrical crystals made of lead zirconium titanate. Piezoelectrical crystals change length when a voltage is applied and this process is fully reversable. A vibration of 15 Hertz was reached, similar to that of a probing mosquito proboscis. When tested on volunteers, they stated that the device caused less pain than a traditional hypodermic needle, although the pain lasted longer. The device needs further improvement.

Vector, sensitivity of the mosquito to infection with plasmodia

Some mosquitoes are sensitive to one species of malaria parasites and resistant to another. Thus for example P. vivax can develop successfully in A. atroparvus from Northwest Europe, but P. falciparum  from tropical regions is not capable of forming oocysts in this mosquito. P. falciparum from Southern Europe which has now been eradicated could develop in this mosquito. Penetration into the mosquito salivary gland cells is receptor-ligand dependant. This specificity explains why certain parasites can only be transmitted by certain Anopheles . For example, the complete sporogony of P. cynomolgi can be completed in Anopheles freeborni , but the parasites cannot penetrate the salivary glands of the insect. Some mosquito species can cause lysis of ookinetes in their intestines or encapsulate them in a melanotic capsule. There are certain peptides (defencins, similar to the cecropins of other insects) in the haemolymph of mosquitoes. These are being studied for their protective effect on the mosquito. Our knowledge of the protective mechanisms of Anopheles sp. against parasites is not complete.

Vector, biting behaviour of mosquitoes

Anopheles stephensi female biting and sucking blood. Malaria vector. Copyright ITM

Different mosquito species have widely varying habits: some bite chiefly animals (zoophilic) and others humans (anthropophilic). They may live and bite outside (exophilic and exophagic respectively) or come into houses and bite there (endophilic and endophagic respectively). Anopheles mosquitoes are good flyers: they can cover several kilometres in one night. This is of course of great importance for their control. If there are lots of animals around the houses, malaria transmission by zoophilic vectors will be reduced. Contact with endophagic mosquitoes can be reduced by mosquito nets. Endophagic mosquitoes will often rest on walls after a blood meal. Residual insecticides which are applied there will kill the vector. There are also techniques for controlling adult mosquitoes outside houses. Vector control of malaria targets the larvae and the adult insects. Eggs and pupae are difficult to control. Hypersensitivity to the saliva of blood sucking insects can lead to formation of itchy vesicles or even bullae ("culicosis bullosa"). Delayed hypersensitivity reactions can lead to chronic and severe localised pruritus.

Vector, how does the mosquito find her prey?

Mosquitoes are attracted by an increasing CO 2 gradient. Yet CO 2 is an aspecific attractant, since all animals breath out CO 2 . This is therefore a problem for mosquitoes which have a preference for a specific host. Dilution in the atmosphere limits the range of this substance to approximately 20 metres. The warmth of the skin, lactic acid and moisture (breath) play a part over short distances. Every animal produces a number of volatile substances in its skin, breath, faeces and urine. A number of the substances (kairomones) are used by the mosquito to find its prey. The skin has various microhabitats (armpits, groin, soles of the feet, scalp, and so on) which are colonised by various bacteria (staphylococci, micrococci, coryneforms, etc.) These bacteria can convert some substances in sweat and sebum into attractants. The presence of Brevibacterium epidermidis  plays a part in the typical smell of sweaty feet (comparable to the smell of Limburger cheese, in which Brevibacterium linens  produces the typical odour). This odour is highly attractive to mosquitoes. The details are complex, however. Thus Anopheles gambiae prefers to land on the feet, while A. atroparvus  prefers to bite the face. Families of odorant receptor genes have been identified in the A. gambiae genome, and one receptor responds to volatile compounds in human sweat. An odorant in human sweat called 4-methylphenol activates olfactory receptor AgOr1. Only female mosquitoes carry AgOr1. The discovery might have potential, for it opens the way to a new generation of mosquito repellents that neutralise 4-methylphenol. Alternatively, 4-methylphenol could be used to bait traps to lure mosquitoes to their death.

Various methods have been developed to find attractants. The substances may be identified by the measuring the activity of receptors in the mosquitoes' antennae when they are in a stream of air containing attractants. In the laboratory the chemicals are separated in a gas chromatograph. With the purified chemicals, one can perform electro-antennography, or single-cell recordings with micro-electrodes on the olfactory receptor cells of the insect. Potential mosquito repellents may also be screened and researched in this way. Perhaps one day certain cocktails of attractants can be used to make mosquitotraps more effective. An interesting mosquito trap is based on the fact that most hematophagous insects are attracted by humidity, warmth and CO 2 (exhaled air). There are battery-operated devices which use propane to generate warm moist CO 2 . This can be augmented with octenol, another attractant. Some are equipped with small blinking LED lights. When these small insects approach, they are sucked up by a vacuum aspirator. A single battery and a single propane tank (5-13 liters) are sufficient for several months, which is useful for insect surveys. In some areas (e.g. campings, gardens), these devices are used to diminish the number of biting insects. In general, big flies are too heavy to be sucked up. Some devices have a replacable wrap-around sticky and striped "fly paper" as an additional trap.

Vector, resistance to insecticides in malaria vectors

Malaria vector control is primarily based on the use of insecticides. Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide uses in malaria control programmes. DDT has gotten its share of publicity, good and bad, and even has a poem dedicated to it: A mosquito was heard to complain that a chemist had poisoned his brain. The cause of his sorrow was paradichloro- diphenyltrichloroethane

Note: Pyrethroids and DDT, two important insecticides used for vector control, block the nerve-impuls conduction by preventing a sodium channel from closing after an action potential. An important mechanism that confers resistance to pyrethroids and DDT, known as knockdown resistance or kdr, was first described in the housefly Musca domestica . It has been reported that a single mutation in the sodium channel sequence is the molecular basis of kdr in Musca domestica . The gene has also been characterized for Anopheles gambiae . PCR tests have been developed for the detection of the kdr-mutation in A. gambiae .

Vector, examples of important vectors

Map: North, Central and South America. Malaria vectors: A.darlingi, A.aquasalis, A.freeborni, A.quadrimaculatus, A.albimanus, A.nuneztovari, A.pseudopunctipennis

As more research is carried out and powerful techniques such as PCR and DNA sequencing are used on newly trapped mosquitoes during surveys , the number of Anopheline species which is recognized increases. Among those "new" species, there are mosquitoes which are positive for sporozoites, such as the recently discovered An. ovengensis from Cameroun. In 2012, a Kenyan survey found that five of 293 unknown mosquitoes tested positive for P. falciparum sporozoites. The epidemiological importance of the newly recognized vectors remains to be determined. The following is not a all-inclusive list. In bold , important species.

1. North American A. albimanus A. freeborni A. quadrimaculatus   2. Central American A. albimanus A. albitarsis A. aquasalis A. argyritarsis A. aztecus A. darlingi A. pseudopunctipennis  A. punctimacula 3. South American A. albimanus A. albitarsis A. aquasalis A. argyritarsis A. bellator A. braziliensis A. cruzii A. darlingi A. neivai A. nuneztovari A. pseudopunctipennis A. punctimacula A. triannulatus 4. North Eurasian A. atroparvus A. messeae A. pattoni A. sacharovi A. sinensis   5. Mediterranean A. atroparvus A. claviger A. hispaniola A. labranchiae A. messeae A. sacharovi A. superpictus 6. Afro-Arabian A. culicifacies A. fluviatilis A. hispaniola A. multicolor A. pharoensis A. sergentii 7. Afrotropical A. arabiensis A. funestus A. gambiae A. melas A. merus A. moucheti A. nili A. pharoensis 8. Indo-Iranian A. aconitus A. annularis A. culicifacies A. fluviatilis A. jeyporiensis A. minimus A. philippinensis A. pulcherrimus A. sacharovi A. stephensi A. sundaicus A. superpictus A. tessellatus A. varuna 9. Indo-Chinese hills A. annularis A. culicifacies A. dirus A. fluviatilis A. jeyporiensis A. maculatus A. minimus A. nigerrimus 10. Malaysian A. aconitus A. balabacensis A. campestris A. dirus A. donaldi A. flavirostris A. jeyporiensis A. letifer A. leucosphyrus A. ludlowae A. maculatus A. mangyanu A. minimus A. nigerrimus A. philippinensis A. subpictus A. sundaicus A. whartoni 11. Chinese A. anthropophagus A. balabacensis A. jeyporiensis A. pattoni A. sinensis 12. Australasian A. farauti type 1 A. farauti type 2 A. hilli A. karwari A. koliensis A. punctulatus A. subpictus

Note: Anopheles gambiae complex

Mosquitoes which are identical as regards size, colour, pattern, morphology, may nevertheless differ in behaviour, adjustment to the environment and DNA. Diptera have specific banding patterns on the polytene giant chromosomes of the larval salivary glands or the ovarian nurse cells of adult mosqitoes. The enormeous length (2 mm in Drosophila ) of polytene chromosomes is much longer than metaphase chromosomes. Polytene chromosomes consist of 100 or more copies of DNA, arranged side by side. The DNA is said to be endoreduplicated. Examination of these chromosomes by Feulgen or antibody staining staining and light microscopy reveals alternating highly and moderately dense regions, called bands and interbands. The expanded length of polytene chromosomes, relative to metaphase chromosomes, allows a much higher resolution mapping of morphological chromosome features than is possible with G- or Q-banding (staining with G iemsa or Q uinacrine). The high local DNA concentration of aligned DNA sequences make polytene chromosomes ideal targets for in situ hybridization with specific sequence probes. The lesser density of interbands suggests that the chromatin in these regions is less condensed and may correspond to euchromatin of normal interphase nuclei. Conversely, the bands probably correspond to the condensed heterochromatin. Electron micrographs allow fine definition of banding patterns. Since now the complete genome of A. gambiae is known, polytene chromosome analysis has become less important.

There are several species complexes among the malaria vectors that are each composed of closely related but reproductively isolated species. More than half of the known malaria vectors belong to cryptic species. It is necessary to know the cryptic species, particularly in the light of the growing drive for the development of bioengineered resistant vectors to control this disease. Failure to recognize cryptic vector species and their separate role in transmission would seriously undermine any such effort at control. Morphological identical mosquito species are in fact different species if genetic exchange between them is no longer possible or leads to sterile hybrids. Such mosquitoes may be found in the same region (sympatric mosquitoes). One can often differentiate sibling species by electrophoresis. Proof of speciation is based on the lack of heterozygosity of the diagnostic iso-enzymes in species which breed in the same environment, and also by culture experiments. Nowadays molecular markers are used on dried specimens. In this way six closely related species were identified in the most important African vector, Anopheles gambiae . These mosquitoes form the Anopheles gambiae species complex. They are mosquitoes that cannot be morphologically distinguished from each other.

Anopheles gambiae   sensu strictu, an anthropophagic and endophilic fresh water mosquito which flourishes preferentially in rather moist regions. An excellent vector for malaria.

Anopheles arabiensis   is anthropophilic, but in the presence of livestock this mosquito prefers cattle. Its behaviour while resting can vary greatly (endophilic or exophilic). If they are exophilic, spraying the inside of the house with insecticides will not be effective. The mosquito lays its eggs in fresh water.

Anopheles quadriannulatus   is zoophagic and exophilic. No control measures are necessary for this fresh water mosquito since it is not a malaria vector.

Anopheles bwambae   occurs only in a very small area, in the Semliki Forest in the Rift Valley. It is an anthropophagic mosquito which breeds in geothermal fresh water streams and is for the most part insignificant.

Anopheles melas , anthropophagic and endophilic, occurs on the coast of West Africa. Its capacity as a vector is considerably lower than that of A. gambiae and A. arabiensis . It is a brackish water mosquito and thus is not found inland.

Anopheles merus   breeds on the coast of East Africa both in fresh and brackish water, and is more exophilic than endophilic. Another example of the importance of sibling species is Anopheles culicifacies . In India, the sibling species A. culicifacies species A breeds predominantly in ponds, while A. culicifacies species B breeds in streams. Species B was found not to be involved in malaria transmission, so vector control can focus on species A, for example with larvivorous fish in ponds, leaving the streams untreated.

Note: Anopheles gambiae and Brazil One could have the impression that the geographical distribution of mosquitoes is stable. However, sometimes insects are imported into new regions where they can flourish and become pest species. Just such a case occurred in 1930. In March of that year larvae of Anopheles gambiae were found in a very limited area in Natal, Brazil. This mosquito originated in Africa, however, and until then had never occurred in the New World. The local authorities were informed but showed no interest and refused to cooperate in control measures. In January 1931 there was a malaria epidemic with 10,000 cases. The size of the infested area increased. In 1938 the number of patients rose to 100,000 cases with 14,000 to 20,000 deaths. There was severe disturbance of everyday life. The cotton harvest was not picked due to the huge numbers of people who were sick, and the food supply was threatened. The President, Getubio Varga, intervened and appointed Fred Soper as leader of a huge eradication campaign. He was an energetic and extremely gifted man. After setting up laboratories, a training school, a cartography department and a surveillance system, the foci were charted and the region was divided into small units. Teams were trained and made repeated visits to every focus and individual dwelling. Spraying was carried out with copper aceto-arsenite (Paris Green) and drainage projects were completed. Oil was poured regularly on water surfaces to kill the larvae. Ships, aeroplanes, trains and cars on the roads were fumigated upon leaving the epidemic region. In one year he was able to drive the mosquito back to two small foci which were then brought under control. The mosquito was eradicated from the continent, a notable achievement in the time before DDT existed.

Vector, extrinsic incubation period of the parasite

The development of plasmodia (from gametocytes to sporozoites) in a mosquito takes at least 9 days (sometimes as much as 30 days, depending on the temperature). After an infected blood meal a female mosquito will pass through at least 4 or 5 egg-laying periods (and blood meals) before it becomes infectious. There is thus ample time over several days to destroy the vector before she can transmit. Mosquitoes which are infectious are already "middle-aged". They may sometimes survive a month or more, but often the life span is much shorter. This long period before a mosquito becomes infectious is a weak point in the cycle of plasmodia. For example: at a temperature of 30°C at least 70% of mosquitoes need to survive every day if more than 1% of mosquitoes are to survive the 10-day development period.

A practical example can be seen in "airport malaria". Sometimes (due to lack of decontamination or resistance to insecticide) infected mosquitoes are brought from an endemic area to northern airports in aeroplanes. If such a mosquito can remain active in its new environment due to high temperatures in a hot summer, bites someone and injects sporozoites, it will already be at least 10 days old. Adding the incubation period for P. falciparum and the time between the first fever and time lost before making the diagnosis (in northern regions one does not think quickly of the possibility of malaria), it can then be assumed that the mosquito which caused the disease will probably already be dead when the diagnosis is finally made. Patient's delay and doctor's delay are important factors in lethal malaria. One mosquito can infect several persons (for example if the blood meal is interrupted the mosquito will bite several times), also possibly outside the airport (carried by the wind, or flying). Also, several mosquitoes may be introduced. Due to these factors, clusters of airport malaria may occur in hot summers. The risk that descendants of an infected (or non-infected) mosquito would survive in Europe and find a gametocyte carrier and cause infections again after the incubation period, is very small.

Note on Diptera

The diversity of insects that can transmit pathogens is large. Some of the biting insects themselves can be large. One of the bigger, impressive and aggresive mosquitoes is Psorophora ciliata ("shaggy-legged galliniper mosquito"), which lives East of the Continental Divide of the USA all the way to Argentina.  Some of the biting insect are very small, e.g. sandflies and Culicoides sp. To be able to place them taxonomically, the following explanation is given (for more details see entomology course). The order of Diptera (two-winged insects) is subdivided into two large suborders: Nematocera ("segmented antennae": antennae with many similar segments; 26 families all together, such as mosquitoes, simulids and ceratopogonids) and Brachycera ("short antennae", with 104 families all together). The Brachycera are further divided in the -  (1) Cyclorapha ("round seams") which refers to a larval characteristic; 85 families, e.g. tsetse flies ( Glossina sp), fruit flies (Drosophilidae), house flies (Muscidae); hover flies (Syrphidae), blow flies (Calliphoridae), Sarcophagidae and tachinids. In the higher flies (Cyclorapha), pupation occurs inside the skin of the third last larval stage, which is known as the puparium. - (2) Orthorapha (19 families), exemplified by the horseflies; tabanids belong to this group, as do the robber flies (Asilidae) and long-legged flies (Dolichopodidae).  About 20% of all fly species, representing more than 20 families, are parasitoids. This means that their larvae develop inside the bodies of hosts, which are killed in the process. Those insects (e.g. tachinids) are of immense importance in the control of natural populations of pest insects.

Nematocera are subdivided into:

1.Culicidae : mosquitoes 2.Psychodidae  : sandflies ( Phlebotomus ) 3.Ceratopogonidae : biting midges ( Culicoides ) 4.Simuliidae  : buffalo gnats or blackflies

Not biting humans and of no direct medical importance:

Tipulidae           : crane flies Chironomidae     : midges or thunder flies Cecidomyiidae    : gall midges Mycetophilidae   : fungus gnats Blefaroceridae    : net-winged midges Trichoceridae     : winter gnats Bibionidae          : St Marks flies, March flies, fever flies

Biting mosquitoes are divided into three large subfamilies, although alternative taxonomic trees exist:

  Anophelinae: Anopheles Culicinae: Aedes, Armigeres, Eretmapodites, Haemagogus, Uranotaenia, Culex, Culiseta, Mansonia, Coquillettidia, Sabethes, Psorophora Toxorhynchitinae: they have no direct medical importance since they don't bite humans. However, the predatory larvae can be used in vector control (they devour other mosquito larvae).

Note: Paradox in insect flight

How is it possible that we can actually hear mosquitoes fly and buzz? The flight muscles of some insects contract faster than what the limits of nerve conduction seem to allow. Each contraction of a mammalian muscle cell is triggered by a single nerve impulse. However, insect flight muscles contract far more frequently than nerve impulses can be conducted. Some insects beat their wings 1000 times per second, generating a high-pitched whine that humans can hear. This is more than 40 times faster than human fast twitch muscles can contract. Insect flight muscles make use of resonance to achieve these high rates of contraction. It turns out that their flight muscles are sensitive to stretch - if you pull on the muscle it virtually immediately contracts, and when you release the muscle it relaxes. The wings are connected to the thorax. The thorax of insects is a stiff box containing two types of flight muscles, one of which moves the wings up, and the other which moves them down. The flight muscles are actually not connected to the wings themselves, but are anchored instead to the walls of the thorax. Movement of the wings, which are attached to the roof of the thorax, is produced indirectly, by altering the shape of the thorax. The thorax in effect acts a resonating box that pulls alternately on the elevator and depressor muscles, stimulating first one and then the other to contract. When the elevator muscle contracts, the roof of the thorax is depressed and clicks into a new position, causing the wings to move up. But the new shape of the thorax stretches the depressor muscles, causing them to contract, and it simultaneously removes the tension from elevator muscles, so that they relax. Consequently, the roof of the thorax suddenly snaps back to its original position, flicking the wings down. This, of course, stretches the elevator muscles, stimulating them to contract, and simultaneously relaxes the depressor muscles, so that the cycle begins all over again. Because movement of the thorax can be achieved with only tiny changes in muscle length, it can occur extremely fast. And because the flight muscles are stimulated by stretch rather than by nerve impulses, they can contract faster than nerve impulses can be conducted. This explains how these insects are able to "break the limits". It does not explain why mosquitoes don't get nuts from their own buzzing. Many insect groups have four wings. In Diptera (flies and mosquitoes) the forewings are used for flying, but the hindwings have been modified to become halteres. These modified hindwings resemble drumsticks. During flight, they vibrate about 400 times per minute and function as a balance organ. They are responsable for the fact that mosquitoes cannot fly in fog and misty weather, as minuscule water droplets (around 5 micron in diameter) hit the halteres thousands of times per second, interfering with their gyroscopic function. This does not happen in ordinary rainy weather, when mosquitoes are hit by a large raindrop on average once every twenty seconds.TSuch impacts do not seem to bother them too much.

Malaria, Physiopathology

Plasmodium falciparum , thick smear. Copyright ITM

The incubation period may be short (minimum 7-9 days for P. falciparum ) to very long (several years for P. ovale ). In falciparum malaria the parasitaemia can be very high: up to 80% of erythrocytes may contain parasites, but even 5% is sufficient to result in severe disease. These situations may be life-threatening. The other malaria parasites produce much lower parasitaemia (maximum 2 %). They do cause severe illness, but are rarely life-threatening. P. knowlesi infections mimics severe P. malariae infections.

The rupture of the red blood cells (haemolysis) is accompanied by fever, muscle pain and general malaise. Massive haemolysis may cause kidney failure. Parasitised red blood cells are removed by the spleen. Splenomegaly will result. Anaemia occurs due to the destruction of erythrocytes, suppression of the bone marrow and excess activity of the enlarged spleen (hypersplenism). In severe falciparum malaria, there is activation of blood coagulation system along with thrombocytopaenia, even before widespread DIC and coagulation failure occur. In falciparum malaria there will often be a drop in glycaemia. The hypoglycaemia can be corrected by administration of glucose.

The details of how cerebral malaria actually happens, are not clear at present, and various researchers have different opinions. More than 100 years ago, the Italian pathologists Bignami and Marchiafava reported on the sequestration of parasitized red blood cells in the brains of people who died of cerebral malaria. Erythrocytes which contain schizonts of P. falciparum , develop small knobs on their cell membranes. These consist, among other things, of a histidine-rich protein, P. falciparum erythocyte membrane protein 1 and rifins. Rifins are clonally variant proteins encoded by rif  genes (“repetitive interspersed family") and are expressed at late ring or early trophozoite stage on the infected red cell surface. Their high copy number, sequence variability, and red cell surface location indicate an important role in host-parasite interaction. The knobs have an overall negative charge, allowing non-specific attraction to positive endothelial ligands, but specific molecular adhesion also plays a part. With these knobs the infected cells cling to the walls of the capillaries and to the vascular endothelium of the post-capillary venules in the brain. The low local O 2 pressure and high CO 2 pressure are optimal for further maturation of the parasite. Infected red blood cells are less easily distorted and more rigid than normal erythrocytes. This impedes the bloodflow, which can lead to cerebral malaria. Other organs too may be affected, for example the placenta and the intestines (resulting in abdominal pain and diarrhoea). Red blood cells which contain schizonts of P. malariae , also develop knobs on their membranes, but these cells do not adhere to the vascular endothelium. When post mortem cerebral sequestration was compared with the peripheral parasitemia, there were about 26 times more infected red blood cells in the brain microvasculature than in the peripheral blood if there were free-mixing. More blood vessels in the cortex and cerebellum than in the brain stem are affected. Some researchers found more sequestration in white matter than in cortex. Coma requires sequestration, but sequestration itself is not enough to provoke cerebral malaria. The rapid reversible nature of cerebral malaria led to the hypothesis that soluble neuroactive mediators might play a role in the pathogenesis, maybe involving reversible disturbances of the blood brain barrier and biochemical disruption of normal metabolism.

There are two groups of parasites in P. falciparum infections: (1) the young forms in the peripheral blood which can easily be observed in a thin blood smear, and (2) the mature group which is attached to small blood vessels and which cannot be seen. Falciparum schizonts are rarely found in peripheral blood, but these are important for the development of cerebral malaria. The whole mechanism of cerebral malaria has not to date been fully explained. As well as the attachment of parasitised red blood cells to the vessel walls (cytoadherence) other mechanisms possibly also play a part. Normal red blood cells sometimes attach to parasitised cells, which impairs the microcirculation. All kinds of released chemical substances (cytokines, oxygen radicals, etc.) may also play a part. Cytokines such as tumour necrosis factor (TNF-a) increase the expression of receptor molecules on the endothelium and will contribute to the cytoadherence and flow obstruction which characterise falciparum malaria. When the schizont is mature and the red blood cell ruptures, glycosyl-phosphatidyl inositol anchors (GPI-anchors) are released, which stimulates the production of TNF-a from macrophages. This led to its description as a "malaria toxin". This mechanism is similar to the release of TNF-a by endotoxins in Gram-negative septicaemia.

Increased intracranial pressure is found in some patients, but certainly not in all. Increased permeability of the vessel wall may play a part, but oedema of the brain is not the general rule. The role of certain immunological mechanisms is being investigated. The final answer will require further study. There is still no good animal model for cerebral malaria, which makes research difficult.

Thin blood smear, sickle cell anaemia. The red blood cells are distorted. Photo copyright ITM

Carriers of the sickle cell anaemia gene (heterozygotes for haemoglobin S) have relative protection against severe infection with P. falciparum and thus have a survival advantage (in homozygous patients, malaria may be fatal and the disease itself tends to kill patients before the reproductive age). The same advantage probably applies to persons deficient in G6PD. This may explain why these two conditions are so common in Africa. Genetic variation in CD40 ligand might also be important. In Papua New Guinea ovalocytosis is common. These red blood cells have an oval shape and cannot be penetrated by P. falciparum parasites. Heterozygotes are thus protected against P. falciparum (homozygosity is not compatible with life). [Distinguish hereditary elliptocytosis, which is only infrequently associated with anemia, from Southeast Asian ovalocytosis, an inherited mild hemolytic anemia]. In West Africa, haemoglobin C is rather frequent. Haemoglobin C contains a glutamate-to-lysine mutation at the sixth position in the beta-globin chain (cfr with sickle haemoglobin, which contains a glutamate-to-valine mutation at the sixth position in the beta-chain).  People with haemoglobin AC or CC can be infected with Plasmodium falciparum and can develop substantial parasitaemias. The presence of Hb C therefore does not protect against infection itself. Haemoglobin C might protect against the lethal effects of P. falciparum malaria by reducing cytoadherence of parasitised erythrocytes. The infected red blood cells tend to be less sticky to the vascular endothelium and display less rosetting. When viewed under an electron microscope, the distribution of the knobs on the surface is different, as compared with infected red blood cells carrying haemoglobin AA. Haemoglobin E (chiefly Southeast Asia) does not protect against P. falciparum infections itself, but more study is needed. The data on the influence of thalassemia on the clinical severity of malaria are contradictory (many different mutations lead to thalassemia, an important confounding factor). Pyruvate kinase catalyses the convertion of phosphoenolpyruvate to pyruvate with the generation of one molecule of ATP. There are some data suggesting that people who lack pyruvate kinase activity in the red blood cells (i.e. homozygous) might be less suspectible to malaria. The effect of the heterozygous state (carrier) on malaria is not clear.

While circulating in human blood Plasmodium falciparum exhibits antigenic variation. On the surface of the infected red blood cell a certain protein is expressed: the P. falciparum erythrocyte membrane protein 1 (PfEMP-1). The parasite is able to make many variants of this protein. By interchanging which variant of PfEMP-1 is present, the parasite can evade the immune response to these immunodominant antigens. PfEMP-1 also inhibits antigen presentation by dendritic cells. The proteins can bind to endothelial receptors [such as ICAM-1 (intercellular adhesion molecule type 1), VCAM-1 (vascular cell adhesion molecule-1), ELAM-1 (E-selectin), CD36 and thrombospondin]. The PfEMP1 proteins are the gene products of what are called var genes, of which there are 50 to 150 present in the genome of the parasite. These proteins are thought to be the major virurence factor found on the surface of infected red blood cells, directly contributing to the pathogenic nature of the infection and placing these genes at the centre of a disease responsible for several million deaths in developing countries. Although there are many var gene copies, only a single var gene is expressed at any given moment (i.e. there is mutually exclusive expression). Over the course of an infection, expression switches from one var gene to another, resulting in antigenic variation of the parasite population and a persistent infection which is difficult to clear by the human immune system. There are also some other variant multigenic families, the products of which can be expressed on the surface of infected red blood cells. Antigenic variation has important implications for the development of vaccines. The repertoire of proteins which are expressed in the Anopheles mosquito is far less pronounced, probably because the vector has no adaptive immune system.

There seems to be several genetic factors that influence the final clinical outcome of an infection. Persons with a gain-of-function mutation in the promoter-region of "inducible nitric oxide synthase" (NOS2), the enzyme which synthetizes NO, have a 75-85% lower risk of severe malaria. NO is a strong vasodilator. High NO levels may be protective against P. falciparum infection by inhibiting cytoadherence. This suggests that the therapeutic potential of NO in the treatment of severe falciparum malaria should be evaluated. Preliminary data suggest that certain TNF-alpha alleles and certain promoters (DNA regios) confer protection against severe malaria. The mean number of complement receptor 1 (CR1, syn. CD35) molecules on erythrocytes in normal individuals is 100-1000 molecules per cell. There seems to be a direct interaction between PfEMP1 on infected cells and CR1 on uninfected erythrocytes. This 'stickiness' between PfEMP1 and CR1 contributes to rosetting, and rosetting probably relates to obstruction of blood vessels. Complement-receptor polymorphism probably influences this interaction and therefore the severity of a malaria attack. Certain blood group antigens (e.g. Knops) are located on CR1. The relationship between malaria severity and Knops blood groups (cfr McCoy, Swain-Langley) is being studied at present. A large case-control study of malaria in West African children showed that a human leukocyte class I antigen (HLA-Bw53) and an HLA class II haplotype (DRB1*1302-DQB1*0501), common in West Africans but rare in other racial groups, are independently associated with protection from severe malaria. In this population they account for as great a reduction in disease incidence as sickle-cell trait. These data support the hypothesis that the extraordinary polymorphism of major histocompatibility complex genes as well as other genes has evolved primarily through natural selection by infectious pathogens. There are some studies which support the hypothesis that infection with helminths would reduce the probability of cerebral malaria with up to 70% and the risk of acute renal failure with up to 84%. These findings need to be confirmed before they can be generally accepted. There is a possibility that activation of CD23 in human endothelial cells by IgE complexes, would increase NO release and reduce ICAM-1 expression, with reduction of cytoadherence of parasized red blood cells. Malaria is very often accompanied by thrombocytopenia, the causes of which seem to be multiple and not completely known. The severity of the thrombocytopenia correlates with the parasitemia and the clinical severity of infection. Activated thrombocytes bind to malaria-infected red blood cells via platelet-expressed CD36. Upon binding, the platelet's alpha-granules release the  chemokine "platelet factor 4" (PF4). This PF4 binds to the red blood cells displaying the Duffy blood-group antigen (Fy). This restricts the growth of the parasite and/or kills it.  

Clinical picture

Clinical picture, classic acute uncomplicated attack

Most clinical episodes of malaria are characterised by fever with aspecific symptoms. Certainly in children the presentation can be very misleading. Any fever should bring the possibility of malaria to mind. There is a danger, however, that in time every fever episode will be regarded as malaria and other important diagnoses are then likely to be missed.

P. falciparum : typical incubation time: 7 to 30 days. If a person is taking preventive antimalarials and if the parasite is partially resistant, there may be temporary suppression of a malaria attack. The fever is generally irregular. If the attack is not treated, after a few weeks a regular fever pattern will develop with peaks every 2 days (tertian malaria, so called because the fever reappears on the third day). This is rare in everyday clinical practice, however. At the beginning of the attack the symptoms are similar to influenza: general malaise, tiredness, muscle pain, headache but in general without respiratory tract problems or runny nose. These symptoms are not very specific. After a while the muscle pain and headache become worse. Sometimes there is also abdominal pain and diarrhoea. Rarely there is a classic attack: this lasts for approximately 12 hours and occurs every 48 hours. At first cold shivers with high fever occur, followed by an intense feeling of heat and fever, leading to a sweating stage with a drop in fever. Most falciparum attacks do not follow this classic pattern. So what is referred to as a classic attack is paradoxically not the general rule.

P. vivax  and P. ovale : the incubation time is a few weeks to years. The awakening of dormant parasites in the liver (hypnozoites) explains late relapses. The fever is sometimes regular (every 48 hours), especially in cases of recrudescence (tertian malaria). In 1922 P. vivax was introduced for the treatment of neurosyphilis. It was thought that the bacterium which causes syphilis had little resistance to heat, so the high fever would kill the bacteria ( Treponema pallidum ). With modern genetic techniques it became clear that P. ovale is subdivided in P. ovale wallikeri and P. ovale curtisi . More study will be needed to understand the implications, such as clinical manifestations, drugs susceptibility and relapse periodicity.

P. malariae : the incubation time is 3 weeks to many years. The very late attacks are probably not due to awakened hypnozoites (to date these have never been detected) but due to the activation of blood parasites which are present at a very low concentration. Fever peaks may occur every 72 hours (quartan malaria).

P. knowlesi is a monkey parasite which can be misidentified as P. falciparum in the early ring stage and as P. malariae in the older stages. It has the shortest asexual life cycle of all, i.e. 24h. The prepatent period is 9-12 days. Till present, no hypnozoites have been found. At present, PCR is needed to firmly identify this species.

Mixed infections: mixed infections do occur, but for reasons which are unclear they are much less common than would be expected based on the prevalence of the individual species. Underreporting may play a part, but this is probably a real phenomenon (partial cross-immunity to heterologous species?, biological interference?).

Clinical picture, natural course of malaria in the autochthonous population

Children are very susceptible to infection. The highest mortality is found in children below the age of 5 years. Gradually, after repeated infections, a partial immunity develops in those who survive. There is a high degree of tolerance to the infection in adults, provided that they live in a stable malaria region. This semi-immunity (premunition) is maintained by repeated infections and mild latent infections. It disappears after approximately 6 to 24 months if there is no further infection (e.g. a stay in a non-malaria region). This partial immunity is reduced during pregnancy. A pregnant woman is at increased risk of hypoglycaemia and cerebral malaria. Malaria is an important cause of severe (sometimes spectacular) anaemia in the mother, low birth weight, premature birth, abortion and increased perinatal death. Chondroitin sulphate and hyaluronic acid, both present in abundance around the syncytiotrophoblasts of the placenta, are mucopolysaccharides (glycosamine glycanes) which act as receptors for red blood cells infected with P. falciparum . Probably there are also other receptor molecules. Infected cells accumulate in the placenta, resulting in reduced placental function. The placental barrier is very seldom passed. Congenital malaria is not common and occurs chiefly in neonates of non-immune women. Neonates of semi-immune women receive transplacental anti- Plasmodium antibodies. Due to this passive resistance in the first 3-6 months they are at a lower risk of malaria.

Several observations of humans infected with both malaria and helminths suggest that co-infection provides a benefit to either parasite. The evidence indicates that malaria patients co-infected with helminths are protected from severe malaria, possibly through skewering of the immune response towards T helper (Th)2 immunity.

Malaria and HIV interact in several, rather complex ways.

Clinical picture, acute severe malaria

Malaria, Plasmodium falciparum . Gangrene of a toe. Copyright ITM

CT-scan abdomen in a case of Plasmodium vivax malaria. Spleen haematoma. Copyright ITM

Acute severe falciparum malaria is a medical emergency. This encompasses:

Coma (the patient cannot be woken) Repeated generalised convulsions Hypoglycaemia: reduced consciousness, aggressive behaviour Severe anaemia: weakness, polypnoea, pale mucosae Tendency to spontaneous bleeding (pronounced thrombocytopaenia) Circulatory collapse (shock); cf. algid malaria Pulmonary oedema (dyspnoea and bilateral crepitations) leading to ARDS Haemoglobinuria (dark urine) Kidney failure: the urinary flow should be monitored and kept above 400 ml/24h. Acidosis (chiefly due to lactic acid): rapid deep respiration. If too many salicylates are given, this may exacerbate the acidosis (not unusual in febrile patients). Other important signs are: marked jaundice, confusion without coma, extreme generalised weakness; very high fever (hyperpyrexia).

The priorities are cerebral involvement, severe anaemia, hypoglycaemia and kidney failure, and the presence of hyperparasitaemia. The degree of parasitaemia correlates with the severity of the symptoms: the higher the parasitaemia, the greater the risk of severe symptoms. It should be borne in mind that the parasitaemia (the percentage of parasitised cells that are found in a smear preparation) changes by the hour. This is because the red blood cells with mature P. falciparum parasites (schizonts) attach themselves to the small capillaries of deep organs, and are not found in a thin blood smear. A parasitaemia of 0.5% is already severe, 2% is pronounced, and patients with a parasitaemia of more than 10% have a relatively poor prognosis. Over 25% is often fatal. Another consideration is that a parasitaemia of 3% in someone who still has a normal red blood cell count, is different from a parasitaemia of 3% in an anaemic patient.

Hypoglycaemia may quickly lead to general deterioration and coma. It is common in children (up to 25%) and pregnant women. Glucose may be life-saving. If the glycogen store in the liver is low (malnutrition) the risk of hypoglycaemia increases [glycogen is converted to glucose = blood sugar]. The conversion of glycogen to glucose is also inhibited by certain cytokines which are released during infection with P. falciparum . [Hypoglycaemic effects of TNF-a and possibly interleukin-1 and TNF-ß]. The parasites themselves also use glucose for their metabolism and contribute to the hypoglycaemia if they are present in large numbers. Quinine can stimulate the secretion of insulin from the pancreas and in this way can also contribute to hypoglycaemia.

The term " algid malaria " (L. "algidus" = cold) is obsolete. The condition is characterised by hypotension with progression to shock. The patient is clammy and often feels cold. There is no fever. Often there is septicaemia with Gram-negative bacteria. Mortality is high. As well as therapy with quinine, treatment with antibiotics and IV fluid administration is of great importance. Shock seldom occurs in malaria if there is no septicaemia. Splenic rupture can also cause hypovolaemic shock, however.

Splenic rupture . This may occur spontaneously or after an unobserved trauma. This complication can occur in P. falciparum, P. vivax , P. ovale  or P. malariae . The presence of intraperitoneal fluid is suggestive in this context. In these cases ultrasound can often detect a splenic haematoma, splenic rupture or intraperitoneal fluid. A diagnostic peritoneal lavage may be indicated.

Plasmodium falciparum. In cerebral malaria, numerous petechiae appear in the brain. Copyright ITM

Plasmodium falciparum. In cerebral malaria, numerous petechiae appear in the brain. Copyright ITM

Cerebral malaria is the main cause of death (80 %) in falciparum malaria. This complication occurs chiefly in non-immune persons (children, travellers). Cerebral signs include confused behaviour, psychosis, convulsions, stupor, coma, paralysis. Unlike meningitis, there is no real neck stiffness (pain) or photophobia (intolerance to light) but neck retraction and opisthotonos may occur. Sometimes the difference between neck stiffness and neck retraction is not clinically clear. It is typical of the coma that it develops swiftly in 75% of cases and also quickly disappears. If a child survives cerebral malaria it has approximately a 10% chance of significant sequelae. Children with cerebral malaria and with a normal eye fundus have a good prognosis, while papiloedema and retinal bleeding suggest a guarded prognosis. Repeated generalised convulsions should not be regarded as "normal" febrile convulsions. Severe convulsions with contraction of the abdominal muscles and compression of the stomach, may cause reflux of gastric acid and food into the pharynx. Aspiration of gastric contents into the lungs is a real danger as this may result in Mendelson's syndrome or aspiration pneumonia. IM phenobarbital is sometimes given as a prophylactic measure (a dose of 3.5 mg/kg up to 10 mg/kg). If there are convulsions, these are stopped by administering diazepam (Valium®) IV or paraldehyde IM. Paraldehyde should be drawn up into a glass syringe (not plastic). A CT scan or MRI scan of the brain of patients with cerebral malaria shows few abnormalities except an occasional increased cerebral volume. Herniation of the brain stem is a rare event.

If confronted by a febrile coma or confusion with fever in the tropics, glucose must be administered (preferably IV), quinine therapy should be instituted and a lumbar puncture carried out without hesitation (to rule out meningitis). Of the persons who will die in hospital due to cerebral malaria, 50% of the fatalities occur within the first 12 hours after admission. At autopsy countless petechiae can be seen in the brain. Small ring-shaped haemorrhages also occur around cerebral blood vessels.

Pale conjunctiva in a patient with anaemia due to repeated malaria.

Icterus secondary to Plasmodium falciparum malaria. Photo Dr Van den Enden, copyright ITM

Note: Febrile convulsions

Febrile convulsions are generalised tonic-clonic convulsions. They only occur in children between the age of 6 months and 5 years and will not be repeated during the same fever episode. They occur during the phase in which the fever is rising fast. They always last less than 15 minutes (including postictal coma) and there is never postictal hemiparesis. It is important to differentiate between febrile convulsions and convulsions during fever (e.g. cerebral malaria, meningitis, cerebral abscess). Approximately 2% of children have a tendency (possibly genetic) for febrile convulsions. The risk that epilepsy will develop in this group of patients is no greater than in children without febrile convulsions. Brief and sporadic attacks have a good prognosis. No maintenance therapy with anti-epileptic agents must be instituted in cases of febrile convulsions.

Severe anaemia   occurs due to haemolysis (of both parasitised and non-parasitised red blood cells - the latter via immune-mediated mechanisms), due to excessive action of the spleen i.e. hypersplenism (until weeks after the infection), due to possible haemorrhages (low blood platelets, splenic rupture) and due to disturbed production of new blood cells in the bone marrow (dyserythropoiesis) due to TNF-alpha. Malaria pigment interferes with the differentiation of blood cells in the bone marrow and can contribute to the anaemia.

Hyperpyrexia should be treated by cooling the patient and administering paracetamol. It is assumed that malaria fever is caused when lysis of the red blood cells releases malaria pigment (haemozoin) as well as GPI-anchors ("malaria toxin") which ere absorbed by the reticulo-endothelial system. This in turn releases endogenous pyrogens (cytokine network). The concentration of tumour necrosis factor in the peripheral blood correlates with the severity of the malaria. In cases of repeated malaria attacks the liver, spleen and bone marrow are stained black by the enormous amounts of haemozoin.

Black pigmentation of the bone marrow in the spine, due to accumulation of malaria pigment (repeated malaria). Photo Dr Gigase. Copyright ITM

Massive increase in spleen size in hyperreactive splenomegaly due to malaria. Copyright ITM

ARDS (acute respiratory distress syndrome) is a feared complication of Plasmodium falciparum malaria. Copyright ITM

"Petechiae on the shins; thrombocytopenia due to hypersplenism in Plasmodium falciparum malaria. Copyright ITM"

Hyperreactive splenomegaly in malaria. Copyright ITM

Hyperreactive splenomegaly in malaria. Copyright ITM

Black water fever is a severe but life-threatening complication. Acute massive haemolysis occurs. It has been observed after taking halofantrine, artemisinin-derivatives and after irregular use of quinine. The precise mechanism is not known. The parasitaemia is generally very low. There is high fever, jaundice, back pain, shock and very dark urine. Renal insufficiency occurs: the urine production is very low (oliguria) or zero (anuria). Mortality is very high. When quinine was no longer used prophylactically, black water fever became very rare. As this product is increasingly back into use, it can be assumed that this complication will again become more common. Differential diagnosis should be made with leptospirosis and viral haemorrhagic fever. Acute renal failure may also be caused by shock, hypovolaemia with reduced renal circulation, DIC (diffuse intravascular coagulation), obstruction of the renal glomeruli by parasitised red blood cells and by the precipitation of released haemoglobin in the kidney (pigment nephropathy). The combination of these factors can result in acute tubular necrosis. Glomerulonephritis may occur in chronic quartan malaria, but this complication plays no part in acute renal problems.

Pulmonary oedema is a common complication of severe malaria. The dividing line between overhydration and dehydration is narrow. Adults easily develop non-cardiogenic pulmonary oedema if there is limited fluid overload, but on the other hand dehydration and hypovolaemia may lead to hypotension, shock and renal failure. As a guideline the central venous pressure should be kept around 5 cm H 2 O. If intensive invasive monitoring is available (e.g. Swan-Ganz catheter in an intensive care unit) an attempt should be made to keep the pulmonary capillary wedge pressure around 15 mm Hg. [The PCWP reflects the pressure in the left atrium]. Pneumonia is observed quite often if coma lasts for longer than 3 days. ARDS (acute respiratory distress syndrome) may occur. This is caused by diffuse damage to the vascular endothelium and the alveolar epithelium. There is a rapid progression towards dyspnoea, arterial hypoxia, bilateral patchy pulmonary infiltrates due to pulmonary oedema with a protein-rich fluid. The treatment is both aetiological and symptomatic: artificial ventilation, with or without intubation or an endotracheal cannula, possibly with NO, high-dosed oxygen and positive end-expiratory pressure (PEEP). Surfactant administered via aerosol might be helpful in this situation, although it is often not available. Further data is needed.

Clinical picture, chronic falciparum malaria

Where P. falciparum is partially resistant to chloroquine, the parasite may be suppressed, but will remain present. This may lead to a whole range of clinical pictures, from asymptomatic parasitaemia through to mild aspecific symptoms, to significant chronic malaise and fatigue. Curative therapy with Malarone®, for example, produces rapid improvement.

Clinical picture, hyperreactive malaria splenomegaly (HMS)

Some adults have a very strong immunological reaction to P. falciparum antigens. The level of IgM in the blood is very high. Due to the polyclonal immune stimulation, all kinds of auto-antibodies can appear. Immune complexes are formed, and are removed by the reticulo-endothelial system, which leads to splenomegaly and sometimes enlarged liver. In these individuals the swollen spleen swells also breaks down normal, unparasitised blood cells. The number of parasites is very low, but very high concentrations of anti- Plasmodium falciparum antibodies can be detected. The splenomegaly disappears after curative therapy with, e.g. quinine + tetracyclines followed by months or even years of adequate malaria chemoprophylaxis (impregnated mosquito net + efficient chemoprophylaxis in a malaria region), but recovery is very slow. In rare cases splenectomy is necessary. Steroids have no place in the treatment.

The disorder may be very similar to a certain indolent splenic lymphoma (e.g. splenic lymphoma with villous lymphocytes). The latter disorder is related to B-cell chronic lymphocytic leukaemia and occurs chiefly in elderly persons. The disease is often accompanied by significant cytogenetic abnormalities and monoclonal "villous" B-lymphocytes in the peripheral blood. It is likely that in HMS, excessive stimulation of the B-lymphocytes by malaria antigens increases the risk that oncogenic mutation may occur, followed by clonal growth of these cells. The extent to which this etiopathogenetic mechanism is similar to the MALT lymphomas (mucosa-associated lymphoid tissue) which are sometimes seen in chronic infection with Helicobacter pylori , is unclear.

Clinical picture, Burkitt's lymphoma

Burkitt's lymphoma. The chromosomal translocation (8q24 - 14q32) displaces the oncogen c-myc and places it against the gene coding for immunoglobulines. Copyright ITM

Burkitt's lymphoma in a Cambodian woman, aspect before chemotherapy. Photo Dr Lut Lynen, copyright ITM

'Burkitt's lymphoma in Cambodian patient; photo copyright Dr Lut Lynen - ITM.'

This malignant tumor originates from B-lymphocytes. It is very agressive with a volume doubling time of about 3 days. The endemic form occurs in sub-Saharan Africa in geographical latitudes 10-15° north and south of the equator in areas below 1500 meter with rainfall more than 50 cm per year and temperatures above 16°C. Outside Africa it is also found in Papua New Guinea. In these areas, it account for up to 50% of childhood tumors.  In the differential diagnosis, the possibility of a deep mycosis (e.g. rhinophycomycosis, mucormycosis) or a chronic abscess of the tooth, jaw or sinuses should be borne in mind. Wilms' tumor (= nephroblastoma), neuroblastoma, lymphoblastic lymphoma and embryonal rhadbomyosarcoma can mimick Burkitt's lymphoma. There is also a sporadic form of Burkitt's lymphoma, but this one is rare in children. BL generally presents with swelling of the jaw and mouth ulcerations (75%, especially maxilla tumors), abdominal swelling with ascites (60%) and central nervous system involvement (30%, including cranial nerve palsies, malignant pleocytosis or paraplegia). Location in testis, ovaries, lungs, kidneys and other organs also occurs.  The age at presentation is usually 4-9 years. The tumor is basically unknown in children less than 12 months old. About 1% of patients is less than 2 years old.  Less than 5% of patients are older than 15 years. Infection with the Epstein-Barr virus (cf. mononucleosis) plays an important part in the endemic form of Burkitt's lymphoma, probably by causing genetic instability. Epstein-Barr viral DNA is found in about 90% of African Burkitt's lymphomas. Only one protein, EBNA-1, [Epstein-Barr nuclear antigen-1] is expressed. In a large prospective study in Uganda, it was found that BL patients seroconverted for Epstein-Barr about 6-24 months prior to tumor appearance. In patients with non-BL tumors, Epstein-Barr genome was not detected in neoplastic cells although these tumors came from patients with positive EB serology. One hypothesis states that repeated malaria attacks may have a mitogenic effect on infected B-lymphocytes (polyclonal B-cell stimulation) increasing the risk of mistakes during chromosomal replication which subsequently would lead to neoplastic behaviour. A small percentge of BL patients test negative for Epstein-Barr, indicating that EBV is not absolutely necessary for the development of Burkitt's lymphoma. At present it is unclear if contact with the plant Euphorbia tirucalli has a role in pathogenesis. This plant is indigenous to tropical eastern and southern Africa and contains toxic milk-like latex. There are no blast cells in the peripheral blood, but in a closely related form there is a leukaemic phase (ALL-type [acute lymphoblastic lymphoma]). Histologically a monotonously uniform picture can be recognised, consisting of small cells with round to oval nuclei containing two or more prominent nucleoli. The basophilic cytoplasm may contain clear fat vacuoles (Oil Red O positive). There are many mitoses. The histological picture is sometimes described as a starry sky. The stars are macrophages. Metastasis occurs chiefly to the brain and bone marrow. In 1972 Manolov reported a specific and characteristic chromosome abnormality in Burkitt's lymphoma. In about 90% of BL, part of the long arm of chromosome 8 is translocated to the the long arm of chromosome 14. This typical translocation places the c-myc cellular proto-oncogene from chromosome 8 very close to the immunoglobulin heavy-chain region on chromosome 14. In other cases, c-myc is translocated very close to the kappa light-chain loci on chromosome 2 or the lambda light chain on chromosome 22. These translocations are observed in all BL tumors irrespective of Epstein-Barr genome status, endemic or sporadic. This repositioning disturbs the control of the c-myc gene and gives rise to malignant growth behaviour.  Nevertheless the pathogenesis of Burkitt's lymphoma is not yet completely clear. A second oncogene appears to be needed to complete the neoplastic transformation.

The tumor is very responsive to chemotherapy. Advise of a specialist should be sought. Treatment will be multidisciplinary, with surgery needed for biopsy, debulking, spinal cord decompression and eventual insertion of an Ommaya reservoir (intrathecal methotrexate). Debulking is clearly beneficial. Althought the tumor is radiosensitive, its very fast growth makes conventional radiotherapy ineffective (the tumor regrows between each day's therapy). Superfractionation (3 sessions per day) can be given but is rather impractical in most circumstances, even in those rare places when radiotherapy is available. The tumor responds well to cytostatic drugs. The alkylating agent cyclophosphamide (Endoxan®) is first choice (the target dose 1-1.5 gram/m 2 IV every 3-4 weeks with 2 doses in remission).  Cyclophosphamide should not be confused with cyclosporin! During treatment haemorrhagic cystitis sometimes occur as complication. Mesna (sodium 2-mercaptoethanesulfonate) is given during chemotherapy with cyclophosphamide to prevent hemorrhagic cystitis and kidney damage. Mesna binds toxic metabolites of cyclophosphamide which are filtered by the kidneys.  Patients with BL, especially those with extensive disease, have a high risk of tumor lysis syndrome because of the rapid tumor cell turnover. Patients should receive prophylactic allopurinol and aggressive hydration with alkalinization starting as soon as Burkitt's lymphoma is diagnosed. Electrolytes, especially potassium, calcium, phosphorus, uric acid and creatinine should be monitored closely. In ideal situations, treatment should be performed at a facility where renal dialysis is available, particularly for patients with extensive disease. Allopurinol protects from the risk of hyperuricaemia during tumor lysis. If available, methotrexate (15 mg/m 2 P.O. for 3 days) and vincristine (1.5 mg/m 2 IV per week) may be given in addition. Citrovorum factor (leukovorin) is not necessary during brief administration of methotrexate. Combination therapy is indicated for the sporadic form of BL. Combinations of several cytostatic drugs are used, e.g. CHOP or hyper-CVAD, cytarabine, L-asparaginase.  CHOP consists of: cyclophosphamide 750 mg/m 2 IV day 1, doxorubicin 50 mg/m 2 IV day 1, vincristine 1.5 mg/m 2 mg IV day 1 and prednisolone 100 mg/m 2 per day for 5 days. Neurotoxicity such as polyneuritis during use of Vinca alkaloids is an inherent danger. Bone marrow suppression should be carefully monitored. Doxorubicin is cardiotoxic. Surgical debulking is an important component of treatment. Monoclonal antibodies (Mabthera®, Rituxan®) aimed at the B-cell specific CD-20 antigen which is expressed by 95% of all B-cell lymphomas, are almost never available in the tropics but can be used in low-grade lymphomas. About 80% of patients can achieve complete tumor regression and 10% have a partial response. About 50% will relapse. If this occurs less than 3 months after initial chemotherapy, the outlook is poor. If a patient remains relaps-free for 1 year, he can be considered cured. The overall 10-year survival is 35-50%.

Clinical picture, nephrotic syndrome in P. malariae

Nephrotic syndrome secondary to chronic infection with Plasmodium malariae . Notice the swollen face and ascites. Photo prof. Gigase. Copyright ITM

Chronic infection with P. malariae may, via immunological mechanisms (chronic immune complex glomerulonephritis) cause a nephrotic syndrome, characterised by oedema and proteinuria (more than 3.5 gram per 24 hours). There is often significant hyperlipidaemia, and lipid bodies are sometimes found in the urine (which appear in polarisation microscopy like bunches of grapes with a Maltese cross pattern). If a kidney biopsy is carried out, it should be borne in mind that severe bleeding will occur in 1% of cases. The treatment of nephrotic syndrome is difficult. A curative malaria treatment is of course indicated, but will not produce any improvement of the kidney problems. Salt restriction and diuretics are indicated (both thiazide and loop diuretics). Albumin IV and treatment with an ACE-inhibitor [angiotensin-converting enzyme-inhibitor such as enalapril] is only possible in better settings. In significant hypercholesterolaemia statins are beneficial but expensive (e.g. simvastatine, HMG-CoA reductase inhibitor). Patients with advanced nephrotic syndrome lose coagulation inhibitors through the urine (protein S, C, antithrombin III) and are therefore at increased risk of thrombosis, chiefly in the vena renalis. Steroids and immunosuppressives are of little benefit in this disorder. An important challenge is to distinguish the entity from minimal change glomerulonephritis (electron microscopy needed to confirm "minimal change").

Diagnosis

Diagnosis, general

When can one assert that someone has the disease "malaria"? There are several problems and the question has still not been fully resolved. The demonstration of malaria parasites in the blood is essential, but insufficient in itself. Most cases are accompanied by thrombocytopenia and normal white count. Many people will develop an acquired immunity after several years of exposure, and may harbour parasites without exhibiting symptoms. The degree of parasitaemia may help, but there is no absolute criterion (the higher the parasitaemia, the more chance that malaria is in fact the diagnosis). There are patients with malaria for whom the thick smear is negative (luckily this is rare in a good laboratory). There are no pathognomonic clinical signs. An accurate diagnosis is becoming more and more important, in view of the increasing resistance of P. falciparum and the high price of alternatives to chloroquine.

Diagnosis, clinical aspects

No single clinical sign permits the diagnosis of malaria. Most cases are accompanied by thrombocytopenia, a normal white count and a positive parasitaemia. Yet malaria must always be considered in cases of fever in the tropics. Since the symptoms can be quite diverse, a clinical diagnosis is unreliable in itself and the diagnosis should be based on identification of the parasite. Microscopic confirmation of the diagnosis is often not possible in many regions and situations. It is of the greatest importance that other important diagnoses are ruled out before instituting a blind anti-malaria therapy. All too often fever is considered as malaria without considering alternative diagnoses. This tendency is reflected in the quote: "if you only have a hammer, you tend to see every problem as a nail" (Abraham Maslow).

The presence of parasites does not rule out an additional diagnosis: e.g. someone with fever may well have some malaria parasites in a thick smear, but this does not rule out meningitis or pyelonephritis. Chronic carriers are people who, in spite of the fact that they have malaria parasites in their blood, have no symptoms of this. When such people develop another infection their symptoms are often attributed to the malaria parasites in their blood, although these are not responsible. The absence of parasites in a single preparation does not rule out malaria, but does make the diagnosis of P. falciparum highly improbable. Where there is strong clinical suspicion it is best to repeat the test 12h later.

Diagnosis, microscopy

Thick smear

Preparation of a thick blood smear. Place a drop of blood in the center of a slide. Use the corner of another slide to mix and spread the blood from the center of the drop outwards. Use a circular motion to spread the blood to about the size of a small coin, circling out, back to the center and back out again several times. Allow to air dry, may take 30 minutes or even more. Do not fix in methanol. Either rinse in distilled water then stain, or stain directly. Use Giemsa. Copyright ITM

Preparation of a thick blood smear.  Copyright ITM

Thick blood smear for the diagnosis of malaria. A correct technique, including labeling, is essential. Copyright ITM

A thick smear concentrates the parasites 10 to 25 times. It is rather more difficult to interpret than a thin smear preparation and often does not permit species identification.

Plasmodium falciparum, thick smear. Copyright ITM

"Positive thick smear, Plasmodium falciparum ; copyright ITM"

A thick smear contains no intact red blood cells (haemolysis due to the distilled water used in the staining). If a thick smear is positive, a thin smear should be examined. Sometimes parasitaemia is estimated in a thick smear and expressed as +, ++, +++. This form of record is of course quite subjective and confusing and is best avoided. If the thick smear is positive, it is then best to count the percentage of parasited cells in a thin smear preparation. The parasitic density can also be roughly determined in a thick smear, by counting the number of parasites per 200 leukocytes and multiplying this by 30. It is assumed that on average there are 6000 leukocytes per µl blood and that there is one leukocyte per 500 red blood cells. For example: 5 parasites per leukocyte (1000 parasites for every 200 leukocytes) corresponds to a density of 30,000 parasites per µl. Roughly 30,000 parasites per µl corresponds to a parasitaemia of 1% (a moderately anaemic person). If the thick smear is found to be negative in a reliable laboratory, and if there is nevertheless strong suspicion of malaria, the test is repeated every 12 hours for 48 hours. One great disadvantage of the thick smear method is that reliable technical expertise is needed which should be monitored (e.g. quality control). The argument that a lab technician has carried out the test for years and thus has plenty of experience, is absolutely no guarantee of quality or reliability. The test also requires plenty of time if the parasitaemia is low, or before a negative result can be concluded. It is possible to concentrate malaria parasites on microscopy slides at least 40-fold using magnets. This approach, which is still experimental, promises to deliver greater sensitivity than classic thick smear preparation. Magnetic deposition microscopy exploits the fact that hemozoin (which contains iron) is sensitive to magnetic fields. The two poles of a magnet (separated by a small gap) are placed near a polyester slide. A cell suspension is pumped across the interpolar gap and the infected cells become trapped. This allows parasitized erythrocytes to be concentrated onto a small region of a microscopy slide.

Thin blood smear

A thin blood film has many advantages : it demonstrates the species present detection of mixed infections is possible distinguish asexual stages from gametocytes assess parasitemia can detect a new or unexpected parasite gives information on red cell morphology allows a white cell differential count inexpensive Other points include Sensitivity and specificity is operator dependent. In a good average lab, the sensitivity is good but limited to about 50 parasites per µL, somewhat better in a reference lab. DNA amplification techniques have better sensitivity and can give information when species is in doubt. Material (microscopes, stains) needs to be maintained properly. Requires internal and external quality control system A blood film shows the presence of undistorted parasites. It permits identification and also calculation of the parasitaemia (% of parasitised red blood cells). This is necessary to start appropriate therapy ( P. vivax is treated differently from P. falciparum ). If the parasite cannot be identified it is regarded as a P. falciparum as a safety precaution. Mixed infections do occur. For staining, Giemsa is used with a slightly alkaline pH. It is a good habit to prepare the buffer solutions each day in the morning (interaction with CO 2 from the atmosphere changes the pH of older solutions). Phosphate buffers are commonly used.

First a stock solution is made of KH 2 PO 4 . This is made by placing 9.078 g KH 2 PO 4 in one litre of purified water. This stock solution can be used for weeks if correctly stored (in a closed bottle).

A stock solution of Na 2 HPO 4 .2H 2 0 is also made by mixing 11.877 g with one litre of purified water. If one uses the anhydrate (Na 2 HPO 4 ) in place of Na 2 HPO 4 .2H 2 0, only 9.474 g per litre is used. This stock solution can also be used for weeks if correctly stored (in a closed bottle).

To obtain a buffer with a pH of 8, add 5.5 ml of KH 2 PO 4 solution to 94.5 ml of Na 2 HPO 4 .2H 2 0 solution and dilute with 900 ml of distilled water. One then has 1 litre of buffered water with a pH of 8. This can then be used for the malaria blood smears for the rest of the day.

For the leukocytic formula it is best to stain with a slightly acid pH of 6.4. For this a different phosphate buffer is used. This requires different ratios. Now 26 ml of the Na 2 HPO 4 .2H 2 0 solution is mixed with 74 ml of the KH 2 PO 4 solution and then 900 ml water is added.

An alternative to Giemsa is acridine orange, but the day-to-day use of this technique is quite unpleasant and it is necessary to have a special microscope (ocular filter, halogen light, interference filter above the condenser). [This latter filter restricts transmission to some narrow spectral bands].

P. falciparum  infection is characterised by:

- small ring shapes, sometimes double chromatin specks - accolé forms forms (parasites adherent to the membrane of the red blood cell) - several parasites per red blood cell - few or no schizonts - banana-shaped gametocytes (the name "falciparum" comes from L. "falx" = sickle and "pario" = bring forth; they are sickle-shaped). Sometimes the red blood cell also contains inclusions (Garnham's bodies) or a gametocyte. - high parasitaemia may occur in P. falciparum and is unusual in the other malarial species (parasitaemia above 2 % is suggestive of P. falciparum ).

P. vivax  preferably penetrates young (therefore large) erythrocytes.

P. ovale  is often found in a thin smear preparation in rather oval-shaped, sometimes distorted red blood cells.

P. malariae  trophozoites sometimes have a typical band shape. The mature schizonts have a daisy head appearance.

Plasmodium falciparum trophozoites in thin blood smear. Copyright ITM

Plasmodium malariae in thin blood smear. Copyright ITM

Plasmodium vivax in thin blood smear. Schizonts have numerous nuclei. Copyright ITM

Plasmodium ovale in thin blood smear. Notice the elongated shape of the erythrocyt. Copyright ITM

In well-stained preparations the nuclei of the parasites are always stained red and the cytoplasm blue. The presence of malaria pigment is very characteristic of the older stages of Plasmodium sp. P. falciparum often contains a single black dot. P. vivax often contains countless fine golden yellow/brown specks of malaria pigment. In P. ovale and P. malariae the pigment inclusions are many and brownish black. Countless fine red spots in the red blood cell (Schüffner's dots) can be seen in P. vivax and P. ovale (the more mature the parasite, the more dots). In P. ovale the dots are sometimes called James's dots. Sometimes a few flecks can be observed in P. falciparum (Maurer's dots or clefts). P. malariae almost never exhibits dots (Ziemann's dots). The visibility of these dots depends to a great extent on the acidity (pH) with which the thin slide preparation is stained (slightly alkaline: pH = 8 is best). The acidity is important because blood smears are usually stained for haematological tests with a slightly acid pH. With such a stain, the dots will not be seen clearly if at all.

Negative thin blood smear. There is a Cabot ring as a red cell inclusion, not to be mistaken for a malaria parasite. Copyright ITM

Babesia microti . These parasites can be mistaken for malaria parasites. Copyright ITM

Thin bloodsmear with Howell-Jolly bodies. These spherical or ovoid eccentrically located granules are approximately 1 µm in diameter. They represent nuclear fragments and stain with dyes which are specific for chromatin. They occur plentiful after splenectomy, in megaloblastic anaemia and in severe haemolytic anaemia. Copyright ITM

Note: Inclusions in red blood cells Malaria parasites Babesia sp. (sometimes not easy to differentiate from Plasmodium sp) Intraerythrocytic Bartonella bacteria Superposition of a blood platelet on a red blood cell (a common source of mistakes) Precipitations of dyes (artefacts) Howell-Jolly nuclear fragments (increased numbers in asplenia, acute haemolysis or bleeding, bone marrow invasion, etc.) Pappenheimer bodies: hemosiderine inclusions. These iron-incontaining bodies tend to be seen in sideroblastic anaemia and post-splenectomy. Basophilic granulation or punctuate stippling: altered ribosomes (contain rRNA, therefore stain), prominent in thalassaemia, lead intoxication, antimitotics, megaloblastic anaemia Reticulocytes: young red blood cells (Crystal violet stain) Cabot rings: single or double ring or figure of 8-like structure left over of mitotic spindle. E.g. in megaloblastic anaemia Heinz bodies: precipitated denatured haemoglobin (G6PD deficiency, unstable Hb), best seen with supravital stain

QBC (Quantitative Buffy Coat)

A special glass capillary tube is filled with 20 µl blood (a droplet via a finger prick). The inner side of this tube is coated with anticoagulants and the dye acridine orange. Acridine orange will bind to the DNA in the nuclei of the malaria parasites, and also to ribosomal RNA. Afterwards the tube is centrifuged (10,000 g x 5 minutes). The blood cells are thus separated according to density. The buffy coat is the part of the centrifuged blood which contains platelets and white blood cells (buff = pale yellow). In the tube is a longitudinal plastic float with the same density as the buffy coat. The float serves to spread the buffy coat and adjacent red cells and press them in a thin layer against the wall. Since parasitised red blood cells are lighter than non-parasitised ones and heavier than white blood cells, infected red blood cells will be found on top of the red cell column, just below the white blood cells, right against the buffy coat. The parasites in this layer can be observed using a fluorescence microscope. However, be aware that Howell-Jolly bodies (nuclear residues) may look similar to parasites. This technique is much quicker than reading thick or thin smears but requires training and appropriate apparatus. Nevertheless, its use can considerably reduce the workload of the laboratory staff especially in larger hospitals where many samples are processed every day. No species identification can be obtained using QBC. With QBC there is quite wide inter-observer variability. The specially prepared disposable tubes need to be available as well as a microhaematocrit centrifuge and a microscope with a UV-lens. The thin tubes sometimes break.

Diagnosis, antigen detection

Malaria diagnosis, antigen detection. Copyright ITM

Malaria, antigen detection. Copyright ITM

Several antigen detection tests have been developed (Parasight®, Malaquick®, ICT Malaria Pf® [Immunochromatographic Test]). The material for the test kit consists of a simple strip (similar to urine dipsticks) and some dropper bottles with reagents. Some tests detect the histidine-rich protein, the PfHRP-II antigen. The abbreviation stands for P lasmodium f alciparum H istidine R ich P rotein. This protein is a constituent of the nodules on the membranes of red blood cells which are infected by P. falciparum . Some tests makes use of two antibodies which are specific for the PfHRP-II antigen. Both are attached to a paper strip. One of the antibodies is coupled to colloidal gold and applied to the place where the blood sample is to be applied. The second antibody is fixed elsewhere on the strip, in a band where the test result is read. Approximately 10 µl blood is applied (a droplet from a finger prick. Some blood may also be applied via a capillary tube containing EDTA). The red blood cells are lysed. If there is PfHRP-II antigen in the blood, this binds to the antibodies labelled with gold. After administration of a buffer the gold-labelled antibodies migrate with the capillary flow along the test strip and then cross the band containing the second antibody. If the blood sample is positive the antigen antibody complex labelled with PfHRP-II binds to the second antibody and a clear purple band is produced. This does not occur with a negative sample. A control band must always be visible. Malaria rapid diagnostic tests are increasingly used in non-endemic settings. They are easy to use, provide results rapidly and require no specific training and equipment. Reported sensitivities vary between different RDT products but are generally good for Plasmodium falciparum, with rapid tests based on the recognition of P. falciparum antigen histidine-rich protein-2 (PfHRP2) scoring slightly better than those which recognise P. falciparum-lactate dehydrogenase. Sensitivity is lower for Plasmodium vivax (66 – 88%) and poor for Plasmodium ovale (55 – 85%) and Plasmodium malariae (21 – 45%). Rapid diagnostic tests have some limitations. The test strips are susceptible to heat and humidity. A positive results can be obtained after correct treatment, when there are no more parasites visible in the thick blood smear. This is due to persistence of the PfHRP2 antigen (up to several weeks) after succesful treatment. Occasional there is cross-reactivity of P. falciparum with the non-falciparum test line and vice versa and rare false-positive reactions due to other infectious agents or immunological factors. False-negative results occur in the case of low parasite densities, prozone effect or pfhrp2 gene deletions. When instruction are not followed (delayed reading, incorrect sample and buffer volumes, not recognizing invalid test results, disregarding faint test lines). errors in interpretation can occur. Rapid diagnostis tests do not give information about parasite density.

The test is quite quick and simple to carry out and needs no technical apparatus. The sensitivity is 90-95% for parasitaemia of more than 100 parasites/µl. Low parasitaemia is thus often missed. The test remains positive so long as there is still antigen in the blood (that is even if the living parasites have already disappeared due to adequate therapy). The ICT Malaria Pf® test can only detect P. falciparum . The presence of rheumatoid factor may lead to a false positive result (a problem with Parasight®). The use of a different buffer (or plain water) as an eluent can also lead to false positive reactions. It is very important that the correct buffer flask is used (this comes with the kit). P. vivax  does not cause cross reactivity. The chief problem, however, is the high price. A curiosity: using Parasight® malaria antigen was found in Egyptian mummies, which is an argument for the presence of this infection in antiquity.

An antigen detection test has also been developed for P. vivax . Using the latter test both P. falciparum and P. vivax can be detected, as well as mixed infections, on the same paper strip (ICT Malaria P.f/P.v®). The result is the presence of one or more horizontal stripes on the strip (as with a urine dipstick). In mixed infections the presence of P. vivax may not be discovered. In view of the simplicity of the test, this technique should in future be of benefit to frequent travellers in isolated tropical regions. It should be noted that these tests do not produce any quantitative information (including no parasitaemia). This is important in regions where chronic carriers are common. An additional difficulty is that in patients with high P. falciparum parasitemia, false positive P. vivax antigen tests (pLDH) are common, as verified by microscopy plus real-time PCR.

Diagnosis, malariapigment

In malaria infections, malariapigment (hemozoin) can be present in some white bloods cells, such as neutrophils. This can generated abnormal light scatter and thereby trigger an automatic warning signal in certain automated cell counters.

Malaria diagnosis, antigen detection. Copyright ITMAutomated cell counters, such as certain Cell-Dyn instruments, use 90° depolarized light scatter to distinguish eosinophils from other leukocytes. Eosinophils are normally the only leukocytes that depolarize light. Some automated hematology analyzers display an alert for possible malaria based on the presence of  activated monocytes (Coulter Counter), hemozoin containing white blood cells (Cell-Dyn series) and an additional peak in the reticulocyte fraction (Cell-Dyn series). During malaria infection, the parasites consume hemoglobin and produce malariapigment, a form of polymerized heme. This pigment, also known as hemozoin, is birefringent. It will be cleared from the bloodstream via the reticulo-endothelial system. This explains the black discoloration of spleen, liver and bone marrow in chronic malaria. Circulating neutrophils and monocytes will also engulf pigment granules. When peripheral blood is analyzed by automated flow cytometry, the pigment will cause atypical depolarization of the laser beam that can be recognized in a scatterplot. Although diagnostic accuracy of these features is too low to exclusively rely on these flags for malaria diagnosis, such an alert is especially useful in situations where the initial clinical suspicion of malaria is low. Since in these situations no specific malaria test (thick smear, antigen test) would be ordered by the clinician, an automated warning signal will be generated. If this deviant scatterplot is detected, a thick smear and antigen test can be performed.

Malaria-antigens used in Rapid Tests

Histidine-rich protein 2 of P. falciparum (PfHRP2) is a water soluble protein that is produced by the asexual stages and gametocytes of P. falciparum , expressed on the red cell membrane surface. It stays in the blood for at least 28 days after the initiation of antimalarial therapy. Several rapid diagnostic tests targeting PfHRP2 have been developed. Plasmodium aldolase is an enzyme of the parasite glycolytic pathway expressed by the blood stages of P. falciparum as well as the non-falciparum malaria parasites. Monoclonal antibodies against Plasmodium aldolase are pan-specific in their reaction and have been used in a combined 'P.f/P.v' immunochromatographic test that targets the pan-malarial antigen (PMA) along with PfHRP2. The pan-malarial aldolase antigen test can also give a positive results in P. knowlesi infections. Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live parasites and it is present in and released from the parasite infected erythrocytes. It has been found in all 4 human malaria species, and different isomers of pLDH for each of the 4 species exist. Cross-reactivity of P. falciparum with P. knowlesi in this pLDH-based test can can occur. With pLDH as the target, a quantitative immunocapture assay, a qualitative immunochromatographic dipstick assay using monoclonal antibodies, an immunodot assay, and a dipstick assay using polyclonal antibodies have been developed.

Malaria diagnosis, PCR

At present, in case of doubt, mixed infections, low parasitemia, forensic questions, suspicion of zoonotic malaria, etc PCR technology (e.g. multiplex real-time PCR) can give answers to several questions, but is in general slower than the traditional methods. Giemsa-stained thick blood films as well as blood extracted from rapid diagnostic tests are a reliable source of DNA for Plasmodium falciparum real-time analysis, allowing applications in reference and research settings in case whole blood samples are not available. Detection of low parasite densities is of particular importance since rapid diagnostic tests have low sensitivities at parasite levels below 100/µl and 500/µl for P. falciparum and the non- falciparum species respectively. The detection limit of real-time PCR is below (better) that of microscopy in reference settings, i.e. close to 10-50/µl. Real-time PCR has several advantages, being faster than traditional PCR, and having no need for agarose gel preparation, gel electrophoresis, detection via ethidium bromide and imaging. In vitro culture is possible for some parasites (blood sample from patient to be taken on ACD anticoagulant, e.g. 3 cc ACD for 17 ml full blood). If the parasite grows in vitro, certain genetic sequencing can be performed.

Diagnosis, serology

Serology can only be carried out in reference hospitals and is of no importance for the individual diagnosis in acute fever. The antibodies are positive from the tenth day, so at the beginning of the attack they will be negative. The presence of antibodies only shows that there has been contact with the parasite. This does not mean that there is sufficient immunity. There will be high titres of antibodies in the tropical hyperreactive splenomegaly syndrome. Malaria type IgG antibodies penetrate the placenta and will give the neonate temporary and partial protection against malaria during the first months of life. Antibodies after infection remain positive for a longer time.

Diagnosis, indirect aspects

Signs of haemolysis include yellow serum, dark urine while faeces have a normal colour, low total cholesterol and HDL, high LDH and low haptaglobin. Often there is thrombocytopenia. Sometimes there is malaria pigment in white blood cells. The percentage of neutrophils containing malaria pigment is a measure of the severity of the situation (e.g. more than 15% is a high percentage). Monocytes which contain malaria pigment have little clinical prognostic value. Pigment-containing monocytes persist in the periperal blood for a median of 216 hours, while pigment-containing neutrophiles persist of a median of 72 hours. It is necessesary to count many blood cells in order to have a good idea of the percentage of the pigment-containing cells. In general, this is only reliably done with automated high-speed cell-sorting machines (flow-cytometry).

Diagnosis, test therapy

In endemic regions fever, muscle pain, or even generally feeling unwell are often attributed to "malaria". An anti-malaria treatment is then instituted, without obtaining confirmation of the diagnosis or often even without considering alternative diseases. The argument given is that such a treatment can do no harm, that the diagnosis of malaria is always probable because the disease is common, and that this is a good strategy for first-line care. Each of these arguments can be defended to a certain extent, but in this way often useless and sometimes expensive treatments with potential side effects are administered. Not recognising and treating other diseases (borreliosis, rickettsiosis, kidney infections, amoebic liver abscess, pneumonia, septicaemia and so on) is a daily reality in many tropical regions. The over-diagnosis of malaria often leads to under-diagnosis of other treatable disorders. It is sometimes stated that fever which does not disappear after three days adequate therapy, is not malaria. The problem with this attitude is of course " adequate" : the problem of drug-resistant malaria and malaria accompanied by complications (e.g. septicaemia).

Treatment

Treatment, specific anti-malaria drugs

General

Most people are not very interested in the history of a particular medicine. Quinine, however, is rather different and occupies a special place. For 300 years this was the only specific treatment for malaria. It has now been used for 360 years. It was used long before Ehrlich wrote down the principles of chemotherapy. The story of its discovery, the important part which quinine has played in the colonisation of the tropics, its role in both World Wars and during the Vietnam war, and the present come-back of this product all make it unique. At present quinine and related products are used in the treatment of P. falciparum malaria, as an antiarrhythmic, as a muscle relaxant and as a flavouring (Schweppes!). There are also some minor applications such as the treatment of babesiosis.

The bark of a certain tree in Peru has been used in traditional medicine since time immemorial. According to the legend in 1638 in Lima, in what is now Peru, Anna del Chinchon, the second wife of Don Luiz Fernandez de Cabrera Bobadilla y Mendoza, count of Chinchon, the Spanish Viceroy of Peru, was successfully treated by the physician Juan del Vega with this bark for a "malaria attack". Some historians doubt the authenticity of this story and state that her actual stay in Peru did not correspond with the period described in the legend. According to the notes of a certain Augustinian priest, Antonio de la Calancha (1633) the "fever bark" had come from Loxa, 700 km further north, in present-day Ecuador. The cinnamon coloured bark was finely ground and then given to the patient with hot water or warm wine. There were also reports of other acts of healing, such as that of a certain Don Juan Lopez de Canizares in 1630. The Jesuits brought the bark of this plant with them to Rome (Father Bartolomé Tafur) and Spain (Father Alonso Messias Venegas). The first load of bark was sent to Europe in 1641, to Seville, the only town which could then accept goods from the New World. In Rome the effect of the bark was tested under the supervision of Cardinal Juan de Lugo and Gabriella Fonseca, the personal physician to Pope Innocent X. In 1654 the Peruvian bark was introduced into England, but the British protestants objected to testing a "Catholic potion". Oliver Cromwell refused to use it, and is said to have died of malaria in 1658. A few years later the British doctor Robert Talbor healed King Charles II with his "secret drink". In 1678 he was sent by the same English King to France where he successfully treated the son of the French king Louis XIV (1638-1715). He revealed his formula to the French (quinine with opium in wine) in exchange for 3000 golden crowns and a pension for life. The formula was published after his death. In 1677 the tree bark was included in the London Pharmacopoeia as "Cortex Peruanus". In 1692 the Chinese emperor K'ang Hsi of the Manchu dynasty was successfully treated with it, due to which the new Westerners were looked upon favourably in his country. This had far-reaching historical consequences for western influence in China, and for Christianity in particular.

It is possible that malaria was a disease imported into the New World by the Westerners. It would therefore be strange if the Indians had already used the bark for this new illness. Nowhere is it reported that the Indians used quinine bark against fever. Although intermittent fevers (ague) were then well known, the difference between malaria and other causes of fever did not really become clear until the germ theory became generally accepted at the end of the 19 th century. No one identified "malaria" as such, no one knew what Cinchona alkaloids were, let alone quinine. It is a fact that Indians who worked in the cold highlands, drank warm quinine tea so that they shivered less. Quinine suppresses striated muscular tissue in two ways: directly by prolonging the refractory period after muscle contraction and indirectly by heightening the threshold at the neuromuscular junction. This is illustrated by the fact that small amounts of quinine increase the severity of myasthenia gravis symptoms. Quinine can also be used in the treatment of myotonia congenita and leg cramps at night. It was probably due to the observation that quinine reduces shivers, and that malaria is often accompanied by fever, that this was administered. It may have been pure chance that precisely this agent also killed the parasite and brought healing.

Cinchona ledgeriana . Quinine is extracted from the bark of this tree.

Quinine and quinidine are diastereoisomers (they have different physical properties, as opposed to enantiomers). Both are active against malaria parasites. Copyright ITM

In the first half of the 18 th century (1735-1738) France organised a scientific expedition in order to determine the form of the earth by measuring the length of an arc of a meridian and thus establishing the flattening of our globe towards the poles. On this expedition, which was led by Charles-Marie de La Condamine, there was a botanist, Joseph de Jussieu. In 1735 he described the tree as a member of the Rubiaceae, the same plant family as coffee. Initially he gave it the name Quinquina condamine . This caused confusion since another tree, the Quina-quina ( Myroxylon balsamum ), the source of peru balsam, could also be used for medical purposes. He brought specimens back to Europe. In memory of the noble lady mentioned above, the quinine tree was named Cinchona after her by Linnaeus (the misspelling was due to Linnaeus in 1739 and was included in his book "Genera Plantarum"). In 1820 the Frenchmen Pierre Pelletier and Joseph Caventou isolated pure quinine from the bark (etymological origin of "quinine": from "Chinchon" or from the Inca "Kinia" = bark?). They received 10,000 francs for this from the Paris Institute of Sciences. Quinine could not yet be synthesised, however, and so its supply remained dependent on continuous importation of the bark from South America. A large proportion of the harvest was transported to the port of Paita to be shipped north from there to Panama. It was then taken overland to Portobello, to again be shipped westwards to Havana and then to Spain. Part of the load regularly rotted and became worthless. Some of the bark was smuggled. Often the cargo was mixed with useless bark from other trees. It was thought that the bitter taste of the quinine bark was important to its healing power. All kinds of bitter tree barks were therefore sold as authentic quinine bark. Massive harvesting of the wild trees endangered further production. In 1795 the explorer Willem Alexander von Humboldt (1769-1859) recorded that in that year alone near Loxa 25,000 wild trees perished. Exhaustion of the natural supplies was partially counteracted when it was discovered that after pollarding new branches formed, which after 6 years had bark containing a higher concentration of the active product. An attempt was made to set up plantations. The export value of Cinchona bark was very high. Bolivia, Peru, Ecuador and Colombia tried to benefit from their monopoly positions. However, this threatened the British and Dutch overseas interests. In 1852 Justus Hasskarl, director of a Dutch botanical garden on Java, organised a secret mission to obtain seeds from the trees in South America. The next year he returned with a bag of seeds, for which he was knighted. The quinine content in the bark proved to be disappointingly low, however. Apparently the different varieties of Cinchona trees also differed in the amount of alkaloids which they produced. It proved a difficult genus for botanists and taxonomists. There were more than 40 species and each had several varieties. With the help of a Bolivian Aymara Indian, Manuel Incra Mamani, in 1865 the Australian Charles Ledger smuggled seeds from the quinine tree ( Cinchona ledger iana ) out of Bolivia. After the British government refused to buy the seeds (they did not trust Ledger), he sold them to the Netherlands for 20 dollars a pound. This was the among best historical investments ever. A huge series of plantations was set up with these seeds on Java. Some plantations were started in the Nilgiri hills in India. After selective breeding of the plant it was possible to produce large quantities of quinine. The amount of quinine in the bark rose from 0.3% to 8%, sometimes in exceptional cases to 13% and even 18%. In 1930 the Dutch plantations produced 22 million pounds of bark on Java, which covered 97% of the world-wide need for quinine. Only five of approximately 40 species of the Cinchona , are of direct practical importance in the production of quinine. Cinchona ledgeriana , C. officinalis , C. calisaya , C. succirubra and to a lesser extent C. pubescens are the chief sources. Traditionally the barks are classified according to colour: yellow Cinchona is obtained from C. ledgeriana and C. calisaya , red Cinchona from C. succirubra and brown Cinchona from C. officinalis.

The plants grow between 10°N and 22°S, with the exception of plantations in India, where they are cultivated up to 17°N. Cinchona is generally cultivated at heights above sea level of between 1000 and 2000 metres. High humidity and a continuous supply of water throughout the entire year are important. As with many plants the trees are short and squat at the outer limits of their growth zone. Below 1000 metres they quickly become diseased. The higher the altitude the trees are growing, the higher the content of quinine in the bark. In the wild they can grow to 20 metres high and live for 80 years under optimum conditions. In plantations they rarely reach more than 6 metres. Approximately 1500 trees are planted per hectare. They are propagated via seed or using vegetative methods (grafting). The plant is a cross pollinator and there is self-sterility. Maintaining genetic purity is therefore a problem. Trees propagated from seed from a tree with a high yield produce a heterogeneous population with a generally low yield. In Indonesia C. ledgeriana is usually grafted onto C. succirubra . This became the basis of the commercial procedure for obtaining clonal material. Infections can have catastrophic results in monocultures (cf. potato blight and the famine in Ireland). Phytophthora palmivora , Pythium vexans and Pellicularia salmonicolor are important pathogens for quinine trees.  Phytophthora cinnamoni has become a major problem in Rwanda and Kenya. The control of such pathogens in plantations is therefore strict. Recognition of this genetic weakness has meant that the question of maintaining the largest possible natural genetic reservoir is taken seriously by protecting the trees living in the wild. Remijia is another genus in the Rubiaceae family. These so called Cuprea trees are actually slender shrubs, native to Peru and Brazil. Remijia bark contains 0,5 - 2 % of quinine and/or related alkaloids (e.g. cupreine), which is lower than what is found in cultivated Cinchona trees, but it is cheaper to process. It is used for making of tonic water. Other plants such as Guettarda noumeana also contain related alkaloids.

Quinine is obtained from the bark of Cinchona trees. The highest concentrations are found in the outer layers, the epidermis and periderm of the branches and trunk. The root bark contains lower concentrations. If careful the outermost bark layer can be scraped off without touching the cambium. The trees will then renew their bark. It was noticed early on that Cinchona -alkaloids develop a red colour after they have been in direct sunlight for a time. It was also observed that the bark on the shady side was richer in alkaloids than that on the sunny side. This led to the technique of "mossing", local application of moss to the trunk. This produced a considerably higher yield of alkaloids. By stripping the bark longitudinally followed by mossing, trees could be harvested continuously. However, this requires a great amount of manual work and is not economic. Nowadays the trees are felled. As an alternative, after heavy pruning two or three new shoots can be left to grow and these can be harvested seven years later, or pruned again. Coppicing leads to higher concentrations of alkaloids in the bark. The bark is removed and dried in the shade. After being ground, the powder is mixed with lime and water and then follows extraction, e.g. with hot toluene. The alkaloids can then be isolated with diluted sulphuric acid. The quinine is purified by means of crystallisation. Nowadays in-vitro callus and suspension cultures can be produced from the plants, but commercial in-vitro synthesis of quinine is not economically attractive.

As well as quinine the bark also contains variable amounts of related alkaloids such as cinchonine, another product with anti-malaria activity. Indeed, 36 different alkaloids have been isolated, but most are present only in trace amounts. Due to the presence of 4 asymmetric carbon atoms in the molecule, 16 stereoisomers can be distinguished, among in particular quinine (8S, 9R), quinidine (8R, 9S), epiquinine (8S, 9S) and epiquinidine (8R, 9R). Diastereomers are stereoisomers differing in the chirality of stereogenic centres, but not enantiomers (mirror images). Diastereomers have different physical properties and can be separated from one another by e.g. crystallization as tartrate salt.  Quinine and quinidine are diastereomers. The stereochemistry proved very important to the activity of the product. In 1908 Rabe was able to determine the chemical structure of quinine. Rabe and Kindler reconstituted quinine from a degradation product, d-quinotoxine. They achieved stereochemical control of the final step in synthesis (conversion of d-quinotoxine to quinine) in 1918, although this was only validated in 2008. Synthetic production of quinine from relative simple starting materials proved to be very difficult, but was eventually achieved in 1944 by Woodward and Doering of Harvard University. They converted 7-hydroxyisoquinoline to homomeroquinine and than to d-quinotoxine. The immediate motivation for this work was commercial rather than military, since it was the Polaroid Corporation which asked to look for synthetic alternatives to quinine as a precursor to light-polarising molecules. However, large-scale production was not economically viable. This led to investigation of alternatives. One alternative to quinine was mepacrine but this product had various side effects. Below are the percentages of the alkaloids obtained via the present standard extraction and via boiling Peruvian Cinchona calisaya bark in either water or 70% alcohol. It has been found that as a percentage of the total number of alkaloids, quinine is more or less constant at approximately 85%:

  Alkaloids  Standard    Water    Alcohol    Quinine  5.40%  3.00%  4.50%  Quinidine  0.75%  0.40%  0.60%  Cinchonine  0.14%  0.09%  0.12%  Cinchonidine   0.09%  0.06%  0.07%  Epiquinine  traces  traces  traces  Epiquinidine  0.08%  traces  traces

Much was changed by the second World War. In 1940 the German army seized the entire European reserve supplies of quinine when they took Amsterdam (Amsterdam used to be the hub of world trade in quinine). For the West, obtaining Cinchona bark from the plantations in Southeast Asia became impossible when in 1942 the Japanese occupied Indonesia. There was a small Cinchona plantation in the Philippines, but a few weeks later this also fell into Japanese hands. The last allied aeroplane to leave the Philippines, however, had 4 million Cinchona seeds on board. These were sent to Costa Rica to be planted. Plantations were also set up later in other countries: Kenya, Rwanda, Congo, and so on. However, it would take several years before there could be any harvest. The American government sent the botanist Raymond Fosberg from the Smithsonian Institute to South America to secure a new supply of bark, originating from trees living in the wild. He succeeded in part -via buying a large quantity from German (!) agents-, but he was never able to find Cinchona ledgeriana . In the meantime alternatives had to be found, for malaria took a huge toll during the conflict in the Pacific.

In 1934 resoquine was discovered by the German H. Andersag, who worked in Bayer's Elbersfield laboratory. This was considered too toxic, however. The following year he synthesised sontoquine, a derivative of resoquine. It was assumed that this would be less toxic. In the meantime the allies had also discovered resoquine and had also not investigated it further due to problems with toxicity. Meanwhile French doctors during the Vichy regime were carrying out clinical trials with sontoquine in Tunis. After the allies took North Africa and found specimens of sontoquine and the data from the studies, there was renewed interest in the product. The product resoquine was renamed chloroquine. [Do not confuse resoquine with resorcin or resorcinol = dihydroxybenzene]. Clinical trials showed that it was clearly superior to atebrine and that the toxicity was not so bad as expected. Preparation of chloroquine in the laboratory was also economically viable. It quickly became the first choice agent and quinine was pushed into the background. In 1950 in Brazil, Mario Pinotti introduced the strategy of adding chloroquine to cooking salt (as was also done with iodine). This method of medicated salt (with chloroquine or pyrimethamine) was known as the Pinotti method.

The synthetic preparation of primaquine was perfected after the war. The British war programme led to the development of proguanil, which itself served as a model for the development of pyrimethamine. Pyrimethamine in combination with sulphadoxine was introduced in 1970 under the name Fansidar. After World War II it was hoped that malaria would be definitively eradicated. The use of chloroquine and the world-wide campaign to eradicate malaria (WHO [World Health Organisation]), led initially to a considerable reduction in malaria infections all over the world. After the anti-malaria campaign diminished due to various circumstances, the resistance of Anopheles to various insecticides and the development of chloroquine-resistant and multiresistant P. falciparum , malaria once more became one of the major problems.

Due to the great initial success of chloroquine, in the late '50s there was no longer so much need for quinine as an anti-malaria agent. The Cinchona plantations would have gone bankrupt and cultivation would have stopped, had it not been that quinidine, the stereo-isomer of quinine, was discovered in cardiology as an anti-arrhythmic agent. It proved difficult to synthesise quinidine chemically. Quinidine occurs in the tree bark, but in small amounts. It was possible to convert quinine chemically to quinidine (via the intermediary quininone and its epimer quinidinone). For this reason the plantations were kept going. By the time quinidine fell out of use due to the development of other anti-arrhythmics, quinine was once more in demand for malaria treatment.

The first soft drink can be traced back as far as the late 1700s when a young Swiss watchmaker and keen amateur scientist, Jean Jacob Schweppe was the first to perfect a process for manufacturing the world's first carbonated mineral water. He founded the Schweppes Company in Geneva in 1783, but soon after moved to England to establish the business there. Originally just producing soda waters and seltzers, the range of flavoured drinks soon expanded, most notably in the 1870s when Schweppes Tonic Water was added to the range. This famous drink, flavoured with quinine, dates from the heyday of the British Empire. Tonic water contains up to 80 mg per litre and some soft drinks (e.g. bitter lemon up to 40 mg/l). Quinine itself is poorly soluble in water but it is readily soluble in ethanol. It is alleged that the British Empire-builders took their daily dose of quinine dissolved in ethanol in the form of gin, using lemon or lime to help mask the bitter taste of the quinine. However, one would have to drink large quantities in order to get enough quinine this way (ref. Editorial: Gin tonic revisited. Trop Med Intern Health 2004;9:1239-40). Quinine can be used for malaria chemoprophylaxis. Fordlandia was an ill-fated rubber plantation with an industrial town attached. It was established at the banks of the Tapajos river in the Amazonian rainforest in 1928. The objective was to obtain a secure supply of rubber for the American Ford Motor Company. Quinine pills were routinely distributed for malaria prophylaxis, but tasted very bitter. The unpopularity of this product was illustrated by the fact that workers who received their daily quinine pill often didn't swallow it, keeping it instead under their tongue, in order to have a spitting contest when out of sight of their superviser, to determine who could spit the pill the farthest.

Whereas World War II led to the discovery of some new anti-malaria agents, the Vietnam war stimulated a huge programme for the discovery of new drugs. The Walter Reed Army Institute of Research of the United States army investigated thousands of constituents. This research resulted in mefloquine (Lariam®) and halofantrine (Halfan®). Research in China produced artemisinin, pyronaridine and benflumetol. In 1976 Trager and Jensen at the Rockefeller University in New York, developed a method of culturing P. falciparum in vitro. This brought research into the molecular biology of the parasite, the resistance patterns and new drugs into the fast lane. In 1997 Claudia Golenda, Jun Li and Ronald Rosenberg of the Walter Reed Army Institute of Research (Washington) and the NIH [National Institutes of Health, Bethesda] described a continuous in vitro culture method for P. vivax. This technique used a high concentration of Duffy-positive reticulocytes in the culture medium. The expected increase in chloroquine-resistant P. vivax could therefore now be studied in the laboratory. No single treatment regimen nowadays gives a 100 % guarantee of cure. Combination chemotherapy, preferably in fixed dose-regimens has become the standard (quinine + doxycycline, quinine + clindamycin, quinine + Fansidar, atovaquone + proguanil, mefloquine + artesunate, artemether + lumefantrine, and so on).

The chronology of anti-malaria medicaments

17 th century: quinine (actually a mixture of various Cinchona- alkaloids administered in warm wine)

1925: Plasmoquine = Pamaquine, Praequine

1932: Atebrine = Mepacrine, Quinacrine

1934: Chloroquine (Germany, Sontochin)

1944: Proguanil = Paludrine®

1950: Pyrimethamine = Daraprim®

1952: Primaquine

1955: Amodiaquine = Flavoquine®, Camoquine®

1971: Artemisinin (only in China)

1971: Sulphadoxine/pyrimethamine = Fansidar®

1979 : Artemisinin is announced to rest of world outside China

1986: Mefloquine = Lariam®

1987: Artemether and Artesunate available also outside China

1988: Halofantrine = Halfan®

1992: Atovaquone (experimental)

1997: Etaquine - Tafenoquine (experimental)

1999: Co-artemether, Riamet (artemether 20 mg + lumefantrine 120 mg / tablet)

1999: Atovaquone / proguanil = Malarone® commercialised 2011: Eurartesim, combination artenimol + piperaquine

Treatment, overview

Broadly speaking, anti-malaria drugs can be divided into four major classes

Blood schizonticides Antifolates Antimitochondrials Redox process-based agents

Blood schizonticides

When the malaria parasite leaves the liver and penetrates an erythrocyte, it can at last begin a haemoglobin diet. However, it cannot use the iron-containing haem group. What is more, released ferriprotoporfyrin IX (syn. = haemin) is toxic for the parasite. It contains trivalent iron (ferric = Fe 3+ ). Normally the parasite polymerises haemin to non-toxic malaria pigment. Chloroquine, quinine, mefloquine and halofantrine interfere with the detoxification of haemin in the digestive vacuole of the parasite. The drugs prevent this detoxification so that haemin can generate free radicals and membrane damage follows. It is therefore logical that the drugs are not active against the parasitic stages which precede the blood forms (sporozoites, liver forms) and which do not consume haemoglobin.

Antifolates

Folic acid is an important metabolic factor. Humans obtain this vitamin from the food they eat. The malaria parasite, on the other hand, must produce it for itself. Para-aminobenzoic acid (PABA) is used at an early stage of the biosynthesis of folic acid by the enzyme dihydropteroate synthetase. This step is inhibited by structural analogues of PABA, such as sulphonamides and sulphones, e.g. sulphanilamide, sulphadoxine and dapsone. The next synthesis step is catalysed by dihydrofolate reductase. This step is prevented by pyrimethamine, trimethoprim and cycloguanil (prodrug = proguanil), to such an extent that tetrahydrofolate - the end product - is not formed. The combination of these two sequential inhibitors forms the basis of Fansidar® (similar to cotrimoxazole). Resistance to both antifolates easily develops. A specific point mutation in each gene ( dhps and dhfr ) is sufficient. Because of its rapid multiplication, the parasite needs to synthesize nucleic acids. Purines and pyrimidines are the precursors for this synthesis. Human erythrocytes cannot synthesize purines de novo. Therefore, it relies on the salvage of preformed purines (e.g. adenosine, hypoxanthine and guanine) from blood plasma, mostly for ATP synthesis. Nucleosides are transported into erythrocytes by means of a specific transport protein in the RBC membrane. In infected erythrocytes, the influx of purines into the red cell is increased. Hypoxanthine seems to be the major purine for the parasite. Malaria parasites must synthesize pyrimidines de novo, because the erythrocyte cannot supply them. The synthesis of pyrimidines is linked to folate metabolism. Dihydrofolate reductase and thymidylate synthetase are two enzymes which in parasites are part of a single bifunctional protein (DHFR-TS). The parasite relies more on the de novo folate synthesis than folate salvage, which explains the synergy of the antimalarial activity of sulpha drugs (which inhibit de novo synthesis) and pyrimethamine (which inhibits the parasite DHFR, an enzyme which is more sensitive to the drug than the host enzyme).

Antimitochondrial products

Although artemisinin derivatives and 8-aminoquinolines cause mitochondrial swelling, this organelle is not their chief target. Some antibiotics such as tetracycline and clindamycin prevent protein synthesis by mitochondrial ribosomes (these are similar to the ribosomes found in bacteria). They are slow-acting. Atovaquone is a naphthoquinone which specifically destroys the electron transport chains of Apicomplexa. The molecule is rather similar to ubiquinone (coenzyme Q) which plays a role in the energy transfer between cytochrome B and C1. The enzymes of Plasmodium falciparum are 1000 times more sensitive to atovaquone than the corresponding enzymes in humans. Resistance can easily develop if used in monotherapy.

Redox reactions

Primaquine and etaquine exercise their action via redox-active quinone metabolites. They are selectively toxic for the pre-erythrocytic stages and are the only medicaments which kill hypnozoites. Etaquine has in addition a pronounced blood schizonticidal action.

Treatment, anti-malaria drugs

QUININE

Quinine and quinidine are stereo isomers. Both are active against malaria parasites. Copyright ITM

This is a powerful product, which acts upon the schizonts of the parasites in the blood (it is a schizonticide). It thus acts chiefly in the second half of the maturation cycle: on the parasites which are sequestered in the small blood vessels (not on the young ring forms in the peripheral circulation). Quinine also possesses gametocytocidal activity against P. vivax , P. malariae and P. ovale (but not against gametocytes of P. falciparum ). Although it was earlier claimed that quinine had a weak antipyretic action (used for example in Latepyrine-quinine®), this is questioned nowadays. Besides treatment of P. falciparum malaria, quinine is also used to reduce muscle cramps. Quinine is available in several forms. It is a substance consisting of 2 parts: a base (the active part) and another chemical group (important for solubility). The 2 components together are called the salt. Some confusion may arise as regards the dosage of quinine, depending on whether this is expressed in mg of the base or in mg of the salt (generally the salt is used).

         

   Salt in mg   Base in mg   Quinine bisulphate  100  59  Quinine dihydrochloride    100  82  Quinine sulphate  100  82

Quinine base is 82 % of the total substance if the salt is the dihydrochloride or sulphate, but is only 59 % if the salt is the bisulphate. Quinimax® is a product frequently used in Africa. Some 100 mg tablets contain only 59 mg of quinine base, however, and a very small amount of other substances. This may lead to under-dosage. Flomaquine adult suppositories contain 500 mg quinine base with 50 mg lidocaine.

Quinine sulphate   is administered orally. It is absorbed well in the intestines. An obsolete product is Arsiquinoforme®. This combination drug contains 62% quinine in the form of quinine formiate and quinine acetarsolate. Each 225 mg tablet contains 18 mg of pentavalent arsenic. Quinine bihydrochloride  is injected, preferably by slow IV (infusion with glucose because of the risk of hypoglycaemia). IM injections may lead to sterile abscesses, but can be used where necessary if there are no alternatives available. For IM injection, it is best to use a diluted solution (60 to 100 mg/ml) instead of the concentrated solution (300 mg/ml). Quinine administered via IM injection is absorbed well even in severe malaria. Treatment with quinine is unpleasant (bitter taste, cinchonism) and poor compliance after the acute phase is common.

Treatment regimens

The basic regimen is 10 mg salt/kg, every 8 hours, orally or slow IV. This should be continued for at least 4 days, preferably 7 to 10 days. This is an unpleasant treatment. Because there is still a risk of relapse if quinine is used in monotherapy, another product is generally combined with it, e.g. tetracycline or vibramycin. Sometimes treatment with Fansidar® is given after a few days, which shortens the treatment period. In the case of cerebral malaria it is best to give a loading dose (first administration = double dose or quinine bichlorhydride 7 mg salt/kg IV over 30 minutes; or once 20 mg/kg IV over 4 to 8 hours). P.S. Please note that the bi chlorhydride is given, and not the mono chlorhydride. The latter is less water-soluble. Sometimes there is a brief increase in the parasitaemia after beginning therapy. This does not directly mean that the therapy has failed.

If a patient vomits within an hour after swallowing the medication, the whole dose should be repeated. If vomiting occurs longer than one hour after ingestion, no new dose is necessary. In case of repeated vomiting, IV administration will be chosen. In hepatic or renal insufficiency the normal dose of quinine is administered for the first 48 hours. The dose is then reduced to half or one third (administration of quinine is then every 24 hours). There is controversy over this, however, (probably much less of a reduction is necessary). ECG monitoring is recommended during quinine therapy in patients with existing kidney failure (watch in particular for QTc-prolongation and arrhythmia). If haemodialysis is required, quinine should be administered after (rather than before) although little or no quinine is removed by this procedure. Dose adjustment during haemodialysis is not necessary. Classic haemodialysis cannot be used therapeutically in quinine intoxication.

Side effects of quinine

Quinine is a substance with highly irritating properties (also for the gastric mucosa: nausea is not uncommon). Capsules are therefore best taken after a meal. Quinine increases the secretion of insulin from the pancreas, with the risk of hypoglycaemia. Quinine allergy is not common. What is common is a range of side effects such as tinnitus, temporary deafness for high frequencies, headache, nausea and palpitations. These toxic phenomena are known as cinchonism. This reduces the patient's compliance. Quinine increases irritability of the pregnant uterus. In case of need one must not hesitate to use quinine in a pregnant woman with malaria (malaria itself can lead to abortion, preterm labour or death in utero). To prevent an impending premature labour, a tocolytic agent can be given, such as the beta 2-mimetic ritodrine, fenoterol or salbutamol. The calcium antagonist nifedipine is as effective a tocolyticum as the beta-mimetics. The oxytocin antagonist atosiban (Tractocile) is also effective, but rather expensive. Quinine and primaquine may be used therapeutically in persons with porphyria (Fansidar® is contraindicated in these patients).  Prolongation of the PR, QRS and QT intervals may occur during the use of quinine (as with quinidine). If the patient has atrial fibrillation, conversion to sinus rhythm may occur, with possibly arterial embolic complications. Atrial fibrillation which has already been present for more than 48 hours is a contra-indication for quinine. Congenital long QT syndrome and Brugada syndrome are equally formal contra-indications for using quinine. Both syndromes are caused by molecular abnormalities in the cardiac Na + ion channels. In congenital long QT syndrome the activity of the channels is increased, while they function less well in Brugada. The latter, an autosomal dominant inherited syndrome, results in episodes of sudden syncope, with a right bundle branch block and ST-elevations in the right precordial leads (V1-3) on the ECG. Isolated episodes of ventricular fibrillation occur without other signs of organic heart disease. The syndrome may be suppressed on the ECG by beta-blockers, and made more prominent by ajmaline, flecainide and procainamide. It is responsible for many cases of sudden death. Quinine is a common cause of drug-associated thrombocytopenia e.g. in patients who take quinine against nocturnal leg cramps.

Overdosage of quinine may lead to very severe situations such as deafness, delirium, bradycardia, hypotension, respiratory arrest or death (lethal dose approximately 8 gram). Overdosage may also lead to blindness via a direct toxic effect on the retina and possibly also due to spasms of the retinal blood vessels and subsequent retinal ischaemia. The half-life of quinine in the blood is short (12 hours). Most of the quinine in blood is bound to proteins. The bound fraction increases from 80% to 90% in acute malaria (increase in acute phase proteins). Overdosage is thus less dangerous in active malaria. The total quinine blood level is of much less importance than the blood level of free quinine, which is much more difficult to measure. 95% of the quinine is converted in the liver and then eliminated via the kidneys.

Note: Quinidine

Quinidine is the dextro-diastereomer of quinine. It has the same anti-malaria properties, but has a more pronounced action on the myocardium. With quinidine there is a narrower margin between the therapeutic and toxic plasma levels. It is used as an anti-arrhythmic agent (e.g. Quinidine durette 250 mg tablet). In emergencies it can be used for malaria treatment. The dosage is the same as that for quinine. A loading dose may be given. N.B.: 10 mg quinidine gluconate = 6.25 mg quinidine base.

Note: quinine and gin

Unlike the majority of other bitter products which occur naturally, the bitter taste of quinine is short-acting with no annoying after-taste. It is therefore used as a flavouring to produce tonic water. The British colonialists in India often drank gin and tonic. The present-day tonic water contains approximately 15 mg per litre, however, only enough to give a bitter taste. Copious drinking of gin and tonic in order to prevent malaria, is thus only an excuse for drinking gin.

Note:  quinine resistance

Why is quinine resistance still rare? The product has been used for 360 years. This is in stark contrast to the resistance to other malaria drugs or antibiotic resistance in bacteria, where the "useful life" of a product is measured in years or a few decades. The concept of a standard dose was only developed in the twentieth century. Earlier the duration of treatment and the dosage were left to the discretion of the doctor. This, together with the fact that the concentrations of alkaloids varied greatly from plant to plant and that quinine was never pure, meant that malaria was treated with a therapy which must have produced the most varied blood levels. Yet no wide spread quinine resistance has been reported. The answer to the question why there is virtually no quinine resistance, could be very important. Is the target molecule of quinine so special that mutation is not possible? It would then be very helpful to know this target. It could also be that there is quinine resistance, but that it was not, and has not been recognised. However, this is doubtful. Is it that the present recommended dose is much higher than that which was formerly necessary? Is it the fact that "quinine" is actually a mixture of various active products, which prevents resistance developing? Resistance to combined therapy requires multiple, simultaneous mutations which is less readily achieved than that to single products. It is, however, possible that quinine has not previously been used at levels which create sufficient evolutionary pressure. The majority of malaria cases in Europe and America were P. vivax infections. Even in British India, P. vivax represented the lion's share of infections. In P. falciparum endemic regions, only a few fortunate people were able to take quinine and then only when they had to (because of unpleasant side effects). Few used quinine as a prophylactic agent (especially among the indigenous population). What is more, quinine has a short half-life, so that the parasite was only exposed to subtherapeutic concentrations for a short time. Probably its limited use is the reason for the absence of resistance, and with continuous use on a large scale, quinine resistance may yet become a reality in years to come Note: quinine and ultraviolet In 1800 William Herschel noticed that the sun's rays gave to different extents the sensation of heat when passed through different coloured glasses. This led him to place the blackened bulb of a thermometer in different parts of the spectrum of the sun, and compare the temperature recorded with that of a similar thermometer in the shade. He found an increasing temperature towards the red, and was astonished to find that the increase persisted well beyond the red, into the invisible region now known as the "infrared". The following year, J.W. Ritter describes how the blackening of silver chloride when this chemical was exposed to light of the visible spectrum was even more pronounced to invisible radiation beyond the violet. This discovery of the ultraviolet was not followed up for many years, largely because the photographic plate had not been invented yet. In 1842, E. Becquerel projected a well-defined solar spectrum on to an iodized silver plate, which was later developed by exposure to mercury, following Daguerre's process. His image showed a number of Fraunhofer lines, and these extended well beyond the visible violet. In 1852 Stokes described some prominent Fraunhofer lines beyond the violet which he was able to observe visually when a spectrum was projected on to a fluorescent quinine sulfate screen. Because the emitted wavelength of fluorescent light is longer than the absorbed one, the presence of ultraviolet could now be observed with the naked eye. Note: quinine, dog urine and sunglasses Herapathite, or iodoquinine sulfate (quinine sulfate periodide), is a chemical which was discovered in 1852 by William Herapath, a doctor in Bristol. During studies of quinine pharmacology, he found that adding tincture of iodine to the urine of a dog that had been fed quinine produced unusual microscopic green crystals. The overlapping portions of crystals which were on top of one another at right angles became ‘black as midnight’, suggesting polarization. The chemical was later used by Land in 1929 to construct the first type of Polaroid sheet polarizer. Such sheets consisted of microscopic crystals of iodoquinine sulfate  embedded in a transparent nitrocellulose film. The very fine crystals are aligned by stretching the film or by applying electric or magnetic fields. Modern Polaroid is made from polymers such as polyvinylalcohol, in which the polymer chains are stretched to force them to align, and then doped with iodine. Polarizing filters have numerous applications, one of which is a certain type of sunglasses.

CHLOROQUINE

Chemical structure of chloroquine. Copyright ITM

The trophozoite in the red blood cell breaks down haemoglobin using lysosomal enzymes. In this digestive process ferriprotoporphyrin IX (haemin) is released from the haemoglobin protein chains. This porphyrin is toxic to the parasite and is usually polymerised to non-toxic malaria pigment. The weak base chloroquine accumulates in the acid lysosome and binds to ferriprotoporphyrin IX. In this way detoxicification of the latter is prevented and the parasite is killed. Since the liver parasite does not feed on haemoglobin and the effect of chloroquine is to prevent detoxification of the haem ring, this drug is not active at the pre-erythrocytic stages of Plasmodium sp. Outside the cell, chloroquine as well as hydroxychloroquine are present mainly in a protonated form, that, due to their positive charge, cannot cross the plasma membrane. However, the non-protonated form can enter the cell. Inside the cell, the molecule gains a proton (H + ) in a manner inverserly proportional to the pH, i.e. the lower the pH, the more chloroquine will bind an extra proton. Chloroquine will be concentrated in acidic cell organelles, such as Golgi vesicles and lysosomes, where due to the low pH, most chloroquine molecules will be positively charged. Since chloroquine is a weak base, the pH will rise. By increasing this pH, several enzymes such as acid hydrolases can be inhibited. Post-translational modification of newly synthesised proteins can be disturbed.

Chloroquine is available in tablet form as chloroquine sulphate  (Nivaquine®) and as chloroquine diphosphate  (Resochine®). Other brand names are Daramal®, Anochlor®, Promal®, Avlochlor®. Hydroxychloroquine sulphate  (Plaquenil®) is different and is used in rheumatoid arthritis. The injectable form is chloroquine dihydrochloride. There is a combination tablet of chloroquine 100 mg + proguanil 200 mg (Savarine®), which makes compliance easier for prophylactic use (1 tablet daily).

The chloroquine dose is always expressed as base (not as salt). This allows easier comparison between the different products. Nowadays most Nivaquine® tablets contain 100 mg. It is also highly advisable to check the current dose per tablet in the region where you are working, so as not to cause accidental overdosage or underdosage.

Chloroquine diphosphate 250 mg tablet = 150 mg chloroquine base

Chloroquine sulphate 200 mg tablet = 150 mg chloroquine base

Chloroquine dihydrochloride 50 mg/ml = 40 mg base/ml 

Chloroquine is a powerful schizonticide. It has strong affinity for various tissues and organs. It is fast-acting and remains in the blood for many days. A brief treatment is therefore possible. The excretion of chloroquine and its metabolites occurs mainly via the kidneys and is slightly improved by acidification of the urine (500 mg vitamin C every 4 hours). Chloroquine may be given orally, SC, IM or SLOWLY IV (infusion). Never inject an ampoule of chloroquine IV rapidly as a bolus. It is essential that rapid infusion is avoided. Chloroquine bihydrochloride IM is well absorbed (>80% even in severe malaria). The injections are not painful.

Psoriasis is a relative contraindication for chloroquine; Photo Dr Van den Enden, ITM

There are several different treatment regimens. Orally 25 mg/kg is given spread over three days. The following is practical for an adult weighing 60 kg: first, 6 tablets of 100 mg base, followed after 6 hours by another 3 tablets, after 24 hours by another 3 tablets and after 48 hours by another 3 tablets: 6-3-3-3. If parenteral therapy is required due to coma caused by chloroquine-sensitive P. falciparum , chloroquine bihydrochloride may be given IM or IV. The total dose should be 25 mg base/kg for an adult of at least 60 kg. There are several regimens, e.g. 10 mg base/kg over 8 hours, followed by 15 mg base/kg over 24 hours. Another frequently used regimen is 3.5 mg base/kg SC or IM every 6 hours. Parenteral administration should be discontinued as soon as oral administration is possible. In moderate renal impairment (creatinine clearance > 10 ml/min) no adjustment of the dose is necessary.

Chloroquine is cheap and not very toxic in normal use. Some people are allergic (pruritus, rash) or suffer nausea. People with psoriasis are more at risk of side effects. A reversible precipitation of chloroquine in the cornea may occur, resulting in small opacities. This may result in seeing haloes around objects, blurred vision or photophobia. This form of keratopathy may become manifest quite rapidly (a few weeks after beginning treatment). After discontinuing the medication it is completely reversible. Chloroquine accumulates in melanin-containing tissues. Chronic use may lead to abnormalities of the choroid and retina (chorioretinitis). This toxic retinopathy is not reversible. The abnormalities are always bilateral and symmetrical. Often there is maculopathy (bull's eye lesion) with central and paracentral scotomata, but constriction of the peripheral field of vision may also occur. The total cumulative dose before such problems occur is generally 100 gram chloroquine or more. Hydroxychloroquine has rather lower retinal toxicity. Tinnitus, hearing loss and neural deafness have been reported as possible rare side effects. Sometimes a proximal "chloroquine myopathy" occurs. The muscular weakness in myasthenia gravis is exacerbated by chloroquine and this disorder is a formal contra-indication for the use of this product. Breast-feeding may be continued without change while taking chloroquine.

Evolution of Plasmodium falciparum resistance in Thailand. Copyright ITM

Chloroquine has a narrow safety margin (just 30 mg/kg may be fatal). In case of overdosage myocardial depression, hypotension, severe arrhythmias and tissue hypoxia may occur. ST-segment abnormalities and T-wave inversion occur. Broadening of the QRS complex (>0.12") and ventricular arrhythmias have a poor prognosis. The patient may become comatose, vomit and aspire the stomach contents. In acute intoxication diazepam is given (Valium® 1 mg/kg) and adrenalin (= epinephrine) or dopamine if these are available. First protect the airways and correct any existing cardiovascular disorders and then consider gastric lavage if the overdose was recent. Introducing activated carbon into the stomach is also beneficial. Acidification of the urine, osmotic diuresis, haemoperfusion and haemodialysis are of little use. After oral ingestion of an overdose the effect is evident approximately 1 hour later. Death follows after a further 2 to 3 hours.

The individuals within a population of parasites are not identical. Drugs do not have an equal effect on each variant. The larger the population and the shorter the life cycle of an organism, the faster mutations will become manifest. Of course Anopheles and Plasmodium have changed more in recent decades than have humans (due to the short generation times and the evolutionary pressure due to insecticides and drugs). The first signs of chloroquine-resistant P. falciparum infections occurred in the '60s, more or less simultaneously in Colombia and Thailand. This resistance spread progressively and is now a significant problem in many countries (see map with zones A, B and C). There are three grades of cloroquine resistance (RI, RII, RIII). In RI the parasitaemia after therapy is so low that it falls below the detection threshold, to rise above it again within 28 days. In RII the parasitaemia is reduced by at least 75 %, but the parasites remain detectabable in the peripheral blood. In RIII chloroquine has no effect on the parasitaemia. In spite of the presence of this resistance, chloroquine still has a place in treatment. It is still active against other plasmodium species. Only some falciparum parasites are resistant and only some have type RIII resistance. In non-urgent situations, therefore, this product may still be used. In 1991 chloroquine-resistant P. vivax strains were discovered in Papua New Guinea. In 2006, already 65% of the Papuan P. vivax strains were chloroquine resistant. P. vivax chloroquine resistance in other areas, such as Indonesia, India, Brazil, Guyana is spreading. To date resistance to chloroquine in P. ovale is very rare. The first chloroquine-resistant P. malariae has been reported (Malaysia, 2002). Note: Chloroquine resistance in Malawi In response to the high rate of treatment failure of chloroquine, Malawi replaced chloroquine with sulfadoxine-pyrimethamine for the treatment of malaria. It was the first country in Africa to do so. At that time (1993), the clinical efficacy of chloroquine was less than 50%. Twelve years after it was withdrawn from Malawi, nearly all P. falciparum isolates in the country had become sensitive again to chloroquine. Although a clinical study in 2005 showed a very high cure-rate with chloroquine-monotherapy in Malawian children, this cannot be used as an argument to avoid combination treatment (e.g. ACT) as a longterm national policy. The remarkable rapid shift in parasite population when the chloroquine-selection pressure disappeared, is most likely due to an inherent lower fitness of chloroquine-resistant parasites as compared to the chloroquine-sensitive wild-type. It cannot automatically be assumed that such a rebound will occur everywhere once chloroquine is withdrawn. In areas where malaria transmission is high, such as in large parts of sub-Saharan Africa, many infections are genetically mixed, with different clones displaying different degrees of fitness. Competition between such strains is high. In areas where the transmission rate is low, such as in much of Asia or the Amazon bassin, most malaria infections are typical caused by a single clone. Distinctive genotypes may predominate over wide areas. In such areas, chloroquine-resistant parasites tend to remain in the population for a prolonged period after the withdrawal of chloroquine.

Note: molecular basis of resistance

The molecular basis of resistance is still not fully understood. Resistance to chloroquine is said to be due to a reduced accumulation of the product in the parasite. In vitro this is reversible with verapamil. On chromosome 7 of the parasite is the gene pfcrt that codes for the protein PfCRT ( Plasmodium falciparum chloroquine resistance tranporter). This is a transmembrane protein in the digestive vacuole. The protein facilitates the transport of positively loaded organic cations (efflux of protonated amino acids). The mutated protein has an increased affinity for chloroquine, so that these molecules are pumped out of the digestive vacuole. The roles of other mutations, e.g. pgh1 and pfmdr ( P. falciparum multidrug-resistance transporter) for example, have not yet been fully clarified.

Note: chloroquine and non-malaria disorders

Besides for malaria, chloroquine is also used for a number of other disorders: rheumatoid arthritis, polymorphous light eruption, discoid lupus erythematosus, cutaneous sarcoidosis, Q fever, cutaneous porphyria tarda. Chloroquine also exerts direct antiviral effects by inhibiting pH-dependent steps of the replication of several coronaviruses, Borna virus, Mayaro virus and retroviruses (there is even a very modest anti-HIV activity). Chloroquine inhibits the uncoating of hepatitis A virus. Certain flaviviruses will be inhibited by affecting the normal proteolytic processing of the flavivirus prM protein (precursor of membrane protein). Chloroquine has immunomodulatory properties, such as suppressing the production and release of TNF-alpha and interleukin-6. Hydroxychloroquine has been succesfully used in the treatment of interstitial lung disease due to genetic surfactant protein C deficiency (probably by interfering with cellular processing of surfactant protein C by changing intracellular pH-gradients).

Example :

The porphyrias are a group of disorders characterised by disturbed production of haem. Most are autosomal dominant and inherited, and there is quite variable expression of the disease. Porphyria cutanea tarda can also be acquired (oestrogens and excessive alcohol play an important role). The disorder generally becomes evident around the age of 40 to 50 and is characterised by hepatic abnormalities and significant skin fragility (chiefly the hands, legs and face. Blisters, erosions and scabs appear on skin exposed to sunlight. A very dark skin (hyperpigmentation) with sclerodermoid changes may occur. It is important for the patient to stop drinking alcohol, and to avoid oestrogen and iron preparations. Hepatic siderosis is generally present and repeated phlebotomies are indicated (400-500 ml every 1 or 2 weeks, until Hb < 11 g%). Generally 4-10 litres of blood are removed before a therapeutic effect is achieved. Chloroquine is used in the treatment of porphyria cutanea tarda. A low dose is used (125-250 mg twice a week). A higher dose of chloroquine causes acute liver toxicity and massive porphyrinuria. Chloroquine complexes with uroporphyrin and increases excretion from the liver. A clinical effect can be expected within 4-6 months (biochemical remission is slower). The risk of hepatoma is probably reduced by the treatment.

AMODIAQUINE

Grey pigmentation of the skin due to chronic use of amodiaquine (camoquine). Copyright ITM

Grey pigmentation of the nail bed due to chronic use of amodiaquine (camoquine). Copyright ITM

Grey skin discoloration due to chronic use of amodiaquine (camoquine). Do not confuse with chronic arsenism. Compare with chronic administration of silver salts. Copyright ITM

Chemical formula of amodiaquine, a 4-aminoquinoline. Copyright ITM

Amodiaquine is closely related to chloroquine. It is rapidly converted by hepatic CYP2C8 to an active metabolite (N-desethyl-amodiaquine).  Amodiaquine is three-times more potent than this metabolite, but the metabolite remains long in blood where it concentrates in red blood cells, and contributes to the prolonged antimalarial efficacy of amodiaquine. 200 mg of amodiaquine base is equivalent to 260 mg amodiaquine hydrochloride . The product has no taste (unlike chloroquine which is bitter). Long-term use causes grey skin pigmentation in white people. Sometimes there are severe side effects (agranulocytosis in approximately 1/2000, liver toxicity in approximately 1/15,000). Amodiaquine (Camoquine®, Flavoquine®, Malarid®) are therefore still only used rarely. There is less resistance to amodiaquine than to chloroquine. Since the product is eliminated slowly, a single dose of 600 mg was (and is) sufficient. A fixed combination amodiaquine with artesunate is known as ASAQ or coarsucam. Because of its low price, it is expected to be widely used in the future. Amodiaquine plus sulfadoxine-pyrimethamine can be used in areas where efficacy of both amodiaquine and SP remains high (mainly the countries of West Africa). This non-artemisinin-based combination therapy is reserved as an interim option for countries that, for whatever reason, are unable immediately to move to artemisinin combination treatment. Amopyroquine (Propoquine®) is an amodiaquine analogue, but is little used and then only IM. Its safety during pregnancy is uncertain.

PYRIMETHAMINE + / - SULPHONAMIDES

Stevens Johnson syndrome after Fansidar, an antimalarial which contains sulfadoxine. Generalised formation of bullae and skin desquamation. Copyright ITM

Stevens-Johnson syndrome after Fansidar, an antimalarial which contains sulfadoxine. The eyes are also affected. Copyright ITM

Chemical structure of pyrimethamine. It interferes with folic acid synthesis by inhibiting the enzyme dihydrofolate reductase. Folic acid is needed for DNA and RNA synthesis. Copyright ITM

Chemical structure of sulfadoxine. Copyright ITM

Pyrimethamine  (Daraprim®) was previously used as a preventive agent, but leads to rapid resistance if it is the only product used. The mechanism of action is the same as that of proguanil. The product is also used in the treatment of toxoplasmosis. In long-term administration megaloblastic anaemia may occur (folic acid antagonism).

Maloprim® or Deltaprim® The combination of pyrimethamine (12.5 mg) with dapsone (100 mg) was and still is used as prophylaxis in some regions, including Zimbabwe and South Africa. It is only slightly effective and the indications diminish from year to year. Dapsone is removed from the body quite rapidly (t½ = 28 hours). It cannot be used as a curative drug.

Fansidar ® is a combination product of pyrimethamine 25 mg and sulphadoxine 500 mg per tablet (Mekalfin® is another commercial name). Sulphadoxine is a long-acting sulphonamide (t½ = 8 days) which in case of allergy may cause severe skin lesions (erythema multiforme and Stevens-Johnson syndrome). For this reason it is best not used for prevention. Plasmodium falciparum has developed resistance to this product in some parts of the world. The curative dose for an adult of 70 kg is 3 tablets taken as one dose. The action is slow. The idea behind the combination of pyrimethamine with a sulphonamide is the sequential inhibition of tetrahydrofolic acid formation which is necessary for purine and pyrimidine (DNA precursors) synthesis. Theoretically there is a risk of icterus (and kernicterus) if pregnant women use it, but this risk is very limited.

MEFLOQUINE

Erythema multiforme after using prophylactic mefloquine. Copyright ITM

Chemical structure of mefloquine. Copyright ITM

Mefloquine (Lariam) is a long-acting product. After 2 to 3 weeks half of the dose is still present in the body. Mefloquine has a rather slow onset of action. Mefloquine can be combined with certain other antimalarials. The combination mefloquine + pyrimethamine + sulphadoxine is known as Fansimef®.

Mefloquine, curative use

The curative dose of mefloquine for an adult is 500 mg orally, to be repeated after 12 hours and after 24 hours (sometimes 750-500-250 mg is also given). In Europe the dose is expressed as the base (250 mg base = 274 mg mefloquine hydrochloride) while in the USA it is expressed as the salt (250 mg salt = 228 mg base). Therefore, a 500 mg dose in the USA gives approximately 10% less active drug than in Europe. There is no injectable form. The action of mefloquine upon the myocardium is the same as that of quinine. There must be an interval of 12 hours between the last dose of quinine and taking mefloquine (quinine is removed rapidly from the body). During treatment of an attack mefloquine may be given if there is renal insufficiency (a common problem in severe malaria). At curative doses there are quite often unpleasant side effects, such as nausea, insomnia, tremor, anxiety and more rarely convulsions or psychotic episodes. Note: there are some data from in-vitro studies that mefloquine has an antiviral effect on JC virus

Mefloquine, preventive use

Mefloquine plays an important role in prophylaxis. The product is 98% bound to plasma proteins. Breakdown to an inactive metabolite occurs in the liver. Excretion is mainly via the liver and biliary route, and to a very limited extent via the kidneys. The plasma half-life is 2 to 3 weeks. Ingestion of 1 tablet per week produces stable blood levels after 7 weeks. This may be drastically shortened by taking a loading dose over 3 days (1 tablet per day x 3 consecutive days). Traditionally it is said that mefloquine prophylaxis should be started before departure. This guideline is based on the consideration that intolerance to the drug can be monitored in this way. It is safe to begin the medication 15 days before departure so that 3 tablets are taken before leaving. In this way 75% of the side effects can be detected. At the prophylactic dosage (adults one 250 mg tablet per week) side effects occur in 2 to 3% of people, which require that the prophylaxis be discontinued. Rarely (1 in 12,000 to 15,000) preventive dosages may trigger epilepsy or psychosis may occur. Epilepsy and arrhythmias (including the use of beta-blockers, calcium antagonists and digitalis) are contra-indications for the use of this product. The product was initially not recommended in pregnant women, but in practice has proved to be safe in the second and third trimesters. The prophylactic use of mefloquine used to be limited to 3 months, but new data indicate that the product is safe if taken for longer. It is best if people who carry out critical motor operations (e.g. aeroplane pilots) do not take mefloquine. In 1994 an anecdotal case was described, of a central anticholinergic syndrome (delirium and stupor, mydriasis, hyperpyrexia) after taking mefloquine. This was very swiftly reversible after injecting 2 mg physostigmine (a cholinesterase inhibitor similar to prostigmine).

Mefloquine, resistance

The first case of mefloquine resistance was described in Thailand in 1982. There is already mefloquine resistance on a small scale in many countries, but this can be significant locally: e.g. the cure rate in East Thailand was only 41% in 1993. P. falciparum malaria can thus sometimes occur in spite of correct prophylactic use of mefloquine. Mefloquine does not kill sporozoites (therefore P. vivax and P. ovale malaria are still possible after leaving an endemic zone and after discontinuing mefloquine).

HALOFANTRINE    (Halfan®)

This is fast-acting, effective and has few, but potentially lethal side effects. The dose for an adult is 500 mg, repeated after 6 and 12 hours. It is recommended that non-immune individuals repeat this after one week. High-dose halofantrine has been used in Thailand. Absorption is highly variable and is considerably (3x) increased if the medication is taken with fatty food. At present, however, the advice is to take Halfan® on an empty stomach. There is a syrup for children. The maximum concentration is reached 6 hours after oral ingestion. The half-life is 1-3 days for halofantrine itself and 2-4 days for the active (debutylated) metabolite (longer in active malaria than in healthy persons). [Desbutyl-halofantrine is being evaluated as a potential prophylactic agent]. Excretion is chiefly via the faeces.

Halofantrine is used for malaria treatment. Copyright ITM

Chemical structure of halofantrine. Copyright ITM

In Southeast Asia resistance is common, but in other regions this problem is still limited. There is limited cross-resistance with mefloquine. Cases of insufficient clinical response (5% of cases) are usually due to inadequate absorption. Sometimes Halfan® is used as the drug of first choice, or as a stand-by treatment for travellers, but this has been superseded by Malarone®. It should be stressed that halofantrine cannot be given IV. Use in severe malaria might therefore be problematic (possibly via gastric tube). During treatment with halofantrine there is prolongation of the PR-interval and the QT time. Severe cardiac problems have been described in people who previously had a cardiac conduction disorder. Prolonged QT c time, thiamine deficiency, conduction disturbances, ion deficiency (hypokalemia, hypomagnesaemia) and concomitant administration of quinine or mefloquine are contra-indications. If there is a falciparum malaria during mefloquine prophylaxis, Halfan® is not a good choice due to possible cross-resistance and cumulative cardiotoxicity. Other products which may also cause QT-time prolongation, such as cisapride [Prepulsid®], terfenadine [Triludan®], tricyclic antidepressants such as clomipramine [Anafranil®] and amitriptyline [Redomex®] are best avoided during Halfan® administration. An ECG is therefore advisable before administration of Halfan®. Halfan® cannot be used as prophylaxis. [A reminder: the QT-time is the time from the beginning of the Q-wave to the end of the T-wave. The correction for cardiac rate using Bazett's formula (QT/square root RR) gives the QTc.] If there is a prolonged QT-time (longer than 440 msec) there is an increased risk of ventricular tachycardia, more specifically "torsade de pointes". This is characterised by polymorphous QRS complexes which vary in amplitude and appear to oscillate around the isoelectric line.

QINGHAOSU and ARTEMISININ derivatives

Artemisia annua in Vietnam. This plant is harvested in order to extract artemisinin from the leaves. Copyright Charles Lugt (with special thanks to prof Kager).

Artemisia annua in Vietnam. This plant is harvested in order to extract artemisinin, used for malaria treatment. Copyright Charles Lugt (with special thanks to prof Kager).

Chemical structure of artemether. Copyright ITM

Malaria treatment with artesunate and artemether. Copyright ITM

Microscopy blood slide : reticulocytes. Copyright ITM

Artemisinin and its derivatives have become essential components of antimalarial treatment. These plant-derived peroxides are unique among antimalarial drugs in killing the young intraerythrocytic malaria parasites, thereby preventing the more pathogenic mature stages. Huang hua hao or qinghaosu ("essence of qinghao") originates from a Chinese plant, Artemisia annua (sweet wormwood). The antimalarial properties of the traditional Chinese medicine qinghaosu were discovered and developed by Chinese scientists in 1971 (secret "project 523"). This research effort was prompted by the requests of Ho Chi Minh to Zhou En Lai for antimalarial drugs for the Vietnamese troops (cfr the efforts of the American forces to develop halofantrin and mefloquin). The Chinese researcher Tu Youyou was central to this work. She was awarded the 2011 Lasker Award for Clinical Medical Research for this work. Artemisinin, which is a sesquiterpene (15-carbon) compound with a peroxide bridge, can be obtained via low temperature ethyl ether extraction. Dihydroartemisinin (syn. artenimol) is a more potent derivative and has largely replaced the parent drug. Artemisinin was first announced to the world in 1979. Full chemical synthesis was reported in 1983, but is very expensive. The plant has composite flowers and is related to absinthe and tarragon. As the name suggests it is an annual plant, from which seeds need to be collected each year for the next cultivation. The leaves and flower heads contain the highest concentration of drug and are harvested (actually the glandular trichomes are richest). The variant which grows in South China and North Vietnam contains a high concentration of active constituents (up to 1.5% dry weight, but more commonly 0.5% yields, as compared with American varienties with only 0.06% dry weight). From it are produced artemisinin and the derivatives artesunate (the hemisuccinate; -CO(CH 2 ) 2 COOH), arteether (the ethyl ether; -OCH 2 CH 3 ), artemether (the methyl ether; -OCH 3 ) and the reduced substance artenimol, syn. for dihydroartemisinin (during chemical reduction, the lactone -a cyclic ester- is reduced to a lactol). A reminder: an ether has the formula R-O-R'. Artelininic acid is a more water-soluble derivative (artelinate), but this compound was not taken beyond animal studies. Anno 2009 is was shown that P. falciparum has reduced in vivo susceptability to artesunate in western Cambodja as compared with northwest Thailand. At present, most Artemisia annua is produced in China and Vietnam. Apart from production on major farms, it can be also grown be grown by small-scale farmers in areas such as parts of East Africa were the plant is foreign. The farmers can be single or be part of an agricultural cooperation.  In a correct environment it is actually an relatively easy crop for the farmers. The plants are not readily susceptable to damage by pigs, monkeys, etc, although rain damage is possible. This crop provides family income and a useful base product for an important disease in the community at the same time. Harvesting the plant is only part of the story. Chemical extraction and purification are essential steps on the road to a practical medical formulation. It is not optimal if the plant is grown e.g. in East Africa, needs to be shipped to China or India for further processing, and the final product needs to be imported into Africa again. It is a pity that a mature pharmaceutical industry which can deal with such problems in Africa itself has not yet been realised. If a commercial monopoly (e.g. a single company bying the entire crop; see "East African Botanicals") would exist, it could prove positive (fixed prize, secure market for farmers) or dangerous (if the company suddenly pulls out or unilaterally changes previous commercial agreements). Regulation is needed. Independent help and advise for farmers can play a big role. At the same time, independent quality control and relevant action if the product would be found below predetermined and accepted standards are essential if such schemes are to be succesful. It is evident that the same applies for the final product. Corruption and the problem with adulterated medication are major items which should not be underestimated. At present there is a world shortage of artemisinine. The future impact of increased artemisinine production via genetic engineering or better farming, development of improved plant cultivars, the changing demand as numbers of malaria cases increase or decrease (e.g. due to increased use of impregnated bednets and -let's hope- an effective vaccine) and the development of reduced sensitivity or full-blown resistance is unclear.

Pharmacokinetics The products are rapidly converted after ingestion to the equally active dihydro-artemisinin. Artemisinin is eliminated by metabolic biotransformation, predominantly by CYP 2B6. Inhibitors of the cytochrome P450 such as grapefruit juice, increase the plasma levels of artemether to a significant extent (e.g. double). After oral administration there is a significant first-pass effect in the liver. The plasma half-life of artemether is very short: 1 hour, both in healthy volunteers and in patients with active malaria. Artemisinin is eliminated by metabolic biotransformation (glucuronidation in the liver). The metabolites are inactive. Artemisinins are weak inducers of some drug-metabolising liver enzymes. They also increase their own clearance.

Structure The active molecule is a sesquiterpene lactone with a peroxide bridge [sesquiterpene = a molecule formed from 3 isoprene units; a lactone = a cyclical ester, R-COO-R']. The endoperoxide bridge (-O-O-) is essential to the activity of the molecule, but not sufficient for antimalarial activity. Nevertheless the mechanism of action is more complicated than purely aspecific oxidative damage to the parasite by free radicals. Ion-dependent alkylation (Fe 2+ ) is the likely mode of action. The enzyme P. falciparum adenosine triphosphatase has been proposed as the primary target for this class of molecules.

Activity Artemisinins are not active upon liver stages, but upon both the immature sexual and the all asexual blood stages. Their broad stage specificity (as opposed to quinine) has several therapeutic consequences. Killing young circulating ring-shaped trophozoites results in a more rapid reduction in parasitemia as compared to other antimalarials and reduces the number of parasites that mature and sequester in the post-capillary venules. Quinine does not stop sequestration since it acts on the mature parasite stages, which adhere already to the vascular endothelium.  Since artemisinin reduces the number of gametocyte carriers, it helps to prevent malaria transmission, although artemisinin does not kill mature gametocytes of P. falciparum . In low-transmission areas, where symptomatic infection constitute the main source of transmission, ACTs reduce gametocyte carrier rate, and if widely employed, is expected to reduce the incidence of malaria.

These drugs were initially chiefly used in Asia (including China, Thailand and Vietnam). Later, they became also available in many African countries, although the problem of fake medicine is a very real one. One important advantage of artemisinin derivatives is the very rapid action (faster than quinine). Artemisinin and artesunate may be administered as suppositories if the patient is too ill to take oral medication (e.g. Plasmotrim Rectocaps®). The bio-availability of the drug via this route is approximately equal to that of the oral form. In monotherapy relapse is frequently seen (depending on the dose and duration of therapy). Neurotoxicity has been observed in animal trials (selective damage to certain brain stem nuclei and to the auditory nuclei in rats, dogs and Rhesus monkeys treated with IM artemether and arteether). Apparently no ototoxicity or neurotoxicity occurs in humans. The drugs also suppress erythropoiesis and reticulocytosis, but the clinical importance is still unclear. When experimental animals receive artemisinins during early pregnancy, fetal resorption may result. The temporary suppression of fetal erythropoiesis is caused by depletion of fetal erythroblasts. The relevance for humans is still unclear, but the medication is best avoided during the first trimester of pregnancy, if a good alternative is available. About 1/3000 patients develop hypersensitivity and urticaria. During treatment with artemether a transitory bradycardia and prolonged QT-interval was observed in approximately 1% of cases, but with no direct clinical repercussions. Artemether and dihydroartemisinin reduce the number of parasites by approximately 10,000 for each asexual cycle. After two cycles (3 day treatment) there is a 10 8 -fold reduction of the parasitaemia. Five day treatment can result in a 10 12 -fold reduction in the parasitic biomass. In functional asplenia artemether is less effective. People who are heterozygous for haemoglobin E (very common in Southeast Asia) have a swifter parasite clearance with artemisinin derivatives than those without this haemoglobin variant.

Artemisinin is poorly soluble in water and oil, but can be given as suppositories.

The derivative artemether  (Paluther®, Arteminth®, Cotexcin®, Artenam®) is oil-soluble and can be used for IM administration (e.g. 600 mg/day x 5 days; recommended dose 3.2 mg/kg IV on day 1 followed by 1.6 mg/kg per day x 5 days). It should be protected from light during storage. The ampoule must be clear (there is sometimes a cloudy precipitate). The absorption from the IM injection site tends to be erratic, especially in very ill patients. There is also an oral form. Absorption improves considerably with concurrent ingestion of grapefruit juice (possible role of intestinal CYP3A4). The fixed combination of artemether with mefloquine produced good results in Southeast Asia.

Artesunate  (Artenam®, Artesunate®, Arsumax®, Artemax®, Arinate®, Plasmotrim®) is the fastest-acting artemisinin derivative. It can be administered parenterally (IV, IM), rectally or orally. The dose for adults is 200 to 400 mg on day 1, 100-200 mg/day once daily on the following days to a total of 5 days. Since the molecule is not stable in water, the dry powder (60 mg) should be dissolved just before the injection with 0.6 ml sodium bicarbonate and 5.4 ml dextrose or dextrose/physiological fluid. Artesunate -which is water-soluble- has more reliable pharmacokinetic characteristics than the fat-soluble artemether. The side-effects are mild and are difficult to distinguish from the effects of malaria itself. There is a lot of evidence that artesunate is superior over quinine. Artemisinins seem to be safe in the second and third trimester of preganacy. If patients with severe malaria cannot be treated orally and access to injections will take several hours because of time needed to transfer the patient to a hospital, a single inexpensive artesunate suppository at the time of referral substantially reduces the risk of death or permanent disability. A single dose of artesunate, given rectally (by e.g. parent), can provide parasiticidal blood concentrations within 10–20 min, and can halve parasitaemia numbers within 6–12 h.

Arteether   (Artecef®, a mixture of a- and ß-enantiomers; beta-arteether is also known as Artemotil®) is at present only available in the Netherlands for IM administration. For children 2.4 mg/kg IV is given on day 1, then 1.2 mg/kg per day from day 2 to day 5 inclusive. Artenimol . Syn. dihydroartemisinin, obtained by reduction (hydrogen addition) of artemisinin. Together with piperaquine it is available as a fixed drug combination, known as Eurartesim. Artenimol has a short halflife, as opposed to piperaquine, which has a long halflife.

ACT : Avoid monotherapy with artemisinin derivatives. It has become clear that monotherapy with a drug is likely to fail earlier in a population than combination treatment (think of HAART in AIDS, cancer chemotherapy, tuberculosis). ACTs are now recommended by WHO as the first-line treatment for all falciparum malaria in malaria endemic countries. The goal is 95% cure-rate assessed at 28 days, and changing antimalarial policy if cure-rates would dive below 90%. Treatment with IV artesunate was associated with a 35% reduction in mortality from severe malaria compared with IV quinine in a randomised trial of 1461 adults and children in Southeast Asia. More and more clinicians will utilise "ACT": artemisinin combination treatment, such as

artemether plus lumefantrine: the first fixed-dose combination (Riamet®, Co-artem®) artesunate plus mefloquine (in areas with low to moderate transmission). This was already employed in 1994 at the northwest border of Thailand. artesunate plus amodiaquine (known as Co-arsucam®) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high).  dihydroartemisinin plus piperaquine, known as Artekin® in China and Eurartesim® in Belgium. dihydroartemisinin plus piperaquine plus trimethoprim, known as Artecom® artesunate-pyronaridine is available as a fixed drug combination, known as Pyramax®

Resistance : Although full-blown resistance has not been noted yet, diminished in vitro sensitivity was recorded in French Guyana, Senegal and the Thai-Cambodian border. Treatment failures rates after artesunate-mefloquine and artemether-lumefantrine often exceed 10%, which is worrying, and higher than anywhere else at the moment. The parasite clearance times are also longer than elsewhere on the planet. If artemisinins are given in monotherapy for 7 days to non-immune patients, about 10% of patients fail treatment. But when the recrudescent parasites are compared with those isolated from successful treated infections, they are not more resistant. Might there be temporarily growth-arrested ("dormant") intraerythrocytic merozoites or young trophozoites, which reactivate weeks later? It is not clear at present. The biological basis of such a phenomenon is not known (see White. Science 2008;320:330 ). Slow parasite clearance is found in a minority of patients (quid reduced splenic phagocytic function?). In asplenic patients, dead parasites may persist for up to a month in the circulation. This needs to be taken into account when trying to determine artemisinin resistance.

Production: Artemisinin is in global short supply. Most of the world production comes from China and Vietnam. Some comes from Africa.  As can be expected, temperature, hours of sunshine, altitude, humidity and chemical soil type strongly affects the yields. Selective breeding without genetic modification might result in higher yielding varieties. Total chemical synthesis of artemisinin is difficult and expensive, but the semi-synthetic of artemisinin starting from artemisinic acid could be cost-effective. The Bill and Melinda Gates Foundation supported the research on transfering the entire biosynthetic pathway that allows Artemisia annua to make artemisinin into the bacterium Escherichia coli . Production via such bioengineered bacteria was a first step.  In the first decade of the 21st Century, Keasling and his collegues at UC Berkeley engineered a yeast strain ( Saccharomyces cerevisiae ) capable of producing artemisinic acid, an immediate precursor of artemisinin. This substance is transported out and retained on the outside of the yeast cell. A subsequent simple and inexpensive purification process can then be used to obtain the desired product. This yeast is already capable of a significantly higher specific productivity than A. annua itself.  At present, researchers are trying to scale up the process to industrial levels. For this outstanding achievement, Keasling received the Heinz Award in October 2012. 

Other uses: Artemisinins also have some activity against other parasites, for example they kill the young stages of trematodes such as schistosomes and Fasciola . They are studied in animal models of clonorchiasis. Artemisinins have some anti-inflammatory properties and inhibit angiogenesis and cell growth in some neoplastic cell lines. They might even find a place in cancer chemotherapy. The rationale for possible antineoplastic activity, is that the endoperoxide bridge in the molecule reacts with an iron atom to form free radicals. Cancer cells have a significant higher iron influx compared with normal cells via the transferrin receptor mechanism, and should in theory be more susceptible to the cytotoxic effect of artemisinin. So far, there are some in vitro data and only anecdotal clinical evidence.

LUMEFANTRINE  or  BENFLUMETOL

Lumefantrine (= benflumetol) was registered in China in 1987 for the treatment of P. falciparum malaria. It is an arylamine alcohol which was synthesised at the Chinese Institute of Military Medical Sciences. The half-life in the blood is approximately 4 days. The product is not active on the liver stages or gametocytes. Lumefantrine, like chloroquine, probably destroys haem polymerisation (a detoxifying pathway for the parasite). It is synergistic with artemether. The combination artemether-lumefantrine is used at a ratio of 1:6. Each tablet contains 20 mg artemether and 120 mg lumefantrine. This combination is also known as co - artemether ( Riamet®, Coartem®). This combined product has been available since 1999 in Switzerland and in various African countries. There is as yet no paediatric syrup or injectable form. For an adult with little or no immunity to malaria the advice is to take 24 tablets Coartem divided over 60 hours.  In patients of 12 year or older, and 35 kg body weight or more, treatment consists of 6 doses of 4 tablets each. If the first dose if give at zero hours, the next doses are taken at 8, 24, 36, 48 en 60 hours later. The possibility of drug-interaction and QTc-prolongation needs to be studied further, especially if this product would be used as stand-by medication in tropical travelers who also might take certain quinolones, azoles or macrolides.

Chemical structure of lumefantrine, malaria treatment. Copyright ITM

Lumefantrine is a lipophilic molecule with very low toxicity. It is a yellow powder that is only partially soluble in water. Absorption in the intestine is highly variable from person to person and is greatly increased (up to 16-fold) by fatty food (similar to halofantrine). Since people who are ill generally do not eat much, this has important consequences. Early in the treatment very little lumefantrine is absorbed. In combinations such as co-artemether, the artemether is responsible for the initial important reduction in the number of parasites and the low residual numbers of parasites is then cleared up by lumefantrine. Lumefantrine has a rather long half-life in patients with falciparum malaria: 4 to 6 days. In HIV-infected children, lopanavir-ritonavir-based ART (Kaletra) was associated with a decreased incidence of malaria as compared wiht ART based on a NNRTI, largely as a result of a medication interaction that increased drug levels of lumefantrine.  

PRIMAQUINE

Primaquine is an 8-aminoquinoline [quinoline = bicyclic N-containing aromatic structure consisting of a benzene ring with a pyridine ring]. It is inactive upon asexual blood forms. It does have an important, though only partial, causal prophylactic effect (on both P. falciparum and P. vivax ), but only if it is taken 24-48 hours (max. 96 hours) after inoculation with sporozoites. It acts upon the exo-erythrocytic stages of the parasites (liver schizonts). The half-life is relatively short (4 hours). For causal prophylactic use a daily dose of 15-30 mg may be taken. These regimens are not, however, very popular and there has been little experience of them. Chemoprophylaxis with primaquine can be stopped 3 days after leaving a malarious area.

Chemical formula of priamquine. Copyright ITM

In cases of P. vivax or P. ovale malaria, hypnozoites still remain in the liver after therapy with chloroquine. These may be destroyed by primaquine. In the past, 15 mg base per day was used for 14 days [26 mg primaquine biphosphate = 15 mg primaquine base], but current medical opinion favors 30 mg per day during 2 weeks. This drug is contra-indicated in pregnant women and in people with a significant deficiency of G6PD (glucose-6-phosphate dehydrogenase), an enzyme in the red blood cells (risk of haemolysis in patient and/or fetus). In significant G6PD-deficiency no primaquine is given, or -if absolutely needed- 0.75 mg/kg once per week for 8 weeks or 30 mg once per week for 15 weeks. There are P. vivax strains (e.g. P. vivax Chesson) which are less sensitive (India, Southeast Asia) or plainly resistant.  Primaquine therapy is certainly sensible for those who are finally returning from a malaria region. Sometimes this is called terminal prophylaxis. Primaquine sometimes causes nausea, especially high doses taken on an empty stomach. Nausea is much less common if primaquine is taken with food. Primaquine also acts on P. falciparum gametocytes. Therefore, in some circumstances (e.g. refugee camps) it may be given to reduce transmission (single dose of 45 mg). Older products which are structurally related to primaquine [8-aminoquinolines], such as pamaquine (Plasmoquine, Praequine), rhodoquine (Plasmocide), quinocide, pentaquine and isopentaquine are almost never used any more.

Mild methaemoglobinemia (usually <13%) is frequent with primaquine, but as long as the concentration of methaemoglobin is less than 20%, there will be no clinical consequences. There is a spontaneous recovery after two weeks. People who have an inborn deficiency in methaemoglobin reductase are very susceptible to primaquine-induced methaemoglobinemia. 

ETAQUINE or TAFENOQUINE

Chemical formula of tafenoquine (etaquine). Copyright ITM

Etaquine or tafenoquine is a new 8-aminoquinoline, derived from primaquine. It has a half-life of two weeks, which is much longer than the half-life of primaquine. It may be taken orally and has low toxicity. It is active against P. falciparum and P. vivax . It is an effective schizonticide and is also active on the pre-erythrocytic stages, including the hypnozoites of P. vivax . The mechanism of action is still unclear, but it probably disturbs the action of the parasites' mitochondria and the Golgi complex. There is also inhibition of the polymerisation of haematin to haemozoin (as with chloroquine). The product is administered as tafenoquine succinate. A dosage of 100 mg base corresponds to 125 mg salt. Ingestion with food increases the absorption by 50% and reduces the gastro-intestinal side effects. Absorption is slow, reaching a maximum plasma concentration after 12 hours. Tafenoquine is concentrated in red blood cells (3 times higher than in plasma). Tafenoquine is not eliminated via the kidneys. The optimum curative dosage has not yet been determined, but 300 mg per day x 7 days yielded a cure rate of 100% ( P. vivax ). A possible role in the treatment of P. falciparum is being investigated, although problems with its slow onset of action have still to be overcome. It is also being studied as a causal prophylactic agent. People with G6PD-deficiency may develop severe haemolysis after ingestion. Development of methaemoglobinaemia (3-15% metHb) is common, but generally subclinical. Nevertheless account should be taken of this by mountain climbers, pilots or individuals with underlying cardiopulmonary disease. The product is not yet on the market.

ELUBAQUINE

Bulaquine (elubaquine, aablaquine) is another primaquine analogue (i.e. an 8-aminoquinoline). Experience with this compound is limited. Bulaquine is currently licensed only for use in India for the radical cure of vivax malaria dosed at 25 mg/day for 5 days, but not (yet) as a gametocytocidal agent in Plasmodium falciparum . A pilot study of the P. falciparum gametocytocidal effect of a single dose of bulaquine 75 mg suggested it may be more effective than primaquine 45 mg.

PROGUANIL  and  CHLORPROGUANIL

Proguanil (Paludrine) and chlorproguanil (Lapudrine) are biguanides which are converted in the body to the active product cycloguanil. The enzyme which catalyses this oxidative activation is probably mephenytoin hydroxylase. Absorption is delayed by simultaneous ingestion of magnesium trisilicate (e.g. stomach powders). The concentration of proguanil in red blood cells is 6 times higher than that in plasma. There is genetic polymorphism for the conversion of the prodrug to the active product. Persons are "extensive metabolisers" or "poor metabolisers". One way to determine this is by measuring the proguanil/cycloguanil ratio in the plasma. Poor metabolisers have a lower plasma level of the active form (= higher P/C ratio) and theoretically are more at risk that the drug will fail. Nevertheless, the importance of phenotype status is not really known. There is also a lack of clarity concerning the various metabolites of the product. Approximately 20% of the population of Southeast Asia are said to convert proguanil to cycloguanil scarcely if at all. In Kenya this is 35%. This does not, however, appear to diminish the efficacy of Malarone®. Chlorproguanil has chlorcycloguanil as its active metabolite. The combination of chlorproguanil with dapsone is also known as Lapdap®. It is used as a cheap, short-half-life antifolate. It may be combined with artesunate (combination known as "CDA"). Proguanil is excreted via the kidneys. There is something strange going on with Malarone. Proguanil acts agonistic with atovaquone, but the metabolite cycloguanil antagonistic. More study is needed to clarify this issue.

Note: Proguanil

Proguanil is a biguanide (cf the antidiabetic drug metformin). What's the origin of this name? The term "pro" refers to the fact that this is a "prodrug". The term biguanide refers to the part of the lateral chains where the five nitrogen atoms are found. During metabolism the side chains are converted to a triazine ring, from which the name 'cycloguanil' comes.

Chemical formula of proguanil. Copyright ITM

Chemical formula of quinine, chloroquine, proguanil. Malaria treatment and prevention. Copyright Wellcome

Cycloguanil  is a powerful inhibitor of dihydrofolic acid reductase in the parasite. That is how the synthesis of nucleic acids in the parasite is disturbed. It has a slow action, and therefore cannot be used in monotherapy as a curative agent in an acute attack. There is swift development of resistance if proguanil is taken as the only prophylaxis. The therapeutic role of proguanil has changed recently, since the confirmation that atovaquone has fulfilled its initial promise (synergistic effect). As a prophylactic proguanil is given as one dose of 100-200 mg per day and chlorguanil as 20 mg per week.

ATOVAQUONE  in  Malarone  

Atovaquone  (Wellvone®, Mepron®) is a lipophilic hydroxynaphthoquinone. A related product (lapinone) was discovered 50 years ago, but at that time was abandoned since it could only be given parenterally. A fatty meal increases the absorption of atovaquone in the intestines. In the blood the molecule is highly protein-bound, but there are probably no significant interactions with other protein-bound drugs. Atovaquone is eliminated by the liver and can therefore be used in renal impairment. Malarone® cannot, however, be used as prophylaxis in renal failure because the blood levels of proguanil/cycloguanil are much higher. Simultaneous use of Malarone® and rifampicin is not recommended (blood levels 50% lower). Its safety during pregnancy is not known.

Chemical structure of atovaquone. Copyright ITM

Atovaquone is a powerful schizonticide for P. falciparum and P. vivax . It inhibits the mitochondria of the parasite (inhibition of the electron transport) and the de novo pyrimidine synthesis. It has a half-life of 2-3 days in adults and 1-2 days in children. On monotherapy recrudescence occurs very quickly. To avoid this problem it is combined with proguanil (brand name of the atovaquone + proguanil combination = Malarone®). These products are synergistic. The combination of atovaquone + doxycycline is also active. Absorption of Malarone® via the intestine improves if it is taken with food, since atovaquone is highly lipophilic. The recommended curative dose is 1000 mg atovaquone + 400 mg proguanil, once daily for 3 days (adults). In practice the curative regimen for an adult is: four tablets once daily for three days. An adjusted dosage is used for children, e.g. children from 11-20 kg: 1 tablet per day for 3 days, children 21-30 kg: 2 tablets par day for 3 days, children 31-40 kg: 3 tablets per day for 3 days. Malarone is not given to children weighting less than 10kg. The combination (250 mg atovaquone + 100 mg proguanil daily) can also be used for malaria prophylaxis. The dose is then 1 tablet per day, beginning the day before travelling and then taken daily until 7 days after return. There is also a causal prophylactic effect. There is no known cross-resistance with other anti-malaria products. The first cases of Malarone resistance were reported in 2001, i.e. very soon after introduction of the drug. The product is also being studied in toxoplasmosis, babesiosis, leishmaniasis, microsporidiosis and in Pneumocystis jirovecii pneumonia. In the treatment of babesiosis it proved more active in some animal studies than the combination of clindamycin/quinine. In general Malarone is very well tolerated. There have been rare cases of hypersensitivity reactions, with eosinophilia, skin rash and systemic symptoms such as pulmonary edema. This is also known as DRESS syndrome (drug eruption, eosinophilia and systemic symptoms). Stevens Johnson syndrome has also been described. Note: in the combination of Malarone®, it is atovaquone which is highly lipophilic and is poorly absorbed if not applied with a fatty meal. In Riamet® the lipophilic component is lumefantrine.

Piperaquine

Piperaquine is a Chinese synthetic drug belonging to the bisquinolines. Halflife of piperaquine is 9 days. Piperaquine is a highly lipid-soluble drug. Its clearance is greater in children than in adults. The combination dihydroartemisinin (artenimol) 40 mg with piperaquine 320 mg per tablet (Artekin, Eurartesim, Duo-cotecxin) is very promising. In 2006 Papua New Guinea became the first country to implement dihydroartemisinin-piperaquine treatment for P. falciparum and P. vivax infection in pregnant women during the second and third trimesters. Because of the slow elimination of piperaquine, this treatment provides up to 6 weeks posttreatment prophylaxis against new infectrions and relapsing P. vivax infection. In other countries this drug is considered a rescue medication, as more information is required regarding its safety profile during pregnancy, pharmacokinetics and optimal dosing. Check ECG for QTc prolongation. Dihydroartemisinin plus piperaquine is marketed as Eurartesim. Eurartesim should be taken on an empty stomach, once per day for 3 consecutive days. Simultaneous use of quinine, quinidine, halofantrine, mefloquine, amiodarone, antidepressants, fenothiazides, macrolide antibiotics, moxifloxacine, domperidone, pentamidine or fluconazole should be avoided. It should not be taken with grapefruit juice.The dose depends on body weight.     5 to     7 kg : 1/2 tablet of 160/20 mg per day x 3 days    7 to   13 kg : 1 tablet    of 160/20 mg per day x 3 days  13 to   24 kg : 1 tablet    of 320/40 mg per day x 3 days  24 to   36 kg : 2 tablets  of 320/40 mg per day x 3 days  36 to   75 kg : 3 tablets  of 320/40 mg per day x 3 days  75 to 100 kg : 4 tablets  of 320/40 mg per day x 3 days    

Pyronaridine

Pyronaridine  is an acridine derivative, just as mepacrine. It is closely related structurally to amodiaquine. It was synthesised in 1970 in China. The formulation known as Pyramax is a fixed drug combination with each tablet containing 180 mg pyronaridine and 60 mg artesunate.  Studies in Africa produced favourable results against P. falciparum malaria.

MISCELLANEOUS  PRODUCTS

Tetracycline , minocycline and doxycycline are antibiotics which are active against malaria parasites, but are very slow-acting. For this reason they are never given as monotherapy, but in combination with quinine. They very much reduce the risk of relapse. Doxycycline has the advantage that it only needs to be administered once daily. It is also more potent than tetracycline itself, probably due to its high fat solubility. It is sometimes used for malaria prophylaxis in mefloquine intolerance, e.g. in Southeast Asia or South America (100 mg per day beginning 1 day before arriving in an endemic zone). Prolonged ingestion of doxycycline can lead to phototoxicity, including photo-onycholysis. Sunscreens do not block ultraviolet A well enough to prevent phototoxic reactions to doxycycline.

Clindamycin  (Dalacin®) is also active against plasmodia, but is a second choice drug (risk of pseudomembranous colitis due to Clostridium difficile ). The dose of clindamycin is 10 mg/kg for 3-7 days. It is given together with quinine.

Sometimes cotrimoxazole  is used as third choice (together with quinine).

Fosmidomycin  is being studied at present for its anti-malaria properties. Fosmidomycin, a phosphonic acid derivative, blocks 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme of the DOXP pathway in the apicoplast of P. falciparum , thereby inhibiting the growth. The drug is well tolerated in people. The optimal treatment schedule is still to be determined, although a dose of 1,2 gram every eight hours for a minimum of 5 days seems promising.

Azithromycin  (Zitromax®) was developed in 1988. It is a macrolide related to erythromycin. The molecule has a ring structure of 15 atoms, including 1 nitrogen (the letters 'az' in azithro refer to the nitrogen). In some studies a prophylactic effect was demonstrated (83% after daily ingestion of 250 mg, 64% after weekly ingestion of 1 gram). Azithromycin is stable in an acid environment (unlike erythromycin) and can easily be taken orally. High intracellular concentrations are reached, even in the white blood cells (100 times the serum concentration) and there is long-term retention in the tissues. The tissue half-life is 50-90 hours, which makes one dose per day possible.

Atebrine  was developed in Germany in the 1930s. It was widely used for malaria prevention during World War II (of strategic importance in the Pacific). In the USA it was named quinacrine and in England it was called mepacrine. It quite often caused stomach problems, nausea, vomiting, dizziness and yellow discoloration of the skin. High doses can cause acute toxic psychosis. Aplastic anaemia occurs in 1/20,000 cases. Psoriasis may become worse during treatment. The product has a disulfiram effect and taking alcohol during its use is not advisable. It passes the placental barrier. The use of atebrine has now been almost abandoned. It is still sometimes used to treat tinidazole-resistant Giardia.

Pentaquine is an obsolete antimalarial agent with important side-effects. It caused orthostatic hypotension and appeared to cause reversal of malignant hypertension in a small number of patients.

Nitroquine  is being evaluated for its activity against various Plasmodium species.

Artemisone is a semi-synthetic artemisinin-derivate ("second-generation"). Initial studies showed a lack of neurotoxicity in vitro and in vivo animal models. It is about 10 times more potent than artesunate. Artemisone is metabolized into an active metabolite. Formulation work has indicated stability problems with the present formulation. These problems have postponed clinical use until they can be overcome. Artelinate is a semisynthetic artemisinin derivative (condensed with benzoic acid), but this product was not taken beyond animal studies.

Isoquine is an amodiaquine-like compound that has been redesigned and synthesized to remove the structural cause of toxicity of its class while retaining full antimalaria activity. This second-generation aminoquinoline retains the easy synthesis of amodiaquine from inexpensive precursors, and promises a new generation of affordable, well-tolerated, and effective antimalarials. It is possible that it will avoid cross-resistance to its chemical cousin chloroquine (and amodiaquine).

Ferroquine . This molecule is derived from an 4-aminoquinoline (cf chloroquine) coupled to an organic iron complex (ferrocenyl). Phase I trails have been completed.

Methylene blue . This thiazine dye was the first known synthetic antimalarial. It is better known for its usefulness in treating methemoglobinemia. Further study is needed to see methylene blue or one of its derivatives / combinations will get a place in malaria treatment.

Arteflene is a synthetic fluorinated bicyclic sesquiterpene peroxide, derived from a substance which is present in the Chinese plant Artabotrys uncinatus (yinghaosu). The half-life is quite short (3 hours). It has an active metabolite. It is not useful as a single dose agent. It was developed in the 1990s and proved an effective antimalarial in clinical trials. It's development has now been abandoned because of high production costs and lack of advantages over the artemisinin derivatives.

Arterolane (OZ227) is a synthetic trioxolane compound containing an endoperoxide brigde like  the artemisinins. (A trioxolane is a structure with a five-membered C-O-O-C-O ring). It can be taken by mouth. It can be mass-produced more cheaply than natural artemisinin, has a longer shelf life than the natural drug, and fewer doses are needed to produce its full effects. Unfortunately, it turned out to be unstable in blood and development was discontinued for use as monotherapy. Research on the combination arterolane-piperaquine is ongoing. Research on hybrid trioxane-aminoquinolines (trioxaquines) is continuing.

Other miscellaneous substances. Iron chelators (deferoxamine), pentoxifylline, mannitol and other products (even cisplatin) have been studied as adjuvants in malaria treatment, but have not to date proved convincing. Naphthylisoquinolines form a new group of experimental anti-malaria agents. Various studies are at present evaluating their potential. Various trioxanes and tetroxanes, highly active in vitro, are being evaluated (an oxane is a cyclic ether). Licochalcone A is a substance obtained from Chinese liquorice ( Glycyrrhiza inflata ) and is at present being studied for its activity against Plasmodium species. It may also be active against Leishmania sp. The bark of Enantia chloranta (Annonaceae) contains some active anti-malaria products, but toxicity studies and in-vivo studies still have to determine a possible therapeutic use. Since the severity of cerebral malaria correlates with the concentration of tumour necrosis factor, and the latter plays a role in the physiopathology, trials are currently being carried out with CytoTAb®, an anti-TNF product. Triclosan, an antibacterial agent which is sometimes used in anti-acne products, deodorants, toothpastes and mouthwash, is an inhibitor of fatty acid synthesis in P. falciparum (and also active against Toxoplasma gondii ). More research is necessary to determine any clinical relevance. Non-infected red blood cells do not produce membrane lipids. When a malaria parasite finds itself in a red blood cell, however, it must synthesise membrane lipids. Phosphatidylcholine is the most important of the phospolipids among the membrane lipids. To produce this, choline is taken up from the plasma by means of a transporter. This step can be blocked by a new experimental agent (G25). Manzamine A is an alkaloid first identified in sponges collected of the coast of Okinawa. It is a promising substance with pronounced antimalarial activity. While the malaria parasite grows inside an erythrocyt, it exports several proteins into the host cell. Proteins destined for export contain a conserved aminoacid motif ("PEXEL"). When this motif is cleaved by the protease plasmepsin V, the parasite protein can translocate into the red blood cell. This protease might be a good drug target for antimalarial agents.

Malarex  is extracted from the Brazilian tree Peschiera fuchsiaefolia , a source of traditional remedies against fever. The active compound is the alkaloid voacamine. The efficacy of this compound is not clear at the moment.

Falcipain  is a critical protease used by the parasite to breakdown proteins in the red blood cells. A treatment based on falcipain inhibitors might be possible in the future if a non-toxic inhibitor would be identified.

Curcumin is the chemical which gives turmeric its distinctive yellow colour, is investigated for its antimalarial properties. Oral administration protected mice against lethal infection with Plasmodium berghei . New derivatives of endochin are examined for their potential use in human malaria. The term endochin derives from its action upon the endothelial forms of avian malaria.

OZ430. The ozonide OZ439 is a new and still experimental synthetic peroxide antimalarial drug. It is fast-acting against all asexual erythrocytic Plasmodium falciparum stages. Low clearance leads to prolonged blood concentrations, allowing single dose administration.  Clinical trials are ongoing.

Note: Apicoplast and new therapeutic drug targets

This cellular organelle may possibly form a binding site for new drugs. Various metabolic reaction chains are present in this organelle. In 1998 it was demonstrated that in some Apicomplexa the "shikimate pathway" is present. The name "shikimate" comes from "shikimi no ki", the Japanese name for star aniseed ( Illicium religiosum ), from which shikimic acid was first isolated. The shikimate pathway is a biochemical reaction chain which occurs in algae, higher plants, fungi and various micro-organisms. It does not occur in mammals, however. It is important for the synthesis of aromatic amino acids such as phenylalanine, tyrosine and tryptophan, as well as for the production of ubiquinone and other substances. In the Apicomplexa it possibly supplies folic acid precursors which are needed for growth. The herbicide glyphosate ("Round-up") is a known inhibitor of an enzyme from this reaction chain and can block the growth of parasites. It also appears that the apicoplast synthesises isoprenoids in a manner which clearly differs from that of mammals. Isoprenoids are used for the production of cholesterol, steroid hormones, coenzyme Q and for enzyme prenylation. Humans synthesise these substances via the "mevalonate pathway", known as a target for cholesterol-lowering substances such as HMG-CoA reductase inhibitors (statins, e.g. simvastatin). The apicoplast on the other hand, uses what is called the "methylerythritol or DOXP pathway" (1-deoxy-D-xylulose-5-phosphate). Fosmidomycin is a substance that actively blocks these reaction chains in P. falciparum . It is hoped that better knowledge of the biochemical details will lead to new therapeutic products for the treatment of malaria as well as toxoplasmosis and cryptosporidiosis.

Treatment, brief survey of side effects

Mefloquine (Lariam®). Not used in cardiac arrhythmias, myasthenia, epilepsy or psychiatric disorders. Insomnia, dizziness, tremor, agitation, psychotic behaviour, nausea. Not to be used together with beta-blockers, calcium antagonists, digitalis or quinine (cumulative cardiotoxicity).

Chloroquine (Nivaquine®). Pruritus is common in black-skinned people (chloroquine has affinity for melanin-containing tissues). Regular eye check-up is advised to detect retinopathy during long-term use (years). Contra-indicated in psoriasis or in myasthenia gravis. Cardiotoxic if given in rapid IV administration. Reduced immune response after rabies vaccination (not for other vaccinations)

Proguanil (Paludrine®). Almost no side effects. Rarely aphthous oral lesions.

Pyrimethamine/Sulphadoxine (Fansidar®). Severe dermal and mucosal lesions (leading to Stevens-Johnson syndrome). Do not give if allergic to sulphonamides.

Quinine (Quinimax® and others). Bitter, tinnitus, transitory hearing loss, nausea. [Thrombocytopaenia has been described as a rare complication, but P. falciparum malaria itself very frequently causes a drop in the blood platelet count]. Do not use with digitalis, quinidine effect on the heart, not in long QT-syndrome or Brugada syndrome, conversion of atrial fibrillation to sinus rhythm possible. Overdosage: blindness, deafness, cardiorespiratory problems, death. Quinine is a common cause of drug-associated TTP-HUS (thrombotic thrombocytopenic purpura - haemolytic uraemic syndrome). Not to be used in myasthenia.

Halofantrine (Halfan®). Almost without side effects, but, rarely, severe and even lethal cardiac problems (conduction disorders). Potential candidates can be excluded by taking an ECG before starting treatment.

Doxycycline . Photosensitivity possible, candidosis of mucosa, not given to pregnant women and young children. Photo-onycholysis can occur during prolonged exposure (opaque nail varnish can help here). Artemisinin derivatives . About 1/3000 patients develop hypersensitivity and urticaria. Suppresion of fetal erythropoiesis (first trimester). CNS neurotoxicity in animals, especially with prolonged administration, but no signs of neurotoxicity in humans with short therapeutic courses.

Treatment, the problem of drug resistance

In chloroquine-sensitive P. falciparum  the drug is concentrated in the parasite. There is slow outflow (t½ = 50 minutes) of chloroquine from the sensitive parasite. In resistant parasites t½ for outflow = 1 to 2 minutes. Resistance is thus not due to inactivation, breakdown or neutralisation of chloroquine. The parasite quickly pumps the product away to the blood, so that the concentration of chloroquine within the parasite is low. At present this cannot be counteracted in humans (in vitro reversible with verapamil). The resistance potential is a function of the total biomass of the parasite. The lower the total biomass, the lower the probability of resistance.

PCR technology [polymerase chain reaction] is required to differentiate a recrudescence (or relapse) in an endemic region from a re-infection with the same species. Several polymorphic loci are analysed. Every combination of alleles that is tested, is in itself rare and permits differentiation between strains.

Malaria. Plasmodium falciparum : different types of chloroquine resistance. Copyright Wellcome

The problem of chloroquine resistance in P. falciparum became manifest in the 1960s in South America (Colombia and Brazil) and in Southeast Asia. Gradually the resistance spread to other continents. Resistance also developed against other drugs, including Fansidar®. The situation is evolving rapidly and is getting worse. The highest resistance is found in some regions in Southeast Asia, including Cambodia, the Thailand-Cambodia border, and the Thailand-Burma border. It is only a question of time before these resistant strains spread further geographically. There are several reasons for this increasing resistance. Important factors include inadequate individual patient compliance, treatments that are are often discontinued prematurely, frequent underdosing, earlier mass-treatment campaigns reaching only part of the population and therapy being sometimes only partly administered, as well as the use of chloroquinated salt [Cambodia, Brazil (the Pinotti method)]. Among the causes of the swift increase in geographical spread are the large-scale migrations of today, and the ability to move rapidly from place to place. Some products are eliminated slowly from the body (e.g. mefloquine t 1 / 2 = 2 weeks) so that for some weeks a subtherapeutic level of the product is present in the body. When malaria parasites are exposed to such low concentrations, partially resistant strains have a selective advantage. The occurrence of subclinical cases functions as a source and reservoir for transmission of parasites with reduced sensitivity. Since the cost price of alternative drugs is generally higher than that of traditional treatments, under-dosing with new drugs will become even more important in future. New drugs are developed only slowly. Combination therapies with for example artemether-mefloquine, co-artemether, atovaquone-proguanil, comparable to the present-day quinine-doxycyclin, will become more common in future. The principle behind this approach can be compared with the present combination treatments for AIDS, tuberculosis and leukaemia.

Direct consequences of the increasing resistance of P. falciparum malaria:

- an increase in mortality and morbidity

- a delayed initial therapeutic response

- a shorter interval before recrudescence occurs.

Example:

Evolution of Plasmodium falciparum resistance in Thailand. Copyright ITM

The use of primaquine could be promoted to counteract transmission (primaquine is a gametocytocide). In practise, drug combinations will be used to counter the growing resistance problem (e.g. artemisinin derivatives plus at least one other component). Stricter control on "fake" drugs (counterfeited medication), some of which contain small amounts of active material, will be an essential component in health programmes. One idea to combat counterfeit drugs and piracy is to tag individual genuine medication boxes with an authentication number (item-unique code) under a scratch-off label on the wrapping. When revealed just before purchase, this number can be send toll-free by telephone text message to an independent certifying company (e.g. Sproxil) which then instantly and automatically replies.

Prevention

Prevention, external agents

"Pyrethrum flowers; Tanacetum cinerariifolium (= Chrysanthemum cinerariaefolium ). This plant is harvested in order to extract pyrethrum. This substance is used for the production of pyrethroids, used e.g. for the impregnation of mosquito nets. Copyright ITM"

A Laotian woman shows her new bed net, which protects her against malaria. Copyright ITM, courtesy Wim VAN BORTEL

Anopheles mosquitoes only bite in the evening and at night. It is possible to protect oneself by wearing protective clothing and using an undamaged mosquito net. Effectiveness is increased by treating the net with pyrethroids (insecticides) such as permethrin (Permas®, Peripel®), lambda-cyhalothrin (IconetT) or deltamethrin (K-Otrine®). This will increase further in importance in the future. The substances are derived from pyrethrum, a product originating from Tanacetum cinerariifolium (= Chrysanthemum cinerariifolium ) and C. coccineum , herbaceous composite-flowered plants (fam. Compositae) which are similar to large daisies. In emergencies where mosquito nets are logistically difficult (e.g. camps after heavy floods), impregnated blankets can be used. These will probably be less effective than impregnated bednets. In most instances, permethrin will be augmented by piperonyl butoxide. Piperonyl butoxide can be derived from safrole, a chemical present in many plants, such as sassafras and brown camphor oil. (Note: Safrole is also illegally harvested from the evergreen tree Cinnamomum parthenoxylon (Saffrol Laurel) for preparing ecstasy or MDMA). Piperonyl butoxide is the most widely used synthetic pyrethrin synergist and there are no reports available on toxic effects on humans resulting from the exposure to it.  Piperonyl butoxide is not an insecticide itself but a cytochrome P450 inhibitor which allows pyrethroids such as permetrin to be much more active (10x). Inhibition of the detoxification pathway allows higher unchanged systemic concentrations of the active insecticide to remain within the target animal for a longer period. Mass produced long lasting insecticide treated nets (LLINs) are replacing older style bed nets. Olyset net was the first LLIN which became commercially available. Sumitomo’s Olyset® technology incorporates permetrine insecticide directly into polyethylene filaments which can be woven into sturdy bed nets to provide long-lasting protection from night-time biting mosquitoes. Olyset Plus, which received WHO approval in July 2012, retains the controlled-release technology and durability, and contains 2% permethrin and 1% of the synergist piperonyl butoxide (PBO). The fibres have been designed to release the two ingredients at a constant ratio of 2:1. The 'bleed rate' at which permethrin and PBO migrate from the internal reservoir in the fibres to the surface of the net has been adjusted in order to make the net active again within 1-2 days of washing. For this work, Sumitomo Chemical became the co-winner of the 2012 'Application of Core Competence' category – Global Business Coalition Health Award. A major production plant has been set up in Tanzania.

Note: Pyrethrum

The term "pyrethrum" refers to the powder made from the dried flowers of Tanacetum cinerariifolium . The term "pyrethrins" refers to the 6 insecticidal components which are found in the powder. All 6 are organic esters formed from one of two acids (chrysanthemic and pyrethric acid) and one of three keto-alcohols (pyrethrolone, cinerolone, jasmolone). Pyrethrins are unstable molecules which degrade within a few days in light and warmth. They can be stored in a refrigerator at -4°C. The term "pyrethroids" refers to the semi-synthetic derivatives of pyrethrins. These products have the advantage that they are much more stable than pyrethrins. The plants with the highest concentrations of active constituents grow well in sunny, semi-arid regions at 1600-2000 metres above sea level. Kenya, Tanzania and Ecuador are important producers. They are best harvested during a warm, dry period when the flowers are fully developed. The plants are dried after harvesting, and finely ground. Extraction may be with kerosene, paraffin or alcohol. Often a synergist is added, such as piperonal butoxide or various oils. Pyrethroids have low toxicity for mammals including humans but are highly toxic, e.g. to fish. They are sometimes used for illegal "chemical" fishing. Fine-mesh gauze can be applied to windows and ventilation shafts. One good argument for using a mosquito net is the fact that it also protects from nuisance insects such as Culex mosquitoes and bedbugs. In regions where there are few Culex , people are not so ready to use a net: after all they cannot see or hear any mosquitoes (anopheline mosquitoes fly with little noise)

Chemical structure of DEET: N,N-diethyl-m-toluamide. Insect repellant. Copyright ITM

Malaria vector control. Spray team preparing insecticides against Anopheles mosquitoes. Copyright ITM

Insecticides based on pyrethrum can be dispersed by means of spraying (spray gun), evoparation (heated electric plate) or burning (mosquito coil, e.g. with esbiotrin). Insecticides can also be applied to the walls or to the curtains by the windows. There are also various insect repellents. DEET (N,N-diethyl-m-toluamide, now called N,N-diethyl-3-methylbenzamide) is moderately active and can be applied as an alcoholic solution to the skin. This produces a sticky effect when the alcohol evaporates, however. The effectiveness is only moderate. DEET is absorbed through the skin and is eliminated quickly via the urine. There is no accumulation in the body. Other repellants include KBR 3023 (Autan active®) which has the same effectiveness as DEET, but is non-irritant and does not affect plastic (spectacles), dimethyl phtalate (low activity) and ethohexanediol. Essential oils such as citronella, cedar, eucalyptus, neem (obtained from the tree Azadirachta indica ) and geranium are short-acting and not very effective. People who need to stay in regions which are heavily infested with mosquitoes, can apply permethrin to their clothing. Taking extra vitamin B and electronic buzzers are of no use.

The product containing IR3535 (ethyl butylacetylaminopropionate) has been subjected to well- controlled field studies. The results demonstrate a protection time for the product of up to three hours or more against mosquitoes. Case studies are not a valid substitute for repellent field studies. Only field studies are to be used to establish efficacy of insect repellants.

Prevention, taking pills

Chemoprophylaxis is in the first instance intended as prevention of P. falciparum malaria. No single drug which is taken preventively, is 100% active against sporozoites and no single drug prevents the formation of liver forms (except primaquine). While taking the drugs no vivax or ovale malaria will occur, but after they have been discontinued an attack with these plasmodia is possible in the following months or years. In view of the extensive resistance of P. falciparum, at present no 100% satisfactory protection against this latter parasite is possible. Advice as to whether or not to take medication, and which kind of drug to take, will depend on the region and differ from person to person (short journeys, resident, local population, pregnancy, young children, allergy, chronic diseases, use of other drugs and so on). Recommendations vary from country to country.

In regions with only P. vivax  and/or sensitive P. falciparum  (WHO zone A) chloroquine 300 mg/week will suffice. In zone B a combination of chloroquine (Nivaquine®) 300 mg/week and proguanil (Paludrine®) 200 mg/d is recommended. If necessary chloroquine 100 mg/day can be taken with proguanil 200 mg/day (= 1 tablet of Savarine® daily). For shorter stays in zone C (<3 months) mefloquine is recommended (Lariam®, 1 tablet per week). An alternative is doxycycline 1 tablet per day (beginning 1 day before departure until 4 weeks after return). Malarone, 1 tablet per day beginning 1 day before departure until 7 days after return, is an easy and effective prophylaxis, but the drug is expensive. For longer stays, the combination Nivaquine®/Paludrine® is advised during the first years, together with the availability of a stand-by treatment in case of a breakthrough. For people who live for many years in such regions it is optional to discontinue the Nivaquine®/Paludrine®, but to still have a treatment on stand-by (e.g. Malarone®, quinine vibramycin, Riamet®). An annual eye examination to check for chloroquine toxicity is recommended for people who take Nivaquine® for many years. In practice this is sometimes difficult to achieve. The local population should not take chronic chemoprophylaxis (most people are semi-immune). There are some high-risk groups: e.g. pregnancy (during pregnancy, in particular in the second and third trimesters and also immediately post-partum, the immunological resistance to malaria falls). The advice that is given here should take into account the general state of health and local guidelines. Intermittent preventive therapy in pregnancy ("IPTp") protects against maternal anaemia and low birth weight, especially in primigravidae and secundigravidae, and its use in areas in medium to high transmission is recommended by WHO. The efficacy of IPTp is reduced in HIV-positive women.

In summary:

Zone A : Chloroquine 300 mg per week Zone B : Chloroquine 300 mg per week + Paludrine 200 mg per day, or Savarine 1 per day Zone C : Lariam 1 tablet per week, or doxycycline 100 mg per day, or Malarone 1 tablet per day

As alternative or supplement:

Personal physical protection (impregnated mosquito net, repellents, clothing that covers, insecticides in rooms) No chimioprophylaxis, but reliable stand-by treatment available (Malarone, Coartem, quinine + doxycyclin)

Prevention, vaccination

Research into a malaria vaccine is based on a number of possibilities. An immune response can be triggered against sporozoites, liver forms, erythrocytic forms and/or gametocytes. However, the immune response does not necessarily have a protective effect. A 100% effective malaria vaccine is not likely to be developed in the foreseeable future, but a vaccine which leads to partial protection is more likely to become available.

SPf66

In 1987 Manuel Patarroyo from Colombia demonstrated that a synthetic peptide vaccine (SPf66) is immunogenic and protects Aotus monkeys against challenge infection with P. falciparum . The vaccine contained a cocktail of synthetic antigens. It also proved safe and partially protective in test subjects in Latin America. It was subsequently tested (double-blind, randomised and placebo-controlled), first in La Tola, Colombia (39% effectiveness) and then (independently) in Idete and Kilombero, Tanzania, regions with high transmission, and in Gambia. In 1994 the results became known. The effectiveness of 3 injections against clinical malaria was 31% (Tanzania) and 0% (Gambia) and the incidence of parasitaemia was the same in test subjects as in the control group. There was no adequate pre-erythrocytic immunity. Other later trials were equally disappointing.

RTS,S/AS02A

Most initial work in developing a vaccine was carried out by studying the proteins found in sporozoites. The idea was that stimulated antibodies, aimed at invariant amino acid sequences which occur in natural proteins, would eliminate the sporozoites, in the hope of preventing infection (stopping the infection before the liver phase). Sporozoites only remain in the circulation for 30 minutes. There is therefore little time to neutralise all parasites by means of antibodies. In 1973, it was demonstrated that protection against malaria in humans is possible by vaccinating them with irradiated sporozoites. This method is not practical to be used in large-scale programmes, because till present, their production requires harvesting them from live mosquitoes. If sporozoites could be cultured en masse, this would be a watershed. In the early 1980s antibodies against sporozoites were used to identify the main antigen, circumsporozoite protein (CSP). This protein contains a repeating pattern of aminoacids which is the main target of the antibodies. The gene coding for this protein was identified and cloned in bacteria, so it became available in large quantities. Antibodies against the repeat pattern represent the humoral branch of the immune response. In order to activate the cellular immunity (T cells), a fragment of the tail end of CSP was added. In the mean time, hepatitis B vaccine was developed via genetic engineering. At high enough concentrations, the recombinant surface antigen of hepatitis B forms virus-like particles that elicit a greater immune respons than individual proteins. In order to make a immunogenic malaria vaccine, it was decided to couple the antibody-inducing repeat (R) part of CSP, the tail-end of CSP which is recognised by T-cells (T) and the hepatitis B surface antigen (S). However, with these additions, the fused protein lost the capacity to form self-assembling larger particles. In order to regain it, "ordinary" surface antigen was added in a ratio of one part RTS to four parts S. In this way the first part of RTS,S was created. The second part is the adjuvant, which is needed to boost the immune response. The adjuvant AS02A is an oil-in-water emulsion with the immunostimulants monophospharyl lipid A and Quillaja saponaria fraction 21. This saponin is extracted from the bark of a the Chilean soapbark tree, and is used in veterinary medicine since the 1930s.  Monophosphoryl lipid A is derived from one of the lipids of the lipopolysaccharide molecule (see Gram negative bacteria, e.g. chapter on typhoid fever). An improved version containing liposomes is under development. The complete vaccine is known as RTS,S/AS02A. Effective therapeutic vaccines contain two primary constituents, antigen and adjuvant. How does one reach such an adjuvant?  The short answer is basically by screening lots of substances. Adjuvants consisting of microbial pattern molecules play a central role in vaccination. Successful vaccine requires efficient induction of antibody, type I interferons, certain cytokines/chemokines, cytotoxic T lymphocytes and/or NK cells. Toll-like receptors in dendritic cells act as adjuvant receptors and sustain adjuvant activity. TLR responses vary with different adjuvants. Monophosphoryl lipid A is a trigger for TLR4. In 1996 the first favourable results became known. Six out of seven individuals were protected. The initial results suggest a safe, immunogenic pre-erythrocytic vaccine which offers significant protection against natural P. falciparum infection. A randomised and controlled study in the Gambia on 306 volunteers showed RTS,S/AS02 to provide significant protection against natural P. falciparum infection. In 2004, a double-blind phase IIb randomised controlled trial in Mocambique in 2022 children (age 1-4 years) showed that the vaccin was safe, well tolerated and immunogenic in this group. Vaccine efficacy for severe malaria was 57%. In 2007, a double blind randomized study in Mocambique in children younger than 1 year showed an efficacy of 65% when 3 doses were given. The efficacy is lower with longer follow-up time. Side effects were minimal. It is possible that combining an adenovirus-based vaccine with RTS,S might give better results, but only the future will tell. The results of phase II trails published in December 2008 (NEJM) were encouraging (efficacy of around 53%). New trials, and the follow-up of current studies, should help determine for how long efficacy will be be maintained. Phase III trials will be conducted in Africa, in areas of varying transmission rates. This may affect efficacy results. Research sites with ongoing malaria trials tend to have lower transmission rates than other parts of Africa, because bednets tend to be more widely available in these places. In a phase II trial of RTS,S/AS01E in children vaccinated at age 5 to 17 months in Kenya and Tanzania, efficacy against a first malaria episode was 46% at 15 months after the third vaccine dose. Malaria vaccine was associated with a decrease in first and all episodes of malaria. In 2013, it became clear that efficacy fell to 16.8% at 4 years.

Protection via sporozoit inoculation

Sporozoites cannot (yet) be cultured in vitro. In 1976 Trager and Jensen achieved in vitro cultivation of P. falciparum trophozoites and schizonts, so that a continuous in vitro cycle could be maintained in the lab. The initial parasites (before culture was achieved) were obtained from a P. falciparum infected South American owl monkey ( Aotus sp). This breakthrough achievement was followed in 1982 by a method for producing gametocytes from these cultures. In 1986 it was reported that humans could become infected by the sporozoites produced in mosquitoes which had fed on these in vitro cultures. In the 1970s, induction of protection in humans was achieved artificially by the inoculation of radiation-attenuated sporozoites. The sporozoites still could invade hepatocytes, but were no longer capable to complete the liver-stage maturation. The acquired immunity was a pre-erythrocytic immunity. This method required more than 1000 infective mosquito bites during five or more sessions and irradiated mosquitoes. The point of these experiments was not to develop an immediate useful vaccine, but to show that protection was possible at all. In 2009, it was shown that inoculation with living non-attenuated chloroquine-sensitive P. falciparum sporozoites via monthly Anopheles stephensi bites while the patient takes chloroquine could achieve protection from homologous challenge. Chloroquine is not active against sporozoites, liver stages or early asexual ring forms of the first generation caused by merozoites released from mature schizonts in infected hepatocytes. With non-attenuated sporozoites, a low dose of blood stage antigens are also exposed to the immune system. In how far this finding will have practical spin-offs in clinical settings is unclear at present. In endemic settings, nonsterile semi-immunity is acquired only after years of natural exposure. Rodent models show that acute blood-stage infection suppresses the induction of protective pre-erythrocytic immunity. It is unclear in how far this finding can be translated to humans.

Prevention, vector control

General

Mosquito control is a speciality in itself and includes the use of insecticides, treatment control of breeding grounds and biological control. Each ecological region requires its own individual approach: savannah, primeval forest, agricultural areas with or without irrigation systems, the margins of uplands, desert margins and oases, city environments, coastal and marsh regions. There are many difficulties: interventions need to be maintained over large areas for very long periods of time, mosquitoes quickly become resistant to insecticides, many people will not allow their houses to be sprayed, high costs, shortage of staff, ecological collateral damage due to insecticides, political instability which interferes with long-term planning. Border regions with rapid expansion and unstable social situations, e.g. mining (precious stones, gold), as in Thailand or Brazil, have their own specific problems. An exclusively technical approach will not be possible without simultaneous improvement in the social and economic conditions of the population at risk. In some areas the spraying of DDT has been reintroduced or is under consideration. This chemical was very effective in the past, but was banned after overuse lead to several problems.

Genetic basis of insecticide resistance

There are three major enzyme families implicated in insecticide resistance: (1) carboxylesterases, (2) glutathione transferases and (3) cytochrome P450s. Genomic analysis of Anopheles gambiae reveals a considerable expansion of these supergene families in the mosquito.

Biological vector control

One way of diminishing Anopheline larvae is the introduction of larvivorous fish (fish which eat mosquito larvae), such as gambusias ( Gambusia affinis ) and guppies ( Aplochelius ). This biological control method is used in several areas, but more study is needed. The are fish of Caribbean orginin. Introduction of ecological foreign fish into a new ecosystem might be dangerous, but so far experience is positive. Gambusia  are the preferred fish in clean water, while Poecilia is better at higher temperatures and can survive in polluted water. Some fish within the Nothobranchius  and Cynolebias genera, have drought-resistant eggs, and can be placed where water has collected temporarily ("instant fish"). Toxorhynchites mosquitoes are large metallic-coloured insects with a curved proboscis [Gr. "toxo" = bowed; "rhinos" =nose) which they use to drink plant juice and nectar. They do not suck blood. The larvae are predatory upon other mosquito larvae, but to date the use of this natural enemy has not produced convincing results. One of the problems is that even low-density vector populations can sustain transmission. 

A female mosquito copulates only once and stores the sperm in a spermatheca. If insemination takes place with a sterile male there can be no reproduction. The massive release of sterile male mosquitoes is a technique which has shown good results against other insect species (screw worms, tsetse fly), but would be of limited use agains malaria transmission.

A certain spider ( Evarcha sp.) are only 8 mm long, but stalk female mosquitoes far bigger than themselves. The young spiders particularly target mosquitoes which are full of blood, puncturing their abdomen, killing the mosquito and siphoning out the blood. Because they target Anopheles gambiae mosquitoes -the most effective malaria vector on the planet- the spiders offer the possibility of biological control of malaria mosquitoes.

Mosquito-killing fungi (biopesticides) such as Beauveria bassiana and Metazhizium anisopliae are being studied for malaria vector control. These fungi are not yet a practical control option, but the approach has at least some major advantages: the fungi are generally safe to people and no insects have been shown to develop fungal resistance.

Genetic vector control (experimental)

Among the 400 species of Anopheles mosquitoes, only a limited number can be infected with plasmodia. The other mosquitoes are genetically resistant to this infection. If the natural mosquito population could be replaced by a genetically resistant population it would stop the transmission of malaria. There are a number of problems, however. First, the mosquito genes which make the parasite's cycle in the mosquito impossible would have to be isolated and understood. Second, it would then be necessary to find a way to bring those genes into a natural vector and to make them function properly. Thirdly, the transgenic mosquito must have a competitive advantage over the natural vector in the Darwinian battle for survival. The mosquito strain selected in a laboratory should be able to compete with wild-type mosquitoes, and displace the wild-type. New, malaria-resistant mosquitoes would need to have a selective survival advantage. Anopheles gambiae populations have various subpopulations which do not mix much. This partially sexual isolation of the natural mosquito populations would make the transgenic vector method difficult. Fourth, they should of course also not transmit other diseases.  In 2007, researchers in Baltimore published results of genetic manipulated Anopheles stephensi , an asian vector, and transmission of P. berghei (rodent malaria). The vectors expressed an oligopeptide which blocks ookinetes from penetrating the mosquito's gut and blocks sporozoites from docking to the receptors on the salivary glands. These altered vectors outcompeted the wild-type mosquitoes after a couple of generations. In the laboratory, they seemed to have a fitness advantage over their infected counterparts. The last word about such transgenic mosquitoes has not yet, however, been said. If the public outcry against genetically manipulated food is a guide, any method which uses transgenic malaria vectors will have to be able to convince public opinion that there is no better alternative. De novo genetic engineering resistance into mosquitoes might be unnecessary. It was found that in an endemic area of Mali, many Anopheles gambiae mosquitoes are already resistant to Plasmodium falciparum . If a technique could be found to selectively kill falciparum-infected mosquitoes, the resistance gene would become more widespread in the natural mosquito population.  Wolbachia pipientis are maternally inherited endosymbiotic bacteria associated with cytoplasmic incompatibility. This provides a reproductive advantage to infected females, since they can mate successfully with infected and uninfected males. In contrast, uninfected females can only mate successfully with uninfected males. Since the Wolbachia infection is transmitted only through females, this can result in an increased frequency of Wolbachia in the subsequent generation and spreading of the Wolbachia infection into the insect host population. W. pipientis infections are common in many mosquito genera but not in natural (wild) Anopheles populations. However, in experimental settings, infections with this endosymbiont can be established in Anopheles gambiae . Such infections could be maintained for many generations in the lab, but much more study is needed before this or similar endosymbiotic bacteria might be used to drive transgenic traits (malaria-resistance) into wild mosquito populations in the real world.

Examples of malaria control measures

AIM OBJECTIVE / ACTION PROTECTION OF INDIVIDUAL / FAMILY PROTECTION OF COMMUNITY   Reduction of human-mosquito contact Screening of houses, mosquito nets, repellents, covering clothing Selection of site for housing Zooprophylaxis Destruction of adult mosquitoes House spraying with insecticides (aerosol) Residual insecticides in houses Ultralow volume spraying (outside) Destruction of mosquito larvae Sanitation around the home Emptying water containers intermittently Larvicides on water surfaces Interrupting irrigation, sluices Biological control Source reduction Sanitation around the home Small scale drainage Environmental sanitation, water management Large scale drainage Destruction of malaria parasites Early diagnosis and treatment Chemoprophylaxis Diagnosis and treatment facilities Chemoprophylaxis of target groups (pregnant women) Mass treatment campaigns Social participation Motivation of individuals Health education, Community participation in projects   Increased immunological defense via vaccination (expected, Mosquirix)

Exercises

Sierra Leone . A child weighing 20 kg suddenly develops fever. The mother decides to wait and see. The next day the general condition is poor and chloroquine is given. The day after the child is in a stupor and is brought to the local hospital. The child has a fever of 40°C. It is comatose. There is no neck stiffness. A thick smear is positive and a thin blood smear confirms parasitaemia with P. falciparum of 7 % (percentage of parasitised red blood cells). What do you do? Indonesia . A pregnant woman presents with P. vivax malaria. What do you do? Niger . A local man comes to ask your advice. He wants protection against malaria but cannot take chloroquine due to eye problems. What do you suggest? Laos . One of your colleagues develops fever every 3 days. A thin blood smear shows some trophozoites of P. malariae . Should Nivaquine and Primaquine be administered? Zambia . A mother brings you a very ill child: fever, tachypnoea. A thin blood smear shows some sparse gametocytes of P. falciparum . What do you do? North Angola . A 40-year-old man comes to ask for your help. In recent months he has felt very weak, is easily tired after normal exertion and has pain under the left ribs. You palpate an enlarged spleen and you notice pale conjunctivae. There is no leishmaniasis in the region. Blood analysis shows haemoglobin at 5 g/dl. No malignant cells are seen. There is no lymphadenopathy. There is no ascites, no previous history of haematemesis, no signs of portal hypertension, also no spider naevi, gynaecomastia or polyneuropathy. The stools contain no eggs of schistosomes. What do you think and what do you do? A Belgian development assistant arrives in Central Africa. It is his first journey abroad. Three days later he develops fever to 40°C. He was taking Nivaquine and Paludrine and thinks that he cannot have malaria. What do you think? Malawi . A patient is admitted with falciparum malaria. He is treated with quinine IV and vibramycin PO. The fever drops but his general condition deteriorates. The blood pressure drops. He remains conscious, the urine production is 700 ml per 24 hours. On the fourth day he is very seriously ill. What would you do? Nicaragua . A woman is brought in with high fever. Her general condition is not so good. Your colleague thinks that it might be severe malaria and quickly injects an ampoule of Nivaquine® IV. The woman then loses consciousness and dies shortly afterwards. What has happened? Can people who are carriers of sickle cell anaemia get malaria? Central African Republic . When the supply of tetracycline has been used up, can you then use streptomycin in the treatment of malaria, while you wait for new supplies? A Bolivian woman has lived all her life in the high mountains. One day she goes to the eastern, low-lying part of the country for 3 weeks. She takes mefloquine as prophylaxis. Five months after her return to the high mountains she develops fever. Can this be malaria? Explain your answer. Angola . A man, 50 kg, is admitted with coma and fever. There is no neck stiffness. A thin blood smear from peripheral blood shows parasitaemia of 2% Plasmodium falciparum (all trophozoites). What do you do? Congo . A 50- year-old missionary develops malaria ( P. falciparum ). She takes 3 x 2 tablets of Quinimax per day for 3 days, yet on the 4th day there is still fever and there are still parasites in the peripheral blood. What do you think? Does a thick smear have high specificity or high sensitivity for the diagnosis "malaria"? Explain. Consider a returning traveller and a person living longtime in an endemic area. Guinea . The symptom "fever" is often equated with the disease "malaria". This sometimes happens without a search for focal signs (pain upon percussion over a kidney, tachypnoea-dyspnoea, neck stiffness, etc.). For this reason chloroquine is often given indiscriminately. Give a number of arguments for and against this approach. Your answer should include frequency of infection, chronic carriers, toxicity and cost price of drugs, resistance, missing other diagnoses, frequency and importance of other diagnoses, cost price and time to obtain microscopic diagnosis, the reliability of this diagnosis, lack of motivation, poor information and training of local staff. Peru . A man has never been in a malaria zone. He is admitted with fever and a thick smear is positive for Plasmodium . Could the fact that a few weeks ago he underwent a bloody operation, be of importance? Are there other possible explanations? If this proves to be P. vivax , should primaquine be given? Gabon . A man is suffering from neck pain and takes an Artenam® tablet. The discomfort disappears. It is usual in the area to regard such discomfort as "mini malaria". What effect might this human behaviour have on the development of resistance of malaria parasites to this drug? Thailand . A German woman travels to the border with Myanmar for several days to visit the hill tribes. She stays overnight in the jungle. As malaria prevention she takes mefloquine, 250 mg per week. Two weeks after her journey she is admitted in Bangkok with confusion, hallucinations and psychotic behaviour. The physical examination is normal. She is afebrile and the glycaemia, the ionogram and the blood count are normal. The thick smear is negative. Are there probable alternative diagnoses apart from mental decompensation due to culture shock? The above-mentioned woman gives birth 6 months after her return to Europe. The baby develops massive haemolysis a few weeks after birth. The diagnosis is congenital P. vivax malaria. Do you require extra information and what treatment do you administer? Other remarks? Tanzania . A married couple travel to various wild parks. The man takes mefloquine as prevention, but the woman takes nothing against malaria. On the tenth day they both develop nausea, watery diarrhoea and abdominal cramps. The man blames this on the side effects of the drug while his wife thinks she is developing a malaria attack. What are your reflections? Rotterdam . A Dutch woman returned from Laos 6 weeks ago. Might her discomfort in the liver region be explained by the presence of hypnozoites? Amsterdam . A Dutch man returned 8 weeks ago from a world tour. Three weeks ago he was successfully treated by you for P. ovale malaria. Is the infection definitively cured? What if this was a P. vivax malaria? Can a P. vivax malaria attack still occur after this treatment, and if so how long afterwards? Is this important if one day he wants to give blood? Gabon . Would you advise a priest who was treated last year in Belgium for tropical splenomegaly syndrome, to take malaria chemoprophylaxis for a long period? Malawi . A Danish development worker has been taking chloroquine/proguanil as chemoprophylaxis for 18 months. She has never had malaria. She feels great, but is complaining of small painful and recurring ulcers on her oral mucosa. Do you need any further information? Vietnam . During the Vietnam war American soldiers took what was called the CP-pill once a week. This contained 300 mg chloroquine and 45 mg primaquine base. The results as regards malaria prophylaxis were disappointing. Can you think of some reasons for this? Southeast Asia . After working for a long time in a rural area, you notice that there are fewer malaria cases in families which keep their water buffaloes and cattle around their houses at night or where the pigs sleep under the stilts of their dwellings. Some ideas of what might be happening here? Vietnam . An important local vector is Anopheles dirus , a mosquito which is very sensitive to light and thrives well in the forest. In many places the forest has been felled and replaced by commercial plantations of fruit or rubber trees. The latex is tapped from Hevea brasiliensis very early in the morning, preferably before sunrise. The latex is placed in coconut shells which are thrown away on the ground after use. What might be the possible effect of this activity upon malaria transmission? India . The anti-malaria product Mekalfin (=sulphadoxine 500 mg + pyrimethamine 25 mg per tablet) is often sold over the counter. Each box contains 2 tablets. What might be the consequences of this on the efficacy of Fansidar on P. falciparum ? Any comments? Germany . After a quarrel a woman swallowed 15 chloroquine tablets in a suicide attempt. Two hours later she is in a coma. Her blood pressure is 80/?. Do you insert an endotracheal tube and organise oxygen and stand-by mechanical ventilation? Do you insert an IV line and give physiological fluid? Do you administer 60 mg Valium IV? Do you give adrenalin? Do you carry out gastric lavage? Thailand . An adult man weighing 65 kg (blood volume approximately 5 litres) has parasitaemia of 5% P. falciparum (not resistant to Riamet®). Say he has an RBC of 4,000,000 per µl. How many parasites does he have in his body? If he is treated with a three-day course of co-artemether and it is assumed that only artemether is responsible for destruction of the parasites in the first 3 days, how many parasites will lumefantrine remain after day three ? Tanzania . A Western couple travelled five times to a rural area in Tanzania in the last three years. It were each time trips of two weeks. Everytime they correctly took atovaquone/proguanil as antimalaria chemoprophylaxis.  On nearly each trip they had a short lasting febrile illness, which was locally considered to be malaria. This made them loose confidence in the Malarone effectiveness. They come to see you with the complain that this medicine is not good at all, and they will not take it anymore in the future. They also heard that if a person takes such a prophylaxis, the correct diagnosis of malaria is more difficult, and therefore the situation becomes more dangerous. Any comment? Congo . During a visit to the local rural hospital, you hear from many patients that they suffer from malaria, typhoid and/or amoebae, in general irrespective of the symptoms they have. What might be going on? Sarawak . A research group studies Plasmodium knowlesi . They discover that the vector Anopheles latens is attracted both to man and to monkeys, with a peak biting time around 18-19h. During a mosquito survey, the researchers are especially interested in parous female mosquitoes. Why would non-parous females not be so interesting to them? Do you agree that only parous females can be infected? Peninsular Malaysia . What do you think of the hypothesis that due to continued human expansion into the wilderness areas, monkeys are now forced to come into more contact with people than before, and that mosquitoes (e.g. Anopheles leucosphyrus group) follow them, and as such increase the risk for simian malaria transmission? You're stuck in Peru. You want to send an email but your local keyboard does not contain the arobase (arrobase) symbol @. How do you create one? Can "Alt 64" help you?

Summary

Difference between Gambian (western) and Rhodesian (eastern) trypanosomiasis Restricted to well defined regions in Africa, determined by tsetse fly vectors Early stage: transient sore, fever, oedema, lymphadenopathy, splenomegaly Late stage: central nervous system symptoms with abnormal CSF (elevated cells and protein, Mott cells, trypanosomes) Diagnosis: always try to detect the parasite Repeated thick smears, Buffy coat, Woo technique, mAECT, lymph node aspiration When screening is positive, lumbar puncture to determine stage Indirect: serology (CATT for West African form), clinical evidence Difficult to treat: Pentamidine, Suramin, Arsobal, Eflornitine, nifurtimox Current several treatment schemes, such as NECT. Fexinidazole oral short course very promising. Importance of early diagnosis and follow-up

General

African sleeping sickness is caused by infection with a unicellular parasite. There are two subspecies of these parasites: the West African or Trypanosoma brucei gambiense and the East African or T. brucei rhodesiense . They cannot be differentiated from each other on morphological grounds. There are also subtypes, which is a bit confusing. Trypanosoma brucei gambiense type 1 is genetically distinct from T. b. brucei en T. b. rhodesiense . T. b. gambiense type 2 resembles T. b. brucei.

Transmission takes place through the bite of an infected tsetse fly (genus Glossina ). Since the parasites are transmitted via tsetse saliva, they are also known as "salivaria", as opposed to Trypanosoma cruzi , which belongs to the "stercoraria" because of its transmittion via the faeces of a bug. In exceptional cases mechani­cal transmission takes place via other biting flies (tabanids). Congenita­l infections are rare. Sexual transmission seems to be extremely rare.

Geographical distribution of foci of sleeping disease (African trypanosomiasis)

African trypanosomiasis or sleeping sickness occurs exclusively in Africa. The area of distribution lies between 14° north of the Equator and 29° south of the Equator. The risk is zero or minimal in South Africa, Lesotho, Niger, Somalia and the Maghreb countries. The risk is maximal in Angola, Congo (ex-Zaire), Uganda and Sudan. It occurs in well-delineated areas within in 36 countries, with a collective population at risk of about 50 million people. There are some 200 areas where the infection is concentrated. The areas of distribution of West African and East African trypanosomiasis show little overlap. Most of the endemic countries have only one form of the disease: or the Western form, or the Eastern form. This facilitates national therapeutic guidelines. However, Sudan and Uganda is examples where both West and East African trypanosomiasis exist. Both forms have their own foci, but these are converging in Uganda. They did not yet overlap in 2005, but were then only 150 km apart. There is a narrow corridor separating the two diseases. If the transmission areas meet (as feared), it would considerably complicate diagnosis and guidelines for management of clinical cases. Countries affected anno 2010: T.b. gambiense : Guinea Conakry, Ivory Coast, Nigeria, Cameroon, Gabon, Tchad, CAR, Rep Congo, DCR, Angola, Sudan, Uganda T.b. rhodesiense : Uganda, Tanzania, Zambia, Malawi, Mocambique. Quid Kenya, Zimbabwe, Botswana? The disease is characterised by fever, lymph node swelling, general malaise and inflammation of the central nervous system. If left untreated, the disease has a fatality rate of practically 100%. There is possibly an increasing resistance to the medications used at present, which is very curious if one consideres the number of cases treated. It was estimated in 1997 that in Africa there were about 300,000 new cases per year. Some estimates for 2003 put the total number at 500,000, but this is probably an overestimate. It seems that since 2005 the total number of cases each year is systematically diminishing.

Note: Other Trypanosoma infections

Trypanosomiasis does occur in South America, but Chagas' disease which is caused by Trypanosoma cruzi is clinically very different from African sleepnig sickness. There are rare human infections with trypanosomes in India and Malaysia. They were due to accidental zoonotic infections with  Trypanosoma lewisi , a rat parasite, or T. evansi , a parasite of buffalo and cattle. A number of human infections with T. vivax and T. congolense have also been reported. Such infections are very exceptional. Herpetosoma sp. are insect parasites related to trypanosomes. A few infections in humans have been described, though such situations are extremely rare. Trypanosoma evansi causes disease (“surra”) in certain animals, such as camels, llamas, horses, buffalo, cattle, dogs, sheep and goats. There is considerable variation in the pathogenicity of different strains and the susceptibility of different host species. The disease ranges from inducing a subclinical infection, mild disease, chronic overt forms (months to years) and rapid fatal infections (esp. in horses and camels). Deer, capybara and coati can become infected and ill and may also constitute a reservoir. Animals subjected to stress such as malnutrition, pregnancy, work, … are more susceptible to disease. Successful treatment by a single dose of diminazene diaceturate has been reported in dogs.

Trypanosoma congolense is the main trypanosome infecting cattle. A few indigenous African cow breeds, such as the N'dama breed, tolerate the parasite's presence remarkably well. However, these trypanotolerant animals are not popular with farmers because they grow slowly and are small. Many farmers prefer Boran cattle, which are more beefy but susceptible to disease. Trypanosoma equiperdum causes a chronic sexual transmitted disease (“dourine”) in horses, mules and donkeys. Infections are endemic in Eastern and Southern Africa, South America, Mongolia, Russia and Kyrgyzstan.

History

African sleeping sickness has apparently been endemic in some parts of Africa for a very long time. The first historical note of this disease comes from the Arabian historian Ibn Khaldun, who described how in 1374 A.D. Sultan Mari Djata of Mali died of an illness which, according to the description, is at least compatible with this disease. "... Jaba had been smitten by the sleeping illness, a disease which frequently afflicts the inhabitants of that climate... Those afflicted are virtually never awake or alert. The sickness harms the patient and continues until he perishes... The illness persisted in Jata's humour for a characteristic of two years after which he died." The disease was observed and described in 1803 by the English doctor Thomas Masterman Winterbottom, who was working in Sierra Leone. He was struck by the frequent occurrence of swollen cervical lymph nodes in sick persons. Slave-traders also knew about this and avoided buying people with cervical lymphadenopathy.

Gabriel Valentin described the first trypanosome in 1841 while examining blood smear from a trout. In 1843 David Gruby in Paris discovered a parasite in the blood of a frog and proposed the name  Trypanosoma sanguinis . The motion of the mobile organism he saw in the blood of a frog reminded him of the action of a corkscrew (cfr etymology "trepaning", the practice of drilling holes in the skull). In 1880 Griffith Evans, a veterinary surgeon in Punjab, India, found trypanosomes in the blood of sick camels, mules and horses. These animals died of a serious disease, known locally as "surra". The 3rd Punjab Cavalry alone lost no less than 300 horses from it. The surra parasite was later given the name " Trypanosoma evansi ". 

The next major step was taken by the British army medical officer Dr. David Bruce, famous because of his work on brucellosis. After the elucidation of Malta fever in 1884, he was transferred to Zululand, South Africa. In 1894 Bruce examined the blood of sick emaciated cattle. The disease was known to the Matabele and Zulus as "nagana". Bruce saw: "...a rapidly moving object... lashing about among the red blood corpuscles. It soon became evident that the rapidly vibrating object was probably a trypanosome". When injected into horses and dogs, the blood from infected cattle caused symptoms similar to nagana. Bruce then send uninfected oxen down into the tsetse fly belt. After a couple of weeks he found in their blood the same trypanosomes as in nagana cattle. Bruce went on to show that the trypanosomes living in the blood of healthy game animals (e.g. antelopes) are the source of the disease when transmitted to domestic animals by the tsetse fly. In order to do so, he traveled down to the lower Ubombo where he shot and bled wildebeest, bushbucks, buffaloes and hyenas. Blood from these animals was inoculated into healthy dogs, which subsequently became infected. In 1899 the trypanosome was named after Bruce, receiving the name Trypanosoma brucei . After the Boer War (1899) he continued his investigation in 1903-1906 in, among other places, Uganda. Prior to 1890 sleeping sickness was unknown in Uganda. The next breakthrough with respect to the etiology of human trypanosomiasis came in 1901, when Dr. Forde discovered a motile parasite in the blood of a sick captain of a river boat in Gambia. He was unable to identify the parasite and the patient was repatriated to England. In 1902 the parasite was identified as a trypanosome by Dutton. Because of the patient's origin, he gave the parasite the name T. gambiense . In 1903 Aldo Castellani found trypanosomes in the cerebrospinal fluid of persons who had died of East African sleeping sickness. This was confirmed by Bruce, by examination of cerebrospinal fluid from 34 patients, finding the parasite in 20 specimens. He contacted numerous missionaries and government employees throughout Uganda and asked them to collect as many tsetse flies as possible and send them to Entebbe. The idea was to plot the distribution of the insects and sleeping disease to determine any overlap in distribution. There was clear sympatry as the two territories coincided. The introduction of the disease has been attributed to the entry of Emin Pasha (the German-born Eduard Carl Oscar Theodor Schnitzer) and his 10,000 followers who arrived from the Congo, a territory where the disease was prevalent. In 1909-1910 Stephens and Fantham described Trypanosoma rhodesiense (after the former Rhodesia). The taxonomy was subsequently modified. Three subspecies of Trypanosoma brucei are now described: Trypanosoma brucei brucei (not pathogenic for man), Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense .

Nagana - Trypanosomiasis. General wasting in cattle. Copyright ITM

Apart from the great rinderpest or cattle plague (viral, resembling a virus related to measles) and nagana epidemics in South Africa at the end of the nineteenth century, there were also epidemics of human trypanosomiasis. It is possible that the great epidemics have arisen through the conflict between two different ecosystems (the natural one with its original vegetation, fauna and vectors and the artificial human ecosystem with its animals, exploitations and cultivations). From 1896 to 1906 there was a major epidemic in the Congo with more than 500,000 fatalities. Another epidemic raged around Lake Victoria and killed two thirds of the local population (between 1900 and 1920 about 250,000 people died of what was probably a T.b. rhodesiense epidemic). Between 1924 and 1926 trypanosomiasis was a major cause of death in Central Cameroon. In the 1980s there was a flare-up in Uganda with many thousan­ds of cases. After an initial phase of decline in the middle of the 20th century, the disease (principally T. b. gambiense ) has now regained much of its former range (Congo, Sudan, Uganda, Angola).

The connection between the terrible rinderpest epidemic and sleeping sickness has been extensively investigated. Rural East Africa and the Sahel before 1887 were dominated by vast grasslands with enormous numbers of cattle. Grazing maintained the grassland biotopes. In 1887 rinderpest was introduced into the continent for the first time via Eritrea. The indigenous fauna had no resistance. This viral disease, related to measles and distemper, had an enormous mortality rate in cattle and in wild (cloven-hoofed) animals. The epidemic spread with lightning speed, resulting in the collapse of practically all cattle stock (for example: Botswana 1891: 400,000 head of cattle; only 20,000 in 1892). The grasslands quickly disappeared and the resulting bush with its thorn bushes and small trees was ideal for tsetse flies resulting in an increase in sleeping sickness. The wild animal stock recovered more quickly than the domestic cattle. An alternative hypothesis states that after the massive die-off of cattle, livestock restocking occurred. The large-scale movement of these cattle could be linked to the introduction of the disease in new areas.

Parasite

Parasite, general

Trypanosoma brucei gambiense in a thin blood smear. Determination of the subspecies (gambiense or rhodesiense) cannot be performed with morphological criteria. Copyright ITM

There are numerous organisms related to trypanosomes that live in the sap of plants ( Phytomonas for example). All kinds of insects drink plant sap. Such parasites were possibly ingested by various insects. It is assumed that later, in the course of evolution, when insects were able to feed on the blood of land animals, a transmission to these animals took place and that Trypanosoma sp. developed.  The parasite's genome has been sequenced. In general among trypanosomes, one can distinguish several morphological forms: amastigote: spherical form without a flagella. Such a stage is present in T. cruzi (see Chagas' disease) epimastigote: fusiform 20-40 µm long with an anterior placed kinetoplast, in front of the nucleus, i.e. on the same side as the flagella is pointing. This stage occurs in the tsetse fly. trypomastigote: the kinetoplast is located behind the nucleus. The parasites are pleomorphic in human blood. Some are elongated and slender ("slender trypomastigotes") and others are shorter and stumpy. Reproduction in man occurs via longitudinal binary cleavage every 7 hours.

Parasite, kinetoplast

The parasite has only one nucleus, is elongated, contains a giant mitochondrion and has a single flagellum. At the base of the flagellum is the basal body. This lies adjacent to the kinetoplast. The latter is a compact DNA (deoxyribonucleic acid) structure, located in the very long mitochondrion. This mitochondrion is almost as long as the entire trypanosome. The name of the Order to which the parasite belongs - Kinetoplastida - refers to this organelle. Between the basal body and the flagellum there is an undulating membrane which is required for motility. The microscopic recognition of all these structures is important, for example when in doubt about a suspect structure in a microscopy preparation. In a buffy coat and/or a fresh blood slide the parasites can be seen to move rapidly ("trypanon" = to drill or bore and "soma" = body). In the form of the parasite such as it occurs in man (trypomastigote), the kinetoplast lies in a posterior position and the flagellum points towards the front, rather like a bowsprit on a large sailing vessel. The parasite occurs in the salivary glands of the tsetse fly as an epimastigote (kinetoplast located just in front of the nucleus). The varying location of the kinetoplast is possibly related to different metabolic requirements in the various hosts.

Structure of a trypanosome. Notice the large mitochondrion. Copyright ITM

Trypanosoma gambiense in cerebrospinal fluid. Copyright ITM

The genome of T. brucei was sequenced and published in Science in July 2005. The DNA in the kinetoplast (kDNA) stains like that of the nucleus (recognizable on a smear). The structure of the DNA in this kinetoplast is very complex. There are numerous (about 40) large DNA loops ("maxicircles") and even more (some 5,000-10,000) small DNA loops ("minicircles"). These form a gigantic tangle. For replication the parasite requires a specific "disentanglement enzyme" (type II topoisomerase; this enzyme cuts both DNA strands, as opposed to type I). This enzyme could be a target in the development of new drugs.

Several mitochondrial genes appear to be incomplete. In 1986 it was discovered that "editing" of the genetic information takes place in pre-messenger RNA (ribonucleic acid) after transcription of the maxicircle-DNA. Certain RNA-bases (uridines) are removed or inserted in order to form a "mature" mRNA. In 1990 it was discovered that very small, so-called guide-RNA or gRNA molecules play a major role in this editing. Most gRNAs are coded in the minicircles. After the discovery of kRNA-editing, RNA-editing was also found in other organisms.

One hypothesis is that kRNA-editing has a regulatory function in gene expression and in mitochondrial metabolism. Some transcripts are found principally in the procyclic (insect) forms, others mainly in the vertebrate blood stages. It seems there is a cyclic activation and repression of various metabolic pathways in the mitochondria, depending on the host. There are significant differences in the energy production of the parasite, according to its point in the life cycle. Glucose metabolism of the parasite while in the vertebrate bloodstream is anaerobic, i.e. via glycolysis, with a non-functional mitochondrion. The mitochondria of the parasites in the bloodstream contain no cytochromes and lack various enzymes of the Krebs cycle. In glucose-rich mammalian blood the parasite uses principally anaerobic glycolysis in glycosomes. These subcellular organelles contain the first 7 enzymes of glycolysis and are unique to the Kinetoplastida. In most Eukaryota glycolysis takes place in the cytosol. In the gut of the tsetse fly, the metabolism is aerobic, with a functional mitochondrion. In the insect, which lacks glucose, the parasite's energy comes mainly from aminoacids (e.g. proline). [In many insects, such as tsetse flies and sandflies, proline is a highly abundant energy source used in flight muscles]. Although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. There is, clearly, insufficient understanding of the details. If a glycosome inhibitor could be developed, this might eventually open up new therapeutic possibilities.

Another illustration of the importance of the mitochondrion in trypanosomes is found in Trypanosoma evansi . This trypanosome causes "surra", a disease in camels and horses. The kDNA of this parasite has a different structure in its mitochondrion (for example, it has no maxicircles). As the maxicircles play a crucial role in the functioning of the mitochondrion, it is reasonable to assume that T. evansi cannot go through a maturation cycle in an insect. A consequence of this may be that a change of host cannot occur here. Indeed, the parasite appears to be transmitted only by a mechanical vector, e.g. biting flies (Tabanidae) or vampire bats.

More than a billion years ago the ancestor of trypanosomes probably merged with a type of green algae. This symbiosis would have enabled it to harness the sun's energy. This would have had a tremendous advantage. When trypanosomes became parasites, they no longer needed to photosynthesize. The symbiont degenerated and some of its genes passed to the trypanosome's genome. At this moment, the genes are vital for the survival of trypanosomes. The leftover plant genes were found by analysing the genomes of T. brucei . So far 16 genes have been found that have their closest relatives in plants. Researchers suspect that more wait to be discovered. Plants use the equivalent genes to photosynthesize, using carbon dioxide to make sugars. Trypanosomes use them to break sugars down, in a unique cellular system. Several microorganisms, including the malaria parasite, seem to have absorbed a symbiont in the past. Both the cellular powerhouses called mitochondria and chloroplasts, which plants use to turn sunlight into chemical energy, are thought to have originally been free-living bacteria.

Parasite, polyamine metabolism

The parasites defend themselves against oxidative stress by means of a polyamine conjugate, trypanothione (two glutathione molecules linked by spermidine). For the parasite, this substance has the same function as glutathione has for man. Studies have been carried out to determine whether trypanothione can be a target for drugs. The polyamines spermidine, spermine and putrescine have various functions in the cell (including synthesis of trypanothione). Polyamines are organic substances that contain two or more amino groups. DFMO (di-fluoro-methyl-ornithine; Eflornitine®) is a therapeutic substance that interferes in the polyamine metabolism of the parasite. DFMO inhibits the conversion of ornithine into putrescine, a precursor of spermidine. Ultimately, the synthesis of trypanothione is disturbed and the parasite is damaged. Trypanosomatids lack catalase (catalase helps to convert 2 H 2 O 2 into 2 H 2 O and O 2 ) and are very dependent on trypanothione-dependent peroxidases for removal of peroxides.

Parasite, sex?

While in the human host, the parasites are diploid. The parasites replicate in humans by asexual mitosis. Diploid and polyploid forms can be found in tsetse flies. Experimental arguments for meiosis and a possible sexual reproduction in T. brucei were first proposed in 1986. In the laboratory tsetse flies were infected with 2 different clones, after which hybrid parasites were isolated, which indicates exchange of genetic material. This could be important for a better understanding of the natural parasite populations, e.g. via the various iso-enzyme patterns that occur in nature. Even if these laboratory data were confirmed, it remains an open question how important this is in nature. Further studies are required.

Parasite, antigenic variation

The notion of antigenic variation in African trypanosomes has been around for a long time. Early investigators would isolate trypanosomes and serum from an animal early in the course of an infection, and then again later during the same infection. Early antiserum would kill the initial strain of trypanosome, but did not affect the trypanosome strain isolated later in the infection. It was apparent that the trypanosome population changed over time. When the parasite is present in an individual, it is covered with a thick monotonous layer of a single type of glycoprotein, VSG or Variant Surface Glycoprotein. These glycoproteins consist of 400-500 aminoacids and various saccharide groups. They are anchored into the cell membrane with a so-called GPI-anchor (glycosyl-phosphatidyl-inositol). Ten percent of the proteins of the trypanosome consist of VSG, estimated at about 10 million molecules per parasite. The entire VSG surface of a trypanosome is recycled every seven minutes by a process of VSG endocytosis and exocytosis. When the parasite is transferred to the tsetse fly, the VSG coating disappears within 4 hours and is replaced by an invariant glycoprotein ("procycline" or PARP). Biochemically and antigenically, the PARP coat becomes the same no matter what variant type is ingested with the tsetse's blood meal. After the parasite has completed its cycle in the fly and arrives into the latter's salivary glands, the VSG coating reappears. The VSG coating is of vital importance for the parasite when it is in the vertebrate host. This explains why only metacyclic trypanosomes (the mature forms in the salivary glands of the insect) are infectious. When an antigenically homogeneous population of parasites is in the human body, antibodies against the VSG of this population are produced. The immune system lyses the parasites which is accompanied by fever. Infections with trypanosomes would be cured quickly, if the parasite population could not constantly change its surface antigens. The switch of VSGs happens about once every 100 cell divisions. The appearance of the VSGs in the infected host is not completely random. Certain VSG appear early while others are expressed later in infection. Compounding the problem of the order of appearance is the immunosuppression of the host by the trypanosomes. The more immunosuppressed the host is, the easier it is for minor variants to hang around.

Trypanosoma gambiense displays different surface antigens in successive populations. Copyright ITM

The parasite has about 800 genes that code for different VSGs and thus has a vast repertoire of surface antigens. Most of these genes are located on some hundred minichromosomes in the nucleus of the parasite. The parasite also has about twenty chromosomes of "normal" size. These do not condense during mitosis. At any one time only one VSG gene per parasite is active.  Invariant antigens on the surface of the trypanosome are shielded by a dense coat of about 10 million copies of a single type VSG. A few trypanosomes in a population have a different VSG (heterologous variants). After destruction of the first, dominant population by the immune system, the heterologous parasites increase in number until the variant VSG has induced antibodies and a new cycle of destruction begins. A third population of minority variants then emerges. This antigenic variation is a very important factor in the development of the disease and explains various symptoms (including its chronic course, fluctuating parasitaemia and fever episodes).

The order in which antigenic variants appear is partially pre-determined. Alteration of surface antigens is not induced by antibodies but occurs spontaneously. The genetic mechanism that the parasite uses for this is very complex. The gene that is to be expressed is duplicated from a locus on one of the chromosomes to a subtelomeric locus (close to one end of a chromosome). The mRNA originating from the gene on this latter locus is subsequently coupled ("trans-splicing") to a small mRNA fragment that is coded elsewhere. This small fragment (mini-exon) is the same for all VSGs. The mechanism for mutually exclusive activation of the VSG genes is still not known. An unusual DNA-pyrimidine base ("J"; compare with A, T, G and C) is present in small quantities in the trypanosome genome and occurs more frequently in the telomeres. The unusual nucleotide is present only in the blood form, not in the procyclic form. The significance of this is not yet known.

It is important for the parasite to keep the population in the host as homogeneous as possible in order to use the VSGs economically. However, when parasites infect a host, diversity is advantageous. Hence, some 20 different variants can be present in the saliva of a fly. This is a major impediment to make a vaccine. If the parasite would revert to the same basic antigen, the prospects for a vaccine would be much better. Any one of these variants is capable of infecting a host provided there are no antibodies present from a previous infection. If the host is immunologically naive to trypanosome antigens, the same VSG eventually dominates the first parasitic population.

Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination (ectopic rather than merely between chromosome homologs). Of 806 analyzed, only 7% were fully functional (i.e. encode all recognizable features of known functional VSGs), 9% were atypical, 66% were full-length pseudogenes with frameshifts and/or in-frame stop-codons, and 18% were gene fragments. VSGs differ significantly in the hypervariable N-terminal domain, which is exposed to the immune system (outside of cell). The C-terminal domain is more conserved and is buried in the coat. Apart from the above mentioned antigenic variation, it is possible that other mechanisms play a role in the variable parasite numbers in human blood. Maybe quorum sensing and mechanisms which rely on induced apoptosis might play a role, but much more study is needed.

Parasite, flagellar pocket

In their mammalian host the parasites have a simple metabolism. They use several biochemical substrates of their host, but this poses a practical problem. Since the parasite is coated with a thick monotonous layer of glycoproteins, how can it absorb the necessary metabolites? At the base of the flagellum there is a small invagination ("flagellar pocket"). This site is accessible to macromolecules, but not to macrophages. The flagellar pocket membrane accounts for about 1 per cent of the surface area of the parasite, but in the bloodstream form at least, it exhibits extremely high levels of cross-membrane traffic, far higher that those seen in multicellular organisms. By contrast, uptake into the cell is much lower in the insect stages of the parasite. Nutrients are taken up and molecules are secreted in the flagellar pocket. The trypanosome takes up material by invagination of the surface membrane and formation of vacuoles (endocytosis). In this way, the pocket functions as a kind of "mouth". Secretion is achieved by the reverse mechanism: vacuoles fuse with the surface membrane and discharge their contents into the outside world (exocytosis). The parasite also uses the flagellar pocket to take up and degrade antibodies. Within minutes of antibody binding, the parasite cleans its surface by transferring the bound antibody to the flagellar pocket where it is rapidly internalized and degraded. Another notable property of the flagellar pocket is its ability to route different proteins to different parts of the cell surface. VSGs, for example, account for 90 per cent of the parasite's surface protein. They are connected to the cell surface via a lipid-based anchor (a glycosyl-phosphatidylinositol or GPI-anchor). But GPIs also anchor other proteins, such as the transferrin receptor, which is restricted to the flagellar pocket. How is this possible? Usually, the destination of proteins depends on short "sorting" sequences within them, but these do not seem to be present in the GPI-anchored proteins. This selective retention in the flagellar pocket raises the possibility that a luminal-based sorting mechanism might operate in the flagellar pocket.

This invagination, since it is the site of an important interaction with the host, may turn out a weak spot of the parasite which can be exploited therapeutically. Trypanosomes have no receptors for the uptake of albumin, but do have receptors for the uptake of LDL (Low Density Lipoprotein). LDL is essential for the parasite. Suramin binds to LDL. This may explain the action of this medicament (interference with the normal LDL uptake in the flagellar pocket) and perhaps also explain the concentration of this molecule in the parasite via this receptor-mediated mechanism.

Parasite, lysis by serum

T. b. brucei cannot infect humans since it undergoes lysis in human serum. Apolipoprotein L-I is a high-density lipoprotein (HDL) component able to kill Trypanosoma brucei brucei by forming pores in the lysosomal membrane of the parasite. Persons with HDL-deficiency (Tangier disease, analphalipoproteinaemia) do not produce this trypanolytic factor and are suceptible to infection with this zoonotic parasite. Haptaglobin-related protein (hpr) allows fast uptake of the carrier HDL particles, probably through their binding to surface receptor of the parasite.  

The hpr -gene is present in the genomes of humans, Old World monkeys and chimpanzees. Of these primates, only chimpanzee serum is not trypanolytic. A functional Hpr is not synthetised by the chimpanzee because the chimp hpr -gene has a single-base deletion that results in a frameshift. Hpr is 94% identical to haptaglobin.

Humans are normally resistant to infection with Trypanosoma evansi . Certain mutations in apolipoprotein L-1 can render a person susceptible to this parasite. Such an infection occured in India, a country outside the traditional trypanosomiasis belt.

 

Note: SRA-protein

The gene SRA encodes a "serum resistance associated" protein. This gene is expressed in T. b. rhodesiense but not in T. b. brucei . The expressed gene can be used to identify rhodesiense group trypanosomes infectious to human beings that are present in animal populations. T. b. gambiense does not contain the SRA gene, which suggests that this organism resists lysis by human serum through a different mechanism.

Parasite, plastid-related genes

Trypanosomes harbour numerous genes which point to apparent common ancestry with bacteria and plants. Many of these horizontally (lateral) acquired genes seem to function in the glycosome. The glycosome is being studied as a possible therapeutic drug target. How did the trypanosomes acquire these "foreign" genes? Trypanosomes fall within the Phylum Euglenozoa, which includes the beautiful euglenid algae. These algae have chloroplasts, green plastids surrounded by three membranes. This is thought to reflect an engulfed eukaryotic algal endosymbiont similar to the apicoplast in Plasmodium parasites. Chloroplasts themselves are thought to have arisen form an endosymbiotic merger of a cyanobacterial prokaryote with an eukaryote. Although there is no evidence of a plastid in trypanosomes, the presence of such genes suggest that lateral gene transfer from some photosynthetic organism(s) occurred in the distant past. It is possible that the ancestors of the currect trypanosomes had plastids, but that they lost their passengers during evolution. There are known examples of plastid loss, e.g. in oomycetes and possibly in ciliates. Bodonids are free-living bacteriovorous kinetoplastids. Some kinetoplastids contain their own bacterial endosymbionts (e.g. Crithidia oncopelti ). It is conceivable that independent endosymbionts could have been an alternative source for some of the "foreign" genes. If further phylogenetic analyses of lateral transferred genes consistently point to a single genetic source, the endosymbiont hypothesis will be supported. If the genes derive from multiple independent lineages, multiple independent gene transfers from e.g. ingested food items will be more probable.

Life cycle of trypanosomes

Anatomy Glossina (tsetse fly), vector of African sleeping disease or trypanosomiasis. Copyright ITM

It was initially suspected that transmission was purely mechanical (as in the case of T. evansi, which is transferred by biting flies or when carnivores bite their prey and get wounded in the mouth). In 1909 the German physician Dr. Kleine carried out transmission experiments from human patients to apes and monkeys. He, like Bruce, showed that infected tsetse flies were vectors. However, he also discovered that the parasite had to be present in the fly for a minimum period before it could cause infection. He subsequently showed that only the metacyclic forms in the salivary glands of the insect were infectious. When a tsetse fly bites an infected human patient, it can ingest parasites. After their arrival in the insect's stomach, many (99%) of the parasites die, possibily because of interactions with specific lectins in the insect's stomach. The few parasites which survive become slender ("procyclic trypanosome"). They must then manage to get into the salivary glands. It was initially thought that there were two ways for the parasites to do this. The first hypothesis was that they first pierce the intestinal wall and then penetrate directly into the salivary glands (this is now no longer accepted as a hypothesis). The second hypothesis states that they undertake a complex migration in the insect. They would then migrate distally in the intestine around the posterior free extremity of the peritrophic membrane, then swim back between the intestinal wall and the peritrophic membrane. When they reach the level of the proventricular valve, they penetrate the peritrophic membrane that is being formed and swim up the oesophagus into the proboscis. Finally, they penetrate the hypopharynx and pass into the salivary glands. During this migration they change morphologically into "mesocyclic forms". In the salivary glands they replicate as "epimastigotes", which undergo a very special asymmetrical cell division. Afterwards, the parasite become infectious as "metacyclic trypanosomes". As well as being important in motility, the trypanosome flagellum is used by the parasite to attach itself to the tsetse salivary gland - without this, it would be quickly washed out. Infected tsetse flies have 100,000 to 300,000 parasites.

After about 3 weeks the fly can transmit parasites into its next victim when taking a blood meal. In man, the parasites multiply outside the cells, namely in blood and lymph. They reach the cerebrospinal fluid, most likely through areas where the blood-brain barrier is absent, such as the area postrema (vomiting centre at the base of the 4rd ventricle) or the plexus choroideus. Some can survive in certain areas (plexus choroideus in the brain), from whence they can cause further flare-ups. It is possible that the testis in men form another sanctuary from where the disease might flare-up (at present only data from mice are available). Note: cerebrospinal fluid circulation CSF is produced by the choroid plexus, which is located in the lateral, third and fourth ventricles. CSF flows from the lateral ventricle to the third ventricle through the interventricular foramen of Monro. The third ventricle and fourth ventricle are connected to each other by the cerebral aqueduct of Sylvius. The CSF flows out of the fourth ventricle into the subarachnoid space through the lateral foraminae of Luschka and the medial foramen of Magendie. Absorption of the CSF back into the blood takes place in the superior sagittal sinus through the arachnoid villi.

Reservoir

Akagera-park in Rwanda. Tsetse flies which transmit East African sleeping disease are common here. Trypanosoma rhodesiense infects antelopes. Copyright ITM

There is a definite animal reservoir for T. b. rhodesiense  in cattle, bushbuck and various antelopes. Buffaloes, antelopes and warthogs often cover themselves in mud in tsetse fly areas to avoid being bitten. The existence of an animal resevoir for T. b. gambiense  was previously unknown, but it is now accepted (dogs, pigs). The epidemiological significance of this reservoir is still not well known. About 5% of the life stock in Africa is naturally trypanotolerant or trypanoresistant. The native Ndama, Baoulé, Muturu and Laguna humpless cattle races are examples. Trypanotolerance is a dynamic state of infection, where pathogenic trypanosomes are present, but the natural immune system keeps them in check without any detrimental effects. Trypanoresistance has a genetic basis, but is different from immunity. Trypanosusceptibility is more prevalent in imported domestic species than in wild species.

Vector

Vector, general

Tsetse fly, Glossina palpalis . Vector of West African trypanosomiasis (sleeping disease). Copyright ITM.

Tsetse fly, Glossina morsitans . Vector of East African trypanosomiasis (sleeping disease). Copyright ITM.

Tsetse fly. Glossina pallidipes female, vector of West African trypanosomiasis (sleeping disease). Notice the hatchet cells in the wings and the forward projecting mouthparts. Copyright ITM

Tsetse fly. Glossina pallidipes male. Vector of trypanosomiasis, sleeping disease. Copyright ITM.

Anatomy Glossina (tsetse fly), vector of African sleeping disease or trypanosomiasis. Copyright ITM

Tsetse fly ( Glossina ), vector of African trypanosomiasis. Notice the hatchet cell in the wings.

Tsetse fly. Glossina morsitans . Vector of trypanosomiasis, sleeping disease. Copyright ITM.

Tsetse fly. Glossina pallidipes male, vector of West African trypanosomiasis. Both male and female bite and suck blood and both can transmit sleeping disease. Copyright ITM

Pupa of tsetse fly, Glossina sp. Vector of trypanosomiasis, sleeping disease. Notice the two polypneustic lobes which have a respiratory function. Copyright ITM.

Pupa of tsetse fly, Glossina morsitans . Vector of trypanosomiasis, sleeping disease. Notice the two polypneustic lobes which have a respiratory function. Copyright ITM.

Geographic spread Tsetse flies ( Glossina sp.) are blood-sucking insects that occur only in Africa (and the Gisan oasis in Saudi Arabia). Four different species of fossil flies were discovered in 20 million-year-old mudrock in Colorado, USA, indicating that the insects once existed in North America. The name tsetse descends from the Tswana language. This name was also used by the Matabele and Zulus, and refers to the sound that the insects make. An English reporter in Southern Africa at the end of the 19th century adopted this name when he wrote about a fly which attacked horses and cows.

Species and subspecies The insects have a prominent elongated mouthparts (proboscis), which explains their scientific generic name ("glossus": tongue). Tsetse flies have typical wing veins, with a "hatchet cell" in the middle. When resting, they fold their wings over their back like a closed pair of scissors. Other flies hold their wings more to the side. There are 31 species and subspecies, but less than half this number are vectors of human trypanosomiasis. The genus Glossina is now divided into three subgenera: the fusca - group (subgenus Austenina ) the palpalis - group (subgenus Nemorhina )  the morsitans -group (subgenus Glossina ) In the fusca -group, there are 15 large-sized species. These insects live in tropical rainforests, gallery forests and mixed forest-savanna areas. They are not important for human disease. The morsitans -group contains 7 medium-sized species that prefer wooded savanna, dense thickets and to a lesser degree open woodland forests and dry bushland. These insects are widely distributed over the East African savanna. They are more zoophilic (they prefer animals to man) and the infection is a zoonosis. Important hosts are the bushbuck, hartebeest and cattle, but also animals such as lions and hyenas. It is possible that the striped pattern of the zebra evolved to diminish bites by this flies, since striped patterns are avoided by the insects (aternating black and white stripes seem to disturb the optical orientation of the flies, but this not explain why only zebras evolved this color pattern). The morsitans group is important in human Eastern African trypanosomiasis and in nagana. The following species belong to this genus: G. austeni, G. longipalpis, G. morsitans centralis, G. morsitans morsitans, G. morsitans submorsitans, G. pallidipes, G. swynnertoni. The palpalis -group contains 9 species. They are rather small insects. These flies prefer dense vegetation in humid areas (e.g. on riverbanks, gallery forests). Their habitat should have exactly the right conditions of humidity, warmth and light intensity, and there should be a blood supply (nearby animals or humans). Humans are frequently bitten when working/standing close to the water's edge. The flies can also be found in cocoa, coffee, mango and banana plantations. They are vectors of human Western African trypanosomiasis and nagana. The following species belong to this genus: G. fuscipes fuscipes, G. fuscipes martinii, G. fuscipes quanzensis, G. pallicera newsteadi, G. pallicera pallicera, C. caliginea, G. palpalis palpalis, G. palpalis gambiensis, G. tachynoides. Fly genitals One of the elements which taxonomists use to distinguish between these species and subspecies, is the structure of the genitals. The male reproductive organ is situated at the tip of the abdomen. The basal part, or epandrium, protects the phallic complex (phallotheca, chelicera and phallosome). Hairy plates known as hectors serve to clasp the female during copulation. The male internal reproductive system includes two testicles and two deferent ducts which meet before joining the phallic complex. Accessory glands secrete fluid into the deferent ducts. This fluid participates in the formation of spermatophores, packets of spermatozoa that are inserted into the female during copulation. Once transferred, the spermatozoa in the spermatophora will survive for up to 200 days, more than the average lifespan of a female fly. This is important since a female tsetse fly will copulate only once in her lifetime. This fact can be exploited in vector control. Copulation between dissimilar species is not succesful, partly because of the structure of the external genitals. The detailed structure of the genitals helps to prevent sterile hybrids from arising. This is not unusual in the insect world.  In females, there are no external projecting organs. The exact structure of the posterior genital plates helps to identify a certain vector. The female internal reproductive system includes two spermatheca, hollow organs where the female stores the sperm for her lifetime. These spermatheca are connected to the proximal uterus via two small canals (spermathecal ducts). There are two ovaries, each of which contains an internal and external ovariole. The paired oviducts meet in a joint oviduct which leads to the uterus. A branched uterine milk gland produces nutrition for the larva during its intrauterine life. This "milk" will reach the larva via a uterine gland duct. Reproduction Young males, a week old at most, are attracted by sexual pheromones emitted by 3-4 day-old females. Female tsetse mate only once in their life. Copulation time varies between species, from 30 minutes to 3 hours. The male injects a spermatophore filled with spermatozoa into the female genital opening. The spermatozoa migrate up towards the female's spermathecae to be stored. The empty spermatophore is ejected through the vagina several hours later. The reproduction is exceptional in that the insect is viviparous or live-bearing (no eggs are laid in the environment). The first ovulation occurs a few days after mating. The ovaries function in a unique way. Each ovary contains two ovarioles. The four ovarioles are alternately active every 10 days in a precise order: right internal ovariole, left internal ovariole, right external ovariole, left external ovariole. This pattern is repeated until the fly dies. The first ovulation occurs when the female is around 10 days old. The next egg is released a few hours after delivery of the previous larva. After each ovulation, follicular debris is shed at the base of the functional ovariole. This physiological scar is used to determine the age of females during fly surveys. The active ovariole produces a follicle which passes into the uterus where it is fertilized. Only 1 egg is fertilized at a time. Three days after fertilization, a first instar larva hatches from the egg. It moults into a second instar larva 36 hours later. It moult again about 50 hours later into a third instar. In total, the larva resides 8-10 days in the uterus. The larva feeds on the secretions of the milk glands that open into the uterus. Part of the food is used for metabolism, part is stored as a reserve food supply for use when it pupates and when it is a young adult before having the first blood meal. The secretions from these milk glands can only continue as long as the mother fly feeds regularly on blood. The pregnant fly searches for a shady spot with loose soil to give birth. The first larva is deposited in the soil when the female is 17-20 days old. "Delivery" takes up to an hour. About 30" after giving birth, the new mother emits a high-pitched 2 KHz sound by vibrating its wings for a few minutes. It is not clear yet why the new mother "sings a short song" after giving birth. After the larva is deposited, it digs itself in very rapidly. Thereafter, the female deposits a new larva once every 10 days on average until her death. In nature, a female tsetse fly deposits only about 6 to 8, exceptionally 12 larvae during her lifetime. Because the larva quickly digs itself in, it is not vulnerable to control measures. Control of the insect must thus be aimed at the adult animals. After digging in to a depth of 2-8 cm, the larva becomes immobile while it molts a third time. Pupation takes place within about 15 minutes.  The tegument hardens, turns brown or black and becomes opaque. Metamorphosis occurs within this shell. The pupa, like the larva, has two typical posterior projections that have a respiratory function (polypneustic lobes). Some 3-13 weeks later the new adult fly emerges. Higher temperatures shorten the pupation period. The larval shell will rupture along a characteristic circular groove. This groove gave the name to the taxonomic suborder to which these insects belong (Cyclorrhapha). The young adult breaks this shell via a projection on its head, the ptilinium. The young adult fly has a soft cuticle and is therefore known as a teneral fly (teneral = soft). The first blood meal generally takes place 24-72 hours after emergence. This first meal is very important in transmission of trypanosomiasis, because it is during this first meal that flies can become infected. If not infected early in life, they will become resistant to infection (see further). Mortality of the larval stages is very low, although abortions are more common in the hot dry season, compared with the rainy season. Mortality of pupae is estimated around 1% per day, and is influenced by flooding, bushfires, soil compaction through trampling by animals, predatory insects and parasites. Mortality of adults is around 3% per day. The maximum growth rate of a fly population is around 2% per day. The whole population can double in size in 35 days when conditions are optimal. The average life span of a female tsetse fly is 2-3 months, and 1 month for males. Life span is influenced by climatic conditions: it is maximal during the rainy season, shorter in the cool season and minimal in the hot dry season. Example: for G. morsitans morsitans the life span is resp 160 days, 110 days and 50 days under these conditions. Record longevity rates are 9 months for female G. palpalis (Senegal, Ivory Coast), 173 days for G. pallidipes and 228 days for G. morsitans in Zimbabwe. The fact than on average females live longer than males means that in fly surveys, the majority of insects will be female. In G. mostitans submorsitans , the sex ratio is distorted for genetic reasons. Depending upon the type of X-chromosome, certain males only sire females, while other sire equal number of sons and daughters. Sensory system Tsetse flies see well with their compound eyes. A prey can be located up to 140 meters. It is attracted to blue and avoids green, yellow and red. Shade contrast is important. This information is important for the design of fly traps. Flies can detect sound, such as the buzzing of an asilid predator, the sound of a host or a bushfire. They have a sense of smell, and each tsetse species has its own preferences. They detect odour gradients (ketones, aldehydes, phenols, CO 2 ). The smell of infected cattle is more attractive to them than the smell of healthy cattle. This increases the probablility of transmission. Females can detect the smell emitted by the anus of a larva that is about to pupate. This is very attractive for nearby females about to depost their own larva. This odour signal fosters the concentration of larvae at sites where they can easily burrow into the soil. When they land on a potential host, the fly analyses the sweat, blood and lymph in the skin. Chemoreceptors which are considered analogue to taste receptors are located on the mouthparts and the legs. As all flies, they have short antennae with 3 segments. This characteristic is used in taxonomic identification. Flies are also known as Brachycera ("short horns"), as opposed to mosquitoes which have antennae with more than 6 shackles: Nematocera (thread-like horns). The distal segment of the tsetse fly's antenna resembles a brush and is known as the arista. It has a sensory function. Biting Both male and female tsetse flies take blood every 3-4 days. After landing on a host, the fly will lower its proboscis to a vertical position, and stab with a rocking motion of the body. The rough dentate part of the proboscis saws through the tissues. Bites of forest flies are less painful than those of savanna species. During a bite, it injects saliva which contains anticoagulant proteins, and sometimes trypanosomes. Feeding time ranges from 20 to 25 seconds. In a single meal, 5-80 mg (max 155 mg) of blood in taken up and stored in the crop. Only a small quantity goes directly to the midgut. A hungry fly can take up a bloodmeal greater than its fasting weight. When satiated, the fly heads to a roosting site to digest at leisure.

Digestive system Two coiled and rather long (15 mm) salivary glands are found in the upper abdominal cavity. They are linked to the mouthparts by two canals that pass through the thorax. When a tsetse fly sucks blood, the blood enters the mouthparts (proboscis). From there it passes to the pharynx and the esophagus. Afterwards, there is a bifurcation, one branch leading to the midgut, the other to a crop, a kind of diverticulum located in the anterior abdomen which functions as a storage organ. This bifurcation is a bit dilated and is surrounded by a muscle. This is known as the proventriculus. Most of the blood will initially be stored in the crop. About 5-10 minutes after the meal, when the fly is safely away from the host on which is was feeding, the crop contracts and the blood will regurgitate from the crop via the proventriculus to the midgut. After 30 minutes the crop is empty. Like many insects, tsetse flies secrete a "peritrophic membrane" ["peri" + "trophe" = "around food"] in the intestine. This secretion takes place at the transition from the anterior intestine to the midgut (proventricular valve). The membrane consists of chitin and a glycoprotein matrix and functions as a very fine sieve. Small molecules can pass through it, but bacteria or large food particles cannot. It offers some mechanical protection to the middle intestinal cells (the intestine contains no lubricating mucus producing cells). An additional function is the compartmentalisation of the digestive process. A first portion of the food is found in the endoperitrophic cavity, a second portion in the ectoperitrophic cavity. Small food molecules diffuse through the peritrophic membrane. Absorption takes place through the surface of the intestinal cells. It was previously assumed that trypanosomes could not pass through the peritrophic membrane, but this assumption has become increasingly doubtful.

Resting areas Adult tsetse flies are airborne for short periods and rest for the remaining time. On average they cover 200-300 meters in the dry season. In savanna areas, they only take flight at times of the day when temperature is suitable. At the hottest time of the day (above 35°C) and during the night, they rest. Farmers take advantage of this trait by driving their herds through infested areas after dusk. In forested areas where temperature swings are less marked, they fly more often. There are several different species of tsetse flies, each with its own ecological preference. Accurate knowledge of the resting places is essential for meaningful control measures. [For example, the maximum height at which the flies rest on tree trunks determines the area where insecticides should be applied. Large numbers of the insects are frequently found on the lower 30 cm, but this varies according to the season].  In an endemic area usually fewer than 1% of the flies are infected.

Gallery forest, typical habitat for tsetse flies which transmit West African trypanosomiasis. Photo prof Eyckmans. Copyright ITM

Gallery forest, typical habitat for tsetse flies which transmit West African trypanosomiasis.

Normal East African Savannah. Copyright ITM

Note: other transmission routes

In areas where there are many other biting flies ( Stomoxys , Tabanus , Haematopota , Haematobia sp.), mechanical transfer of parasites can occasionally occur via these insects, i.e. without the parasite undergoing maturation in the vector. Overal, these mechanical vectors play a minor role in transmission of human trypanosomiasis. Transplacental transmission, accidental inoculation (lab-infections) and contaminated blood transfusions are other possibilities. Regarding non-sleeping disease trypanosomes (e.g. in veterinary medicine), other transmission routes are known besides tsetse fly bites and mechanical transfer. Tsetse flies can leave droppings infected with T. grayi on the mucous membranes in the mouth of crocodiles. Lions and hyenas can get infected through the mouth when eating infected prey and injuring their mouths on broken bones. Transmission via food contaminated with T. cruzi -containing faeces is known for Chagas' disease. Sexual transmission of Trypanosoma equiperdum in horses leads to dourine, a SOA of these animals.

Vector, bacterial endosymbionts of tsetse flies

Intracellular associations between bacteria and insects are widespread in nature. Study of such associations have led to the conclusion that they are commonly found in insects that utilize diets containing an excess of one class of compounds but are deficient in some essential nutrients. It is thought that the function of the endosymbionts is to rectify this imbalance by the synthesis of these essential nutrients for the host. The relationship is mutual benificial. The bacteria get housing and food, and in return produce panthotenic acid and other B vitamines. Tsetse flies contain three different genera of bacterial symbionts: Wigglesworthia , Sodalis and Wolbachia . All three symbionts are acquired by the larva from the mother.  Wolbachia bacteria are present in the ovaries and are transovarially transmitted through maternal lineages. The name Wigglesworthia glossinidia refers to the British entomologist Sir Vincent Brian Wigglesworth, who described the germ, and to the host insect. These Gram-negative endosymbionts live in the midgut in specialised insect cells, the bacteriocytes or mycetocytes. The bacteria are transmitted maternally (intrauterine transmission via the "milk"). Horizontal transmission does not seem to occur. Fly and bacterium have become co-dependent in the course of 50 million years of evolution. Different species of tsetse flies have each their own symbiont. The bacterium cannot survive outside the insect and the flies are sterile without their symbiont. The bacterial genome contains 62 genes that are essential for the synthesis of 10 vitamins (e.g. folic acid, pyridoxine, biotine, ...). This fact led to the supposition that the fly has become dependent upon this bacterium to supply these vitamins, especially because the blood meal of the insect contains very little of these vitamins. If one kills the bacteria with antibiotics the flies become sterile. When these sterile flies get vitamine supplements, they become fertile again. If one could eliminate or alter those endosymbionts, one might find a new tool in vector control. Endosymbiotic bacteria have a well defined population structure, with a very limited number of bacteria (about 10 8 bacteria per tsetse fly). The bacterium has a very small genome, which was mapped and published in 2002. Seemingly, the bacterium has lost many genes which are found in most other free-living bacteria. Most likely, these genes were not essential in the specific environment of the host cell. Still, Wigglesworthia 's genome contains  remnants of genes typical of free-living bacteria (such as Escherichia coli or Salmonella typhimurium ), e.g. genes needed for mobility, such as synthesis of flagellae. Flagellae itself have not been found (yet) in the bacteria, but if they would be present in a certain life stage, they might help Wigglesworthia to swim from adult fly to the larva. The primary endosymbiont W. glossinidia lives exclusively in the mycetome, a U-shaped organ which contains specialized epithelial cells (bacteriocytes) and located in the anterior gut. A second symbiont, Sodalis glossinidus , is present in midgut cells. In certain insect species, it can also be detected in muscle, fatbody, hemolymph, salivary glands, and milk glands. Wolbachia are related to the Rickettsia, and live in the reproductive tissues (ovaries, vitelline gland). It is transmitted to offspring by the mother through the cytoplasm of the egg.  In G. morsitans and G. brevipalpis infections are restricted to the gonads, but in G. austeni they can be detected in various somatic tissues. When trypanosomes in the blood meal enter the midgut of the fly, epithelial cells produce defensive lectins, although there is some controversy over their exact importance. Lectins are proteins which binds to certain carbohydrates. Endosymbiotic bacteria such as Sodalis produce chitinase enzymes to hydrolyse the peritrophic membrane into D-glucosamine. This sugar inhibits the lectin defense system and therefore promotes infection with trypanosomes. The degree of sensitivity to trypanosome infection depends on the number of endosymbionts. A teneral fly which carries few endosymbionts from birth will be trypanoresistant. A trypanosensitive fly that is not infected during the first blood meal will likely not become infected thereafter because the enzymes involved in blood digestion boost trypanocide activity by increasing intestinal lectin secretion. The complete biochemical mechanisms which enable trypanosomes to colonize tsetse flies are not yet clear.  Via genetic engineering, modulation of vectors is being explored to change the competence of the transgenic insect. It might be possible to drive a certain phenotype (such as trypanosome resistance conferred by gut symbionts) into an insect population. A so-called paratransgenetic manipulation technique employs manipulation of a symbiotic organism, not the fly itself.One of the approaches being studied is a Sodalis -based delivery system for single-domain antibodies, so-called nanobodies, in order to create a trypanosome-resistant tsetse fly. Nanobodies correspond to the antigen-binding variable domain of camelid antibodies (camelid antibodies contain only a heavy-chain, as opposed to e.g. human antibodies, which contain two heavy and well as two light-chains (Lambda or Kappa). Such a trypanosome-resistant vector would be highly valuable in integrated control programs against African Trypanosomiasis, as a variant on the classic "sterile insect technique". . Consequences of infection for the vector When infected, tsetse flies develop a primitive immune response, including the production of several different anti-microbial peptides, such as attacin(s), defensin, diptericin and cecropin. Attacin(s) interfere in trypanosome establishment in the tsetse midgut. The protective role of midgut lectins is controversial. Certain molecules ("reactive oxygen species") and proteases help the fly to defend itself against trypanosome establishment in the midgut. Stressed flies (e.g. hungry) are more prone to infection with trypanosomes than healthy flies. Infected flies can develop partial necrosis of the salivary glands. The normal transparent saliva becomes milky, and its chemical composition changes. The energy available to the fly as well as its life span are diminished by ± 15%. Infected flies are more susceptible to insecticides. Once infected, they alter their feeding behaviour. In the epimastigote stage, trypanosomes adhere to the lateral walls of the alimentary duct. They hamper blood movement and force the fly to increase its suction force. The fly tires more quickly during bloodsucking. It is like using a drinking straw with small diameter instead of a large-bore tube. The fly is obliged to feed more often, as the meals are smaller. This increases the risk of transmission because -for an equivalent quantity of blood sucked- an infected fly takes five blood meals as compared to two for an uninfected fly. Thereby the parasite promotes its own transmission.

Clinical

Clinical, infection by T.b. gambiense

Inoculation

Trypanosoma gambiense, inoculation chancre in the neck. Copyright ITM

East African trypanosomiasis with an important inoculation chancre. Copyright ITM

Trypanosoma gambiense sleeping disease. Swollen posterior cervical lymph nodes are known as Winterbottom's sign.

Trypanosoma gambiense sleeping disease. The face is slightly swollen. The patient looks absent-minded. Copyright ITM

Trypanosoma gambiense sleeping disease. Swollen posterior cervical lymph nodes are known as Winterbottom's sign. Copyright ITM

Trypanosoma gambiense sleeping disease. Notice the typical facial expression. Copyright ITM

Cachectic child with Trypanosoma gambiense sleeping disease. Photo prof Eyckmans. Copyright ITM

Trypanosoma gambiense sleeping disease. A group of patients with advanced late stage disease. Copyright ITM

Any bite from a tsetse fly, whether infected or not, produces a local reaction. When the bite is infected, a small local wound can appear after 1 or more weeks, in general after 5-15 days (trypanosomal chancre or sore or trypanoma). This often remains unnoticed in the local population, though it can sometimes reach quite substantial dimensions (2-5 cm). In infected Europeans, it is described at a frequency of 25-40%. It involves a central blister or ulcer surrounded by red infiltrated skin. The lesion tends to be minimal painful. When it has healed after 1-3 weeks a depigmented scar can remain. The infection develops slowly if there is no medical intervention. The patient's condition gradually deteriorates, ultimately leading to his/her death, sometimes as short as a few months, sometimes much later. There are two quite artificially separated stages: a preliminary haemato­lymphatic stage and a second stage with symptoms of meningoencephalitis. The boundary between these two stages is determined by the findings in the cerebrospinal fluid. The distinction is important for treatment. Asymptomatic human carriers are rare.

Haematolymphatic stage

The haematolymphatic stage lasts 6 to 12 months, but sometimes longer. It is characterised by intermittent, unpredictable bouts of fever separated by irregular intervals of days to a month or even more, headache and general malaise. The lymph nodes swell, especially those in the neck (Winterbottom sign). These glands are soft, mobile and not painful. In early West African trypanosomiasis, swollen posterior cervical lymphnodi are found in 50-85% of early stage patients and in fewer than 25% in the late stage. Oedema sometimes occurs (face), as well as pruritus (itching) and transient red spots or a circinate rash (trypa­nides). This rash can be seen without difficulty on a white skin (reported in 50%) but is difficult to see on a dark skin, The liver and certainly the spleen can be clearly enlarged. There is moderate to severe anaemia. Neurological disorders (personality changes, increased sensitivity to pain, especialy deep hyperaesthesia ("Kerandel sign") can already be present in the first stage. This condition gradually evolves into increasing neurolo­gical collapse, characteristic of the meningoencephalopathic stage.

The condition is characterised by a chronic course with flare-ups and quieter periods. These flare-ups are to be interpreted as destruction of the trypanosomes, followed by the development of a new population of parasites carrying a different surface antigen. Lysis of the parasites releases large quantities of antigen into the bloodstream. These form immune complexes with circulating antibodies which then precipitate, resulting in perivascular inflammatory symptoms (including vasodilation with increased vascular permeability and oedema). Successive generations of parasites each have a different glycoprotein on the outer membrane. It is to this outer membrane that the antibodies attach themselves. Whenever a new glycoprotein emerges, the immune system always has to start again from scratch, with the production of new antibodies. This explains the pronounced increase in the immune globulins (especially IgM) in blood and cerebrospinal fluid. The high IgM serum concentration thus results from chronic polyclonal B cell stimulation. Aspecific cross-reacting and auto-antibodies can also be produced, making the diagnosis more difficult. Meanwhile time goes on and the infection worsens.

Meningoencephalopathic stage

In the meningoencephalopathic stage there is a progression over 3-6 months, ending in death. Personality changes increase and the patient usually loses interest in his/her surroundings. Psychosis sometimes occurs. The patient develops tremor, paraesthesia, increased sensitivity to pain, gait disorders, speech­ difficulties and reversal of the diurnal wake/sleep rhythm. Ataxic dyskinesia is present in most patients. Basal ganlia involvement can produce clinical features which overlap with those of Parkinson's disease. Weight loss and endocrine abnormalities with e.g. impotence are common. Damage to the hypothalamus (paraventricular and supraoptic nuclei) may lead to disturbance of the normal sleep pattern. The patient progressively deteriorates and develops stupor ( sleeping sickness!). The patient can still be woken up, but will quickly go "back to sleep" again. This is finally followed by coma and the patient dies of malnutrition, concomitant infections and destruction of the central nervous system. This disease is not to be confused with neurosyphilis, tuberculosis, AIDS with cerebral toxoplasmosis or cryptococcal meningitis, alcoholism or schizophrenia.

Histopathological changes include leukoencephalitis with demyelinisation and accentuation of the periventricular areas. There is a characteristic infiltration of lymphocytes and plasma cells around cerebral blood vessels (perivascular cuffing).

Clinical, infection by T.b. rhodesiense

Infection with T. b. rhodesiense evolves much faster than West African trypanosomiasis. The incubation phase is shorter (1 to 3 weeks). An inoculation chancre often occurs, and is present some days before the onset of pyrexia. There is high fever and most patients have signs of damage to numerous organs. Hepatitis leads to jaundice, elevated liver enzymes and coagulation disturbances. Myocarditis is common and often gives diffuse T-wave inversions. Heart failure can occur. ARDS can be detected on chest X-ray. Encephalitis leads to neurological symptoms, such as confusion and stupor. There is usually no obvious lymph node swelling. The disease evolves to a fatal outcome within a few weeks or months.

Diagnosis

Diagnosis, detection of parasites

West African trypanosomiasis, lymph node aspiration. Motile trypanosomes can be present in the fresh, unstained fluid. Photo prof Gigase, Copyright ITM

African trypanosomiasis, sleeping disease. Trypanosoma sp. Photo WHO

In the peripheral blood there is usually a normal white blood cell count (no leukocytosis or leukopaenia), a normal blood platelet count and a slight normocytic anaemia. The erythrocyte sedimentation rate is quite high. The diagnosis is best made by detection of the parasite. The sensitivity of the conventional parasitological techniques is, however, quite low. The parasite can be found in fluid from the inoculation chancre, blood (direct examination, thin smear, thick smear, buffy coat), lymph node fluid (needle aspiration) or cerebrospinal fluid (lumbar puncture). In a wet blood smear, the motility of the parasites attracts the eye, but the sensitivity of the technique is too low. A Giemsa-stained thick blood smear is more sensitive, but parasites are frequently deformed in this preparation and are therefore easily missed. Lymph node aspiration is done with a dry needle. After puncture the needle is left in place for a while and the node is massaged. A syringe is then fitted to the needle and after aspiration the fluid is put on a microscope slide for direct examination (the motile trypanosomes can then be observed). Several samples will often be needed, as the parasites are not present in large numbers and appear in the blood in intermittent waves. Concentration techniques facilitate the diagnosis: Woo technique, buffy coat from a centrifuged microhaematocrit tube or quantitative buffy coat test (QBC). In well equipped laboratories a miniature anion exchange centrifugation technique (mAECT) is used (Lanham or Lumsden method). Such a column contains diethylaminoethyl-cellulose (DEAE-52). The separation of blood cells from trypanosomes depends on a difference in surface charge of the blood cells and the parasites. This charge is pH-dependent (importance of iso-electric point). Blood is mixed with a particular buffer (PSG = Phosphate-Saline-Glucose) and gently layered on top of the column. The blood will penetrate the gel on top of the column, and red and white blood cells adhere to the DEAE gel particles. In this buffer, the trypanosomes are at their isoelectric point (=neither positive nor negative charge) so flow through the column. The eluate containing the parasites is collected and centrifuged. The sediment is examined microscopically to determine if parasites are present. The type of buffer and the temperature at which the test is carried out are of very great importance. The more the disease advances, the less frequently are trypanosomes found in the blood, though they are then found more often in the cerebrospinal fluid. The parasites can be cultured in vitro in a specific medium (KIVI; Kit for in Vitro Isolation). In theory as few as 1 trypanosome can be detected in 5 ml, though in 50% of the tested cases the culture remains sterile. The blood is initially mixed with the toxic anticoagulant polyanethol sulphate. The latter also has anti-complement activity. An antigen-capture ELISA (Enzyme Linked ImmunoSorbent Assay) also exists, but there are at present too many false positive results with this.

Comparison of detection thresholds

Technique Sensitivity Fresh blood preparation (10 µl) 6000 trypanosomes/ml Thick drop (10 µl) 2000 trypanosomes/ml Buffy coat (70 µl)          600 trypanosomes/ml QBC (Quantitative Buffy Coat Test) 16 trypanosomes/ml (to be confirmed, probably less sensitive) MAECT (500 µl)  usually 100/ml required PCR (Polymerase Chain Reaction) 10 trypanosomes/ml (depends upon gene target and specific technique) KIVI      1 trypanosome per 5 ml.

Diagnosis, serology

Antibodies can be detected serologically. Several techniques (immunofluorescence etc.) have been developed. There are also methods for use in primitive rural conditions. A cheap and practical method is a direct agglutination reaction of trypanosomes on a plastic card, with macroscopic read-off (CATT = Card Agglutination Test for Trypanosomiasis). This is a good screening method for T. b. gambiense in most areas. The sensitivity of the CATT test in areas (e.g. Cameroon) with T. gambiense  strains which do not carry the variable surface antigen LiTat 1.3 is low. A drop of blood (finger prick) and a drop of reagent that contains blue-coloured parasites of a known serotype are mixed on a white plastic card. The card is mechanically shaken for 5 minutes and then immediately read. When the test is positive (presence of antibodies) the trypanosomes agglutinate and form a blue clot. The CATT has no place in the diagnosis of T. rhodesiense infections, except in a chronic form of T. rhodesiense which exist in Malawi. CATT must not be confused with the CIATT (Card Indirect Agglutination Test for Trypanosomiasis, an antigen-detection test). Another method is to take a blood drop on very small filter papers (confetti) and examine this later in a laboratory. The patient should be called back later if the result is positive. Antigen-detection methods (ELISA) have also been developed, but are not yet in routine use. A problem arises in persons who have a positive serology, but who are asymptomatic and in whom no parasites are found (wait and see with follow-up or treatment with suramin or pentamidine?). After successful treatment the antibodies remain for years. Antibody detection therefore cannot be used for detecting relapse or reinfection. It is hoped that in the future we shall be able to prove a cure by monitoring reductions in the levels of circulating antigens. In 2013, two rapid diagnostic antibody-detection tests were developed for T. brucei gambiense . The HAT-Sero-Strip is a dipstick using blood (30 µL). The HAT Sero-K-SeT test is a lateral -flow device for testing plasma (15 µL). Both tests provide results within 15 minutes. The tests contain variant surface glycoproteins (LiTat 1.3 and LiTat 1.5). Initial evaluation showed excellent sensitivity and specificity. Further field tests in different geographical settings will determine their (eventual) place in diagnosis. Parasite clones which do not contain the LiTat 1.3 antigen have been described from Cameroon.   Genomic tests The first PCR was developed in 1983 by Kary Mullis (Nobel Prize Chemistry 1993). Since then several PCR variants have been developed. Most techniques consist of an amplification step followed by amplicon electrophoresis in agarose gel, but there are other approaches. The sensitivity and specificity largely depend on the DNA sequence targeted by the primers. Prefered genomic sequences are those which are conserved and unique for the parasite and that occur as multiple copies in the genome. Tests based on extra-nuclear minicircle kinetoplast DNA have failed to live up to expectations. With PCR, formal molecular differentiation between T. brucei gambiense and T. brucei rhodesiense is possible. T. brucei gambiense -specific glycoprotein is only present in T. brucei gambiense , while the gene encoding the serum-resistance-associated protein (SRA) is only present in T. brucei rhodesiense . Both however are single copy genes. Techniques vary from single PCR (thermic cycling needed), nested PCR (using two subsequent PCRs), real time PCR (single tube, quantitative and fast), multiplex PCR (several distinct primers), LAMP (isothermic) and NASBA (RNA amplification). Other developments include NASBA followed by oligochromatography (dipstick) and peptide nucleic acid fluorescence in situ-hybridisation (PNA-FISH) which uses battery powered light emitting diode (LED)-based fluorescence microscopes. Improvement of genetic detection methods might facilitate the detection of resistant parasites, which may then of course influence treatment. Also other steps need to improve, such as stabilisation of blood specimens on filter cards or guanidine-based storage buffers before transport. Further development to simplify the techniques and adapt them to field conditions is needed, and validation is necessary. Simplification, standardisation and proper test evaluation might move powerful molecular tests from the research labs into point-of-care use. LAMP is an isothermic nucleic acid amplification technique, amplifying DNA. This technique avoids the need for thermic cycling, but still requires DNA extraction, electricity, a cold chain and certain infrastructural measures to avoid cross-contamination. In contrast with classic PCR, Nucleic Acid Sequence Based Amplification (NASBA) amplifies RNA sequences. The technique uses continuous amplification of nucleic acids in a single mixture at one temperature. NASBA's main advantage is that it works at isothermic conditions, usually at a constant temperature of 41°C. NASBA gives quicker results than PCR and is more sensitive. However, extra caution should be taken during extraction and specimen storage because RNA is more liable to degradation than DNA.  NASBA works as follows: The first primer in the reaction mixture attaches to the 3' end of the RNA template at its complementary site Reverse transcriptase synthesizes the opposite, complementary DNA strand RNAse H destroys the RNA template (RNAse H only destroys RNA in RNA-DNA hybrids, but not single-stranded RNA) The second primer attaches to the 5' end of the DNA strand RNA polymerase produces a complementary RNA strand which is used again in the first step, so the reaction is cyclic.

Note : CATT Supply

For CATT supply, one can contact

Unit of Applied Technology and Production

Institute of Tropical Medicine, Nationalestraat 155, B-2000   Belgium

Tel : 32.3.247.63.68

Diagnosis, clinical

A correct diagnosis can sometimes be reached even though parasites cannot be detected. These "clinical cases" are patients from an endemic area, with clinical symptoms of late stage trypanosomiasis and lymphocytes in the cerebrospinal fluid. Such "clinical cases" may amount to no more than 5% of the total number of trypanosomiasis patients.

Diagnosis, IgM in cerebrospinal fluid

Antibodies should if possible be detected in the cerebrospinal fluid (technically difficult). Determining the IgM content in the cerebrospinal fluid can be very difficult or even impossible to carry out in endemic areas and under field conditions. An experimental latex agglutination test for detection of IgM was developed at the Institute of Tropical Medicine, Antwerp, Belgium. Blood-CSF barrier dysfunction is usually absent or mild and occurs in very advanced late-stage disease. It is possible to calculate and plot diagrams of the quotients CSF/serum concentration for IgG, IgA and IgM (demonstration of intrathecal production of antibodies). Especially intrathecal IgM production will be present in late-stage sleeping disease (occurs in 98% of people with leukocyte counts higher than 20/µl). Similar patterns do occasionally occur in Lyme neuroborreliosis, neurosyphilis, mumps meningoencephalitis and in non-Hodgkin lymphoma involving the central nervous system.

Usefulness of the lumbar puncture:

A lumbar puncture is important :

sometimes in order to make a diagnosis in order to determine the stage (main purpose) in order to monitor therapy

In the 2nd stage the cerebrospinal fluid is characterised by:

white blood cell count (WBC) > 5 per mm 3 , (normally <3) protein > 45 mg%  (normally 15-45 mg%) IgM increase (difficult to carry out; Latex IgM) sometimes trypanosomes and/or Mott cells  

Spinal tap in African trypanosomiasis. Copyright ITM

In African trypanosomiasis, the cerebrospinal fluid can contain numerous lymphocytes. Some of them will have developed into plasma cells containing cytoplasmic vacuoles filled with accumulated immunoglobulines. These cells are also known as morula cells or Mott cells.

Control lumbar punctures should be carried out for late stage West African trypanosomiasis every 6 months for 2 to 3 years after diagnosis and therapy. In East African trypanosomiasis they should be carried out more frequently (every 3 months, certainly in the first year). Good follow-up is essential. Recurrence often presents as a deterioration in results obtained with the cerebrospinal fluid (increased lymphocyte count). Parasitological proof of recurrence is often lacking. Immediately after treatment the lymphocyte count in the cerebrospinal fluid increases (so-called "cerebrospinal fluid storm"). This should not be regarded as a recurrence. There is no permanent protective immunity. Reinfection can occur.

MRI brain in trypanosomiasis

Most cases of HAT occur in poor areas, therefore, there is very little experience with neuroimaging of this condition, especially regarding MRI. When caring for a patient with African trypanosomiasis, one can encounter the following situation regarding MRI of the brain: normal findings meningeal enhancement due to the effect of a recent lumbal puncture meningo-encephalopathy due to the infection itself. Meningeal enhancement (thickening), focal or diffuse lesions on T2-weighted images, but without edema. In late stage, demyelinisation in white matter and choroid involvement. On CT low-density areas in the centrum ovale and periventricular areas. On MRI scanning of HAT itself, one can detect symmetrical focal lesions in the deep white matter of the internal capsule, cerebellum and splenium of the corpus callosum. Diffuse hyperintensity in the basal ganglia, external capsule, and extreme capsule are possible. Perivascular cuffing of lymphocytes occurs both in the brain parenchyma and the arachnoidea. The blood vessels are surrounded by areas of demyelinisation and axonal damage. Severe demyelinisation without hemorrhage suggests HAT as the cause of the lesions, instead of arsenic encephalopathy. arsenical encephalopathy secondary to the administration of melarsoprol: diffuse lesions with hemorrhagic component, and edema as a result of hypoxia residual scarring and cerebral atrophy

Treatment

Treatment, general

There are several different treatment schemes that are determined by the vertical control programme that is or was in place in many areas. Anaemia and any other infections, such as malaria, as well as any vitamin deficiencies should be treated first. The specific therapy is not simple. Drugs that do not penetrate into the cerebrospinal fluid and the brain are useful in the early stage (prior to invasion of the central nervous system). Drugs that do penetrate the blood-brain barrier must be used in the late stage. A number of drugs were developed many years ago. There is an urgent need for better, cheap drugs. There is also a need for clinical studies to compare different therapeutic regimens.

In May 2001, the pharmaceutical company Aventis signed an agreement with WHO to guarantee a gratis production of pentamidine, melarsoprol and eflornitine for at least 5 years. Médecins Sans Frontières (MSF) would be responsible for the logistical part of storage and transport of the drugs. Shortly afterwards, Bayer joined this partnership, providing suramin and reconsidering a decision to halt production of nifurtimox. At present all drugs are donated to WHO by Aventis and Bayer.

The blood-brain barrier is the boundary formed by the continuous "tight junctions" between the endothelial cells of the brain capillaries. There is a basal membrane between endothelial and neighbouring cells. Pericapillary astrocytes surround the whole complex. This barrier restricts the migration of polar substances from the blood to the brain. The barrier is important for maintaining the composition of the fluid surrounding the brain. There are special transport systems that allow certain aminoacids, glucose and specific macromolecules to enter the brain. In addition there is also passive diffusion of lipophilic low molecular weight substances. This high degree of access control provides substantial protection for the brain, but also has implications for drugs. However, the blood-brain barrier is not inert. Although fenestrated capillaries in the choroid plexus allow a more direct exchange between blood and cerebrospinal fluid, their area occupies only 1/5000th of the total exchange surface. There are also a few other small regions of the brain where there is no barrier, namely the area postrema, organum vasculosum of the lamina terminalis, the subfornical and subcommisural organs, the eminentia mediana of the hypothalamus and the neurohypophysis.

Treatment, drugs

Skin desquamation as side effect of suramin, a drug used for treating sleeping disease. Copyright ITM

Skin desquamation as side effect of suramin, a drug used for treating sleeping disease. Copyright ITM

Chemical structure of suramin. Suramin is used in the treatment of trypanosomiasis. In the past it was also used in onchocerciasis. Copyright ITM

 African trypanosomiasis  First stage   Second stage    T.b. gambiense  pentaminine 7 days   Melarsoprol 3 x 3 days or 10 days sequential  Eflornitine 14 days (IV, four or twice per day)    T.b. rhodesiense  suramine 14 days  melarsoprol 3 x 3 days or 10 days sequential Suramin (Bayer 205®, Germanine®, Naphuride®, Moranyl®, Antrypol®). The compound was developed in 1920. It is best administered by slow intravenous infusion, as intramuscular administration (10% solution in distilled water) is very painful. Suramin is excreted extremely slowly by the body. This is important in allergy (exfoliative dermatitis). It can cause substantial proteinuria. When a test dose of 200 mg is tolerated well, the daily dose is 20 mg/kg (max. 1 g per dose; 1 g of the base = 10 ml of a 10% solution). This is to be repeated 5 times with intervals of a few days. There are several treatment regimens, such as 1 g on days 1, 3, 7, 14 and 21 (in adults) or on days 3, 10, 17, 24 and 31. Fever sometimes initially occurs due to lysis of trypanosomes. Suramin also kills Onchocerca volvulus filaria. Patients with active onchocerciasis can exhibit severe side effects to suramin. Suramin is frequently used for reducing the total number of trypanosomes, in order to reduce toxicity when Arsobal is administered.  

Pentamidine  was developed in 1941. It is less active than suramin and not active against T. b. rhodesiense. There are two water-soluble salts: pentamidine isethionate  (Pentacarinat®), to be dissolved in sterile distilled water (not with physiological saline) and (hard to obtain): pentamidine dimethane sulphonate  (Lomidine®), already dissolved as 4% base. Intramuscular injections are painful and are best given with the patient lying down since they cause a drop in blood pressure. If possible, slow IV administration is better. Rapid IV injection causes acute hypo­ten­sion and should be avoided. Hypoglycaemia can sometimes occur (release of insulin from the pancreas). This medicine is also used in pneumocystosis in AIDS patients. The dose is 3-4 mg base/kg/day for one week or every 2 days with a total of 10 injections (in adults). Alternatively, injections can be given on days 1, 3, 5, 13, 15 and 17. To calculate the dose: 1.7 mg salt is approximately equivalent to 1 mg of base. The usual dose for an adult person is between 150 and 300 mg per injection. Adrenalin and fluid are given intravenously if acute hypotension occurs.

DFMO  (Eflornitine, Ornidyl®). Di-fluoro-methyl-ornithine  or DFMO was first used for trypanosomiasis in 1985. It is very water soluble. This substance penetrates quite well into the cerebrospinal fluid. An IV treatment for 2 weeks should be sufficient. This used to be followed by 4 weeks of oral administration, though the additional benefit of this is probably limited. Oral administration is an alternative (second choice) if no venous route of administration is possible. Oral ingestion frequently causes osmotic diarrhoea. The drug is expensive and if supplies are limited it is best reserved for melarsoprol-resistant cases. Unfortunately it is active only against T. b. gambiense . The dosage regimen is 100 mg/kg/6 hours IV x 2 weeks via physiological fluid infusion. This requires a large quantity of product per patient (about 1/3 kg per person). Concentrations in cerebrospinal fluid in children seem to be lower than in adults. Children possibly require a higher dose. [ Note: topical eflornitine is also being studied as a depilatory product in cosmetics.]

Melarsoprol  ( Arsobal ®) was developed in 1949. This trivalent arsenic compound is insoluble in water or alcohol. It is therefore dissolved in propyleneglycol. This solvent is highly irritant to tissues. It causes phlebitis and chemical cellulitis when administered paravenously. Melarsoprol is available in ready to use vials (3.6% solution). This viscous fluid may only be given by very slow IV infusion. The syringe must be perfectly dry, as otherwise flocculation occurs. Melarsoprol penetrates into the cerebrospinal fluid to only a very limited extent. It can nevertheless be used in late stage disease. Melarsoprol as such is quickly eliminated from the plasma. It also has a significant trypanocidal activity (as can be measured via bioassay) in plasma and cerebrospinal fluid for up to several days after administration, although melarsoprol can then no longer be detected with HPLC (high performance liquid chromatography). The molecule is probably transformed into biologically active metabolites such as melarsene oxide, etc. The pharmacokinetic properties are not sufficiently known. Resistance to melarsoprol occurs. Melarsoprol should never be given without first giving suramin (East African Form) or pentamidine (West African form), in order to decrease the risk and severity of severe side-effects). Toxicity results in polyneuropathy and reactive encephalitis. Encephalopathy tends to manifests itself as a sudden violent neurological deterioration at the end of the first series or during the second series of injections. Some studies suggest that a higher number of lymphocytes in the cerebrospinal fluid leds to a greater the risk of very severe neurological complications (fatality-rate approximately 50%), but other studies could not confirm this. At present, there seems to be no way to predict which patient will develop encephalopathy. An (auto?)-immune reaction, rather than a direct toxic effect, is possibly involved here, but details are far from clear. Melarsoprol-related encephalopathy also occured in patients with leukemia who were treated with this drug, and where of course no trypanosome-antigens were present. Corticosteroids diminish the risk and severity of the encephalopathy. It is therefore imperative to administer prednisolone before using melarsoprol. In general, one can expect lethal reactive encephalopathy after administration of melarsoprol in about 3-5% of cases. Clinically, there are three syndromes of reactive arsenic encephalopathy: convulsive status associated with acute cerebral edema, due to diffuse lesions with hemorrhagic encephalitis. rapid progressive coma without convulsions acute nonlethal mental disturbances without neurological signs (e.g. psychosis) Another toxic effect of melarsoprol is polyneuropathy (analogous to heavy metal intoxication). This results in diminished sensitivity and/or paraesthesias in hands and feet, as well as motoric signs. In this case melarsoprol should if possible be stopped and vitamin B (e.g. thiamine) administered. There are several quite complicated treatment schemes. One injection per day for 3-4 days is sometimes given, followed by 1 week rest. This can be repeated three times (total = 9 to 12 vials; usually 3 x 3 ampoules for an adult person). A more cautious scheme uses half the dosage at the beginning. In April 2000 it was shown in quite a large study (500 patients) in Angola that a regimen with ten daily injections of 2.2 mg of melarsoprol/kg bodyweight in combination with prednisolone was easier and cheaper and just as good as the conventional interrupted schemes for treatment of T. b. gambiense .

Chemical structure of melarsoprol (Arsobal), used in treatment of Africal trypanosomiasis. Copyright ITM

Sleeping disease. Treatment with melarsorpol (Arsobal) resulting in dark arsenic skin pigmentation. Copyright ITM

Trypanosoma gambiensis sleeping disease. Treatment with melarsorprol resulting in dark arsenic skin pigmentation. Copyright ITM

Note 1: BAL

The letters "BAL" in Arsobal refer to British Anti-Lewisite (B.A.L.). W. Lee Lewis was the inventor of lewisite, a war gas that was used together with chlorine gas, mustard gas (yperite) and phosgene in the First World War and later by Japanese troops in Manchuria. This blistering and deadly substance is chlorovinyl dichloroarsenene and hence contains arsenic. Arsenic binds SH-groups in various proteins. A metal-chelator, dimercaptopropanol = dimercaprol = B.A.L., was used by the British as antidote. The molecule contains two SH-groups that bind to arsenic (competition with the SH-groups on proteins). This is the origin of the five-membered ring in the above formula. The damaged enzymes can subsequently be repaired (e.g. by glutathione, see G6PD-deficiency).

Note 2: Acute arsenic toxicity

Acute arsenic toxicity must not be confused with chronic arsenic poisoning. The latter occurs in some regions such as Bangladesh because the ground water there often contains high concentrations of this substance.

Note 3: Drug Supply

One can contact

Dr Jean Jannin, WHO CDS/CSR, 20 Avenue Appia, 1211 Geneva 27, Switzerland

tel 41-22.791.3779; fax 41-22.791.4878

TREATMENT  SUMMARY

T. b. gambiense  early stage:

Or DFMO 100 mg/kg IV 4 times per day for 14 days

Or Pentamidine 4 mg base/kg/day IM on days 1 to 7

Or Suramin test dose 5 mg/kg, then 20 mg/kg/day (max. 1 g) IV on days 3, 10, 17, 24, 31

Or Pentamidine 4 mg base/kg IM on days 1, 3, 13, 15, 17 + Suramin 20 mg/kg on days 1 and 13

T. b. gambiense  late stage:

Or DFMO 100 mg/kg IV 4 times per day for 14 days

Prednisolone 1 mg/kg, starting 1 day before the rest of the medication, followed by

Suramin or Pentamidine 1 to 2 injections, followed by

Arsobal 3-4 series of one injection per day for 3-4 days with 7-10 days' rest period in between

Or Arsobal 2.2 mg/kg/day for 10 days with prednisolone throughout the whole period

T. b. rhodesiense

Suramin test dose (check urine for protein and cylinders), then 1 g per week for 6 weeks. The first dose can be given fractionated over 2 days. Quicker alternative scheme: days 1, 3, 6, 14, 21

Arsobal should be administered under cover of prednisolone

A high (double) dose of DFMO gives a favourable result in 50% of cases, if combined with suramin

MEDICATION  SUMMARY

Suramin:

First test dose + ivermectine if onchocerciasis is suspected.

Check urine. Then 10% solution by slow IV infusion, maximum 1 g per infusion

Maximum 5 injections. Example: Days 1, 3, 7, 14, 21

Pentamidine:

4 mg base/kg/day IM or slow IV infusion; max 300 mg base per infusion.

Glucose if hypoglycaemia. Example: 10 daily injections

Ornidyl®

100 mg/kg/6 hours daily IV infusion for 14 days or second choice-orally. Expensive.

Melarsoprol:

Prednisolone before and during treatment!

To be preceded by Suramin or Pentamidine.

Exclusively by IV infusion; usually 3 series of 3 ampoules (adults): 3 x 3

1 week's rest between series.

Dose = usually 3.6 mg/kg, maximum 200 mg. (various schemes)

New scheme: 2.2 mg/kg for 10 consecutive days.

Stop in case of polyneuropathy or reactional encephalopathy

Medications, seldom used

Diminazene   ( Berenil ®). Discovered in 1955. Officially registered only for veterinary use. Can be administered intramuscularly (less painful than IM Pentacarinat®). Daily dose 5-7 mg/kg, to be repeated after 2 and 4 days. Can be useful in cases of resistance, but in view of the official restriction it is used very rarely. It was initially considered to be quite neurotoxic, but in practice this does not appear to be such a big problem. Other commercial names for diminazene are Veriben®, Ganasegur®, Ganaseg®, Azidine®. Cross-resistance between diminazene, used in the treatment of animal trypanosomiasis, and melarsoprol used in the treatment of African trypanosomiasis in man can be selected with relative ease. Both drugs, or their active metabolites, can enter trypanosomes via the P2 aminopurine transporter, and loss of this transporter can impair uptake of the drugs into parasites. It is therefore possible that melarsoprol resistance is linked to selection of diminazene resistance through treatment of animals. At present, this is still a hypothesis. Since selection of resistance to diminazene in animals might lead to melarsoprol resistance in humans, caution must be exercised in the choice of trypanocide used to clear cattle of trypanosomes. Therefore, although resistance to isometamidium and homidium can arise more quickly in cattle than can resistance to diminazene, these drugs could be better choices in terms of implications for human health.

Chemical structure of nifurtimox. Copyright ITM

Nifurtimox   ( Lampit ®): Cf. Chagas' disease. Success of nifurtimox-eflornithine combination therapy (NECT) is hope giving. In the past. Combination treatment eflornitine + nifurtimox gave good results in a phase II trail in 2009. Nifurtimox was difficult to obtain outside South America in the past. Its place in therapy is not very clear, but promising. Sometimes used in cases of melarsoprol resistance (several schemes, including 15 mg/kg/day PO for 60 days). Toxic to the central nervous system (more toxic at higher doses such as 30 mg/kg/day). The efficacy of NECT is non-inferior to that of eflornithine monotherapy. Since this combination treatment also presents safety advantages, is easier to administer (ie, infusion every 12 h for 7 days vs every 6 h for 14 days), and potentially protective against the emergence of resistant parasites, it is suitable for first-line use in HAT control programmes.

Cymelarsene . Structurally very closely related to melarsoprol.

Veterary : Homidium, isometamidium and diminazene are drugs used in cattle, sheep and goats. Suramin, quinapyramine and melarsomine are used in horses, camels and buffalos.

 

Medications, experimental

IMOL 881  (experimental). Is yet to be evaluated. Fexinidazole Fexinidazole is a product related to megazol, tinidazole and metrinidazole. It is a very promising and very stable 2-substituted 5-nitro-imidazole. It was first developed in the late 1970's. In 1983 in vivo trypanicidal activity was demonstrated but this information was not followed up, so that the drug languished in obscurity for more than 25 years. In African trypanosomes, nitro drugs are reductively activated by a bacterial-like nitroreductase. Modulation of nitroreductase levels in T. brucei directly affected sensitivity to nitro compounds, with reduced concentrations of the enzyme leading to moderate nitro-drug resistance. Fexinidazole was "rediscovered" as an oral drug which might be used in early and late stage sleeping disease. It is the first antitrypanosomal drug developed in 30 years. In lab animals, fexinidazole is well absorbed after oral administration and distributed widely in the body, including the brain, which is very important in late stage HAT. In African trypanosomes, the mode of action of nitro drugs involves reductive activation via a NADH (reduced form of nicotinamide adenine dinucleotide)-dependent bacterial-like nitroreductase. In vivo, fexinidazole is rapidly metabolized to at least two active metabolites with equivalent biological activity: the sulfoxide [containing a R- S(=O)-R'- group] and a sulfone derivate (containing a R-SO2-R' group), the kinetics of which are different but overlapping. These metabolites are likely to contribute to the therapeutic effect. They are in animals models active against stage I and II disease. Although fexinidazole is Ames-positive, is is devoid of any genetic toxicity in mammalian cells. Fexinidazole has the potential to become a safe, efficacious, affordable, oral short-course (less than 14 days, oral treatment with a suitable shelf life in the tropics. A Phase I clinical study started in 2009 and phase II trials began in 2010. If fexinidazole successful completes clinical development, it will represent a major breakthrough. Hopefully it will become the first oral drug allowing for a short cheap oral treatment of HAT phase I and II. Compared with 10 days of the dangerous IV melarsoprol, the very complicated IV eflornitine monotherapy (56 infusions over 14 days) and the current NECT (10 days of oral nifurtimox and 7 days of 12-hourly eflornitine IV infusions), it would be a major improvement. This reciprocal cross-resistance between nifurtimox and fexinidazole has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Trybizine . This is a diamidine that can cure acute trypanosomiasis in cattle and rodents. It was developed in China. Attempts are being made to synthesise analogues that are less toxic and that pass through the blood-brain barrier

Megazol.  A fairly active, but still experimental 5-nitro-imidazole derivative. However, the substance is mutagenic in the Ames test. Megazol does not contain arsenic. [The Ames test screens possible carcinogens. The test uses a specific histidine synthesis deficient Salmonella strain. If the tested substance causes mutations that restore the ability to produce histidine, the substance is regarded as potentially carcinogenic].

Enzym blockers. The surface glycoproteins are anchored in the cell membrane with a glycosyl phosphatidylinositol (GPI) anchor (see above). When GPI synthesis is blocked by elimination of any single gene along the biochemical pathway, the parasite dies. A search is now being made for suitable inhibitors of an enzyme from this reaction chain that is specific to the trypanosome. Transsialidase is another enzyme that is being closely investigated. Trypanosomes are themselves unable to produce sialic acid. In trypanosomes, many enzymes of the pentose phosphate pathway and several other pathways are related to cyanobacterial isoforms rather than to those of eukaryotes. This association has led to a suggestion that the ancestors of trypanosomatids harboured a now lost endosymbiont at some time in their evolutionary past. The pathway's third enzyme (6-phosphogluconate dehydrogenase) is essential to trypanosomes. The search is on to determine if structural differences to its mammalian counterpart could be exploited in design of selective inhibitors.

DB289 is an experimental, orally available prodrug (pafuramidine maleate), a diamidine which is converted after absorption into a trypanocidal dicationic form. The Bill and Melinda gates foundation sponsored the development of DB289. Development was stopped in 2008 because of nephrotoxicicty concerns. Acidocalcisomes are membrane-bound acidic cell compartments rich in calcium and pyrophosphate. Inhibitors of such acidocalsisomes (e.g. pamidronate, a bisphosphonate) could be useful, but have still to be tested in formal trials.

It is important to know which molecules are essential for the parasite. Example: the parasite's surface coat proteins are studded with a complex arrangement of sugar molecules, including galactose.  But the parasite cannot take up galactose. Instead, it converts glucose into galactose during the construction of the surface coat, a task carried out by an epimerase. When the enzyme's gene was identified, gene-knockout methods were used to prove that the gene was essential. The structure of epimerase was determined by X-ray crystallography. With these data, the search is on for inhibitors as drug leads. An alternative for finding inhibitors is high-throughput screening, where robotic screening systems can test more than 100.000 compounds against chosen targets.

Trypanosomes do not have the enzymes necessary to make purines, essential components of RNA and DNA. Instead, they must import them from their surrounding environment. Trypanosomes have at least five different purine transporters, so they would be difficult to target with specific inhibitors. An alternative approach would be to use the transporters to deliver toxic compounds into the cell. Purine analogs are already used as antivirals and in anti-cancer therapies.

Treatment, follow-up

After treatment, the patient should have a regular follow-up for 2 to 3 years for possible relapse ( T. b. gambiense : check every 6 months). The first sign of relapse is often an increase in the cell count in the cerebrospinal fluid, followed by a rise in its protein content. Recurring fever, drowsiness and chronic headache are also signs of relapse. Unfortunately, lately it has become clear that obtaining a CATT negative result after treatment for HAT cannot be relied upon to confirm succesful treatment. Further works needs to be done to address this question. New algorithm (to be validated): It might be possible to shorten the period of follow up after treatment for T. b. gambiense infection, when : at 6 months trypanosomes or CSF WBC > 50 are considered failures; CSF WBC < 5 are considered cures. The remaining patients are evaluated again at 12 months. at 12 months : cure if no trypanosomes and CSF WBC < 20; failure if > 20 WBC in CSF or trypanosomes present. 

Prevention and control

Equipment of the organisation responsible for the fight against West African sleeping disease. Vertical control programme.

Blue tissue impregnated with deltametrin, an insecticide. Simple type of tsetse fly trap. Copyright ITM

Glossina sp. (tsetse flies) are attracted to tsetse traps. When they come in contact with the material (impregnated with insecticides), they will be killed.

Tsetse fly trap used in vector control (sleeping disease). Copyright ITM

Tsetse fly trap. Copyright ITM

African trypanosomiasis is a very serious disease for the individual patient. A major danger of sleeping sickness is the risk of attaining epidemic proportions. Systematic detection and treatment of cases requires active searching (specialised mobile service, vertical strategy). An initial screening with CATT (Card Agglutination Test for Trypanosomiasis), and detection of suggestive clinical signs is generally used for Trypanosoma brucei gambiense . If one or both of these is positive, a lymph node aspiration is carried out. If negative, this is followed by a parasitological blood examination. If positive, a lumbar puncture should be carried out.

Passive treatment of patients who come to the hospital is less expensive but also less effective. There are in fact infected persons who can sometimes remain asymptomatic for a long time. Their role in the epidemiology is yet to be elucidated. It is practically impossible to control the disease by treating only the ill patients. Chemoprophylaxis with pentamidi­ne is not used, except in very exceptional situations.

Vector control can yield good results. Active participation of the population is needed in order to set up and maintain tsetse fly traps. These usually consist of a piece of textile of a certain colour, size and shape, that may or may not be impregnated with an insecticide ("imitation cows"). Various models have been developed. One of the more frequently used types is the biconical Challier-Laveissière trap. Other types used for control include for example Vavoua, Ngu, Lancien, pyramidal and bipyramidal traps. Some traps are preferentially used for sampling (glue traps, epsilon and F3 traps). Mobile electric traps can also be used for this latter purpose. Older, no longer used traps include Morris, Harris, Moloo, Swynnerton and Langridge traps. The effectiveness of various traps is increased by baits and attractants (octenol, acetone, butanone, carbon dioxide (CO 2 ), propylphenol, methylphenol). The use of pour-on insecticides on cattle looks very promising. The insecticide is applied directly to the hide. Cattle-dips, where cattle walk or swim through insecticide-containing water, were primarily developed for tick control (control of "East Coast fever", a severe infection caused by Theileria parva ), but could also play a role in tsetse fly control.

PATTEC (Pan-African Tsetse and Trypanosomosis Eradication Campaign) is a sharply focussed campaign committed to tsetse eradication. It was launched in October 2001. PATTEC has as objective the eradication of tsetse flies and trypanosomiasis from Africa through the progressive creation and subsequent expansion of tsetse-free zones. On February 19, 2002, the International International Atomic Energy Agency, responding to a declaration calling for the eradication of trypanosomiasis from the Organisation of African Unity in July, 2000, stated an intention to release sterile males in 37 countries.

PAAT (Programme Against African Trypanosomiasis) is a broad based international inter-UN agency forum, which embraces all those concerned with tsetse and African trypanosomosis research and intervention.

Sterile Insect Technique   (see also control of New World Screwworm)

Female tsetse flies copulate only once in their lifetime. If this is with a sterile male, they cannot reproduce. The mass release of sterile male tsetse flies to control the insect population raises ethical problems. The sterile males are, indeed, not refractory to trypanosomes and their release thus temporarily increases the number of potential vectors. This technique was nevertheless used, for example in Zanzibar from 1994 to 1998. After a preliminary reduction of the tsetse fly population ( Glossina austeni ) with traps and screens, several million insects were produced over several years in the Tsetse Trypanosomiasis Research Institute in Tanga, Tanzania. Approximately 70,000 sterile male flies were released weekly. This led to the complete disappearance of the natural vector population. The intention was especially to control the animal disease nagana. Since Zanzibar lies tens of kilometres off the continental African coast, future reinfestation is unlikely.

Genetic refractory tsetse flies

Tsetse flies carry several bacterial endosymbionts (see higher). It is possible to genetically engineer the Sodalis endosymbiont so that the bacterium secretes a trypanolytic nanobody within the insect host. Such an antibody could be based on the special type of antibodies which camelids produce. About 50% of the antibodies which camels, dromedaries, llamas etc produce only a heavy chain, no light chain. Further more, the heavy chains are not linked. Such a protein carries mainly the critical chemical fingers known as complementarity determining regions, that determine what target an antibody will recognize). Such a structure was nicknamed a nanobody because it is about 10 times smaller than a normal antibody. However, the release of genetic engineered organisms into the wild will be prohibited as far as currect regulations is concerned.

Exercises

Central African Republic. A patient is given a rapid IV injection of pentamidine. He suddenly falls down. Is this probably the consequence of an allergic reaction to the product? Congo . In an epidemic of sleeping sickness it is proposed that all pigs in the region should be slaughtered. What do you think? Belgium . A technician receives a blood smear in the laboratory. She has no clinical information. After a few minutes she notices an elongated blood parasite. Her answer is: " Trypanosoma rhodesiense ". What do you think? Endemic area . Why do a lumbar puncture in trypanosomiasis? Congo . In any screening programme for trypanosomiasis a lumbar puncture should be done in every investigated person. Do you agree? Congo . A man has had fever for 10 weeks. He is emaciated. You notice swollen lymph nodes in the neck. Do you think of malaria, trypanosomiasis, tuberculosis or HIV (human immunodeficiency virus) infection? Give arguments for your opinion. Gabon . Does using a mosquito net offer good protection against African trypanosomiasis? Tanzania . Your friend visited a wildlife reserve in Tanzania 11 days ago. She now has a painful ulcer on her right arm, and also has high fever. What do you think and what do you do? Danmark. Export-import. Because of increasing international air travel there is the possibility that sleeping sickness could be brought into Europe and in time could give rise to local european epidemics. What do you think about this statement? Zambia . A mission sister has high fever. She has constantly taken malaria prophylaxis. Should one think first of sleeping sickness? Congo . An Italian businessman visits the Equator region for the first time. He stays there for 6 weeks and gets fever. He is treated for suspected P. falciparum malaria. The spleen is swollen to 17 cm (total length). There are no swollen cervical lymph nodes. When examining him you notice 2 structures on a thick smear that morphologically resemble trypanosomes. What do you do? Should you give melarsoprol? In earlier times numerous slaves from trypanosomiasis areas were transported to America. Why did T. b. gambiense not become endemic there? Trypanosomiasis is currently spreading in areas with civil unrest. Any connection? Madagascar . A colleague reports on a blood smear from a local patient with fever: "gametocytes of T. brucei brucei". What is your interpretation of this information? North Angola . A 30-year-old woman is diagnosed as having late stage trypanosomiasis. The cerebrospinal fluid contains 50 white blood cells per mm3. One week after the start of the treatment a new lumbar puncture is carried out. The fluid now contains 100 leukocytes per mm3. Is this to be interpreted as failure of the therapy? Why was a lumbar puncture carried out? Sudan . Patients A and B both have trypanosomiasis. You are told that the cerebrospinal fluid of patient A contains 20 WBC (white blood cells)/mm3 and that of patient B 2000 leukocytes/ml. What do you think? How would you outline the management of the clinical problem of "confused patient", based on information about blood pressure, body temperature, history of acute infections or chronic problems, relevant medication, neurological evaluation including eye movement, glycaemia, thick smear test, blood examination, sickle cell test, CATT, HIV-status, lumbar puncture, VDRL results and cranial X-ray?

Summary

Trypanosoma cruzi, only in the New World Transmission via bugs, blood transfusion and congenitally, orally (bug faeces in food / drink) Importance of poverty (housing) in transmission Acute (especially children): chancre, Romaña's sign, fever, lymphadenophathy, myocarditis, hepatosplenomegaly Chronic: cardiac arrhythmias, heart failure, emboli, apical aneurysms Chronic: dysphagia, constipation (mega-syndrome) Diagnosis: clinical + thick smear/buffy coat (early), serology, xenodiagnosis, ECG, X-ray (late), PCR Treatment in the early phase still reasonably successful with medication; in the late phase difficult Benznidazole: problems with bone marrow toxicity, hypersensitivity, peripheral neuropathy. Prevention: much progress in recent years via vector control and control of blood banks.

Introduction

Trypanosoma cruzi. Notice the typical C-shape. Copyright ITM

Trypanosoma cruzi in thick smear. Copyright ITM

In 1907 the physician Carlos Chagas (1879-1934) was working in Lassance, a small poverty-stricken town on the Sao Francisco river in the state of Minas Gerais, Brazil. The town had been built along the railway from Rio de Janeiro to Belem. Chagas treated the workmen for injuries, syphilis, malaria etc. He noticed that cardiac arrhythmias occurred frequently. One day an engineer brought him an insect, of the type which was known to often suck the blood of humans at night. Chagas wondered if this creature could also transmit malaria, like the Anopheles mosquitoes. In the bug he discovered a unicellular parasite. In April 1908 he found the same parasite in a sick cat. Two weeks later, in the same house, the parasite was found in the blood of a 3-year-old child (Rita), who was ill with fever. Her face, liver, spleen and lymph nodes were swollen and the child died shortly afterwards. In the house there were countless bugs which tested positive for the parasite. He sent bugs to Rio, to Oswaldo Cruz, his former teacher (Brazilian physician 1872-1917). In the laboratory the parasite caused an infection in marmoset monkeys ( Callithrix sp. ), rodents and puppies. The disease caused by this parasite, American trypanosomiasis, was named after Chagas. The parasite was given the name Trypanosoma cruzi . The parasite did not always trigger disease, however. In 1908 Chagas also discovered the parasite in another person (Bernice). This woman died in 1989, still infected, but without signs of organ involvement.

The infection apparently already existed before contact with the West. In 1985, 22 mummies were found in the Andes mountains. These were 1500 years old. In approximately half of them the heart, colon and/or oesophagus were clearly enlarged (lesions typical for Chagas' disease). Trypanosoma cruzi  DNA was found in 1999 in a 4000 year-old mummy in Northern Chile. In one of his books Charles Darwin describes how in 1835 in South America he was bitten by the bugs. It is possible that he incurred infection and later developed a chronic form of the disease.

Charles Darwin, Voyage of the Beagle .

March 1835.

   " We slept in the village of Luxan, which is a small place surrounded by gardens, and forms the most southern cultivated district in the Province of Mendoza; it is five leagues south of the capital. At night I experienced an attack (for it deserves no less a name) of the Benchuca, a species of Reduvius, the great black bug of the Pampas. It is most disgusting to feel soft wingless insects, about an inch long, crawling over one's body. Before sucking they are quite thin, but afterwards they become round and bloated with blood, and in this state are easily crushed. One which I caught at Iquique, (for they are found in Chile and Peru,) was very empty. When placed on a table, and though surrounded by people, if a finger was presented, the bold insect would immediately protrude its sucker, make a charge, and if allowed, draw blood. No pain was caused by the wound. It was curious to watch its body during the act of sucking, as in less than ten minutes it changed from being as flat as a wafer to a globular form. This one feast, for which the benchuca was indebted to one of the officers, kept it fat during four whole months; but, after the first fortnight, it was quite ready to have another suck. "

Distribution

The infection only occurs in America in endemic regions. It is a disease associated directly with poverty. The severity varies from region to region. In the South of Texas there are very few cases. Infections occur in Central America sporadically. Although the disease is endemic in large areas of South America, the majority of those infected have no symptoms. Until recently it was thought that approximately 16 million persons were infected, but these figures are under review (see Prevention). The disease is transmitted via the faeces of an infected bug. These are blood-sucking insects which are widely distributed. The illness is characterised by an acute and a chronic phase. Not all infections lead to disease. Untreated, the infection can lead to a great deal of suffering.

Map of areas endemic for Chagas' disease. Adapted from ACM

Reservoir

The parasite, Trypanosoma cruzi , occurs in more than 100 species of mammal (opossums, guinea pigs, goats, dogs, cats, rats, mice, and so on). There are several known (and probably also some unknown) subtypes, each of which has its own distribution and probably also its own pathogenic features. In view of the extent of the animal reservoir, eradication of the parasite will not be possible. This does not mean that the disease and the transmission cannot themselves be controlled. At present the strains are divided into two groups. Trypanosoma cruzi I has an extensive sylvatic reservoir, of which opossums appear the most important. This group is not very common in the "Southern Cone" countries (Argentina, Brazil, Chile, Paraguay, Uruguay), but it is virtually the only form which occurs north of the Amazon region. T. cruzi II seems to be chiefly associated with rodents and is common in the Southern Cone.

Rural Bolivia, area endemic for Chagas' disease. Photo Cochabamba, Bolivia

Transmission

Eggs of Triatoma infestans , vector of Trypanosoma cruzi . Photo Cochabamba, Bolivia

Assasin bug, Reduviidae. The faeces of this insect sometimes contain Trypanosoma cruzi . Infection results in Chagas' disease. Copyright ITM

Chagas' disease. Reduvid bugs, vectors of American trypanosomiasis. Copyright ITM

Triatoma sp, vector of Trypanosoma cruzi . Photo Cochabamba, Bolivia

Transmission occurs chiefly via infected bugs. These large insects like to bite sleeping humans at night (a mosquito net gives protection). They have a sharp proboscis which at rest is folded below the head like a jack-knife. When biting they inject anticoagulants and an anaesthetic substance into the wound. Since this makes their bite quite painless (kissing bugs), people seldom wake up and several bites may take place unnoticed in the course of one night. The parasite is not inoculated directly by the bite, as Chagas initially thought. In 1913 Brumpt showed that the parasite is found in the faeces of the insect. While the animals suck blood, they defecate. By scratching, a bitten person can bring the faeces into the bite wound or rub them into the conjunctiva. The parasites multiply in humans and appear in the blood. The cycle is completed when a subsequent bug drinks infected blood. In the bug the parasite undergoes further changes and after 2 to 3 weeks is excreted with the faeces during a subsequent bite. It is estimated that the risk per bite by an infected Triatoma is one in a thousand. The existence of oral tranmission has been supected for quite a while. It was demonstrated in animals and has now been confirmed in some human cases. How frequent oral transmission happens is not clear yet. Food or drink contaminated with the liquid faeces of infected bugs or containing (crushed) dead bugs may lead to infection in experimental animals.  It was shown that contaminated edible palm ( Euterpa oleraceae ) can become contaminated with T. cruzi. The parasite coudld withstand short periods of freezing, but not decontamination with sodium hypochlorite or heating to 80°C. Congenital infection (1 to 2 % risk) and transmission via blood transfusion also occur (poor people often sell their blood). To give an idea of the scale, this implies for example that several thousands of babies are born wich congenital Chagas each year in the USA, and a lesser number in Europe (from immigrant mothers from endemic areas). Transmission via transfusion is particularly important in urban zones. The risk of infection after an contaminated blood transfusion is estimated at one in five. There are sporadic cases of accidental contamination of laboratory staff (finger prick, aerosol) and after organ transplantation.

Vectors

The bugs are also known locally as "vinchucas" or "barbeiros". The latter common name refers to the blood-sucking (since in the olden days the barber carried out blood letting as well as shaving). Of the approximately 120 vector species only about 7 are important. Each species has its own region of distribution:

Central America and northern South America: Triatoma dimidiata  and Rhodnius prolixus

South America (south of 5° S): T. infestans , T. braziliensis , T. sordida , Panstrongylus megistus

The bugs mentioned here are the main vectors. Other bugs also play a part ( Triatoma barberi  [Mexico], Rhodnius pallescens  [Panama and Colombia], T. phyllosoma  group [Mexico] and T. protracta . Twelve species of triatomines are known to occur in the United States, the most important being Triatoma sanguisuga  in the Eastern United States, Triatoma gerstaeckeri  in the region of Texas and New Mexico, and Triatoma rubida  and Triatoma protracta in Arizona and California. The bugs each have their own preferred biotopes. T. dimidiata , for example, is often found inside houses on the floor or the lower 150 cm of the walls or immediately outside in dung heaps, hollow trees, etc. In contrast, R. prolixus prefers to live in palm leaves either in the roof of the house or in the tree itself. In and around the house the bugs can feed on animals (e.g. dogs are important because they sleep at night, when the bugs are active). The vectors often live in chicken runs, but chickens themselves are not infected (they do eat bugs). During the day the insects hide in all kinds of cracks and crannies (importance of earthen or adobe walls) and in the roofing (straw, wood, etc). It can be seen immediately that the key word in Chagas' disease is "poverty". These are insects which reproduce slowly and whose geographical spread is slow. Migration of bugs, by migrating birds for example, still needs to be studied. In view of these characteristics and the fact that the important vectors live around houses, they can easily be reached by eradication campaigns.

The adult insects measure 2-3 cm. The front wings (hemi-elytra or hemelytra: "half wing sheath"[Gr. elytron = cover]), consist of a hardened foremost part (divided into corium and clavus) and a rearmost membranous part. This order is named "Hemi-ptera": "half-wings"[Gr. pteron = wing]) after their wing construction. The name Heteroptera is used for the suborder which includes the bugs, while the other Hemiptera are classified in the suborder Homoptera. Each species has a typical morphology and colour pattern which includes connexival markings. The latter are markings (red, orange, yellow) of the lateral edges of the abdominal tergites [Lat. "tergum" = back; tergite = sclerosed dorsal segment]. They are visible because the wings are folded over each other so that the back is not fully covered. Naturally the identification of bugs is very important in the evaluation of vector control programmes. Differentiation between re-infestation from neighbouring areas or insufficient local control can be made by genetic analysis of the bugs.

A fertilised female lays several hundred eggs in her lifetime. From the egg comes a nymph which always needs a blood meal for its subsequent development stages (both sexes suck blood). The last instar will develop into an adult insect. During a blood meal they suck more than their own weight in blood. This takes 10-25 minutes. Infestin is an anticoagulant protein from the midgut of Triatoma infestans . Genetically engineered recombinant infestin molecules are studied for possible clinical applications, e.g. in cardiovascular medicine. The insects may live for up to 2 years (5 years for T. barberi ). Rhodnius prolixus has a relatively short generation time (3-5 months), while for T. dimidiata this time is quite long (1 year or longer). Long generation times make the development of resistance to insecticides difficult.

Nitrophorins Rhodnius prolixus is capable of ingesting 300 mg of blood in 15 minutes. The insect has a sophisticated mechanism of avoiding blood coagulation, platelet aggregation and vasoconstriction. Nitrophorins are ferric hemoproteins found in saliva of certain blood-feeding insects. Saliva of  R. prolixus contains several varieties. In the slightly acidic (pH 5) environment of the insect saliva, these hemoproteins contain nitric oxide (NO) ligated to the ferric heme iron (Fe 3+ ). During feeding, dilution, binding of histamine and decreasing acidity (pH host tissue = 7.4) facilitate the release of NO into the tissue where it induces vasodilatation and inhibition of platelet aggregation. This facilitates feeding of the insect. Histamine is released by the host in response to the insect bite. Histamine is also a nitrophorin ligand. Nitrophorins therefore act as antihistamines, diminishing itchyness at the bite site and promoting vasodilatation at the same time. 

Note : Taxonomy Hemiptera

The Hemiptera are divided into : Heteroptera : true bugs Homoptera : aphids, spittle bugs, whiteflies, scale insects, mealy bugs, cicadas, leafhoppers,  treehoppers

Note : K- and r-selection

The bugs exhibit strong K-selection, unlike for example mosquitoes which exhibit r-selection.

K-selection means selection for maximum competitive fitness, the strategy of species which are in balance with their environment. In stable surroundings rapid population growth is not important. K-selected species are often characterised by large individuals, a small number of offspring, long-term survival of the offspring, slow development even in a stable environment or where the changes are foreseeable. There is little random fluctuation and the competition between individuals is very intense. There will be heavy investment in survival strategies.

r-selection , on the other hand, is selection for maximum biotic potential (reproductive potential), in such a way that when favourable circumstances arrive, the species may increase explosively and a biotope may be rapidly colonised. This type of species (opportunists) has many offspring and/or a short generation time. Such a strategy is favourable in fast-changing and unforeseeable circumstances. The population size may show extreme fluctuations over short periods of time. The mortality of adults and immature animals is highly variable, unpredictable and independent of the population density. The animals invest little in survival strategies, but more in maximising the number of offspring in the short term.

The terms r-selection and K-selection were proposed by MacArthur and Wilson (1967) and are derived from the so-called "logistical equation", a term originating from a certain Verhulst in 1838. When populations increase, in the course of time the growth speed can be represented by differential equations: increase in the number of individuals (dN) over a certain time span (dt) or: dN/dt. The extent of this increase compared to the total number of individuals (N), gives the intrinsic growth: (dN/dt)/N = r. But a population cannot of course continue exponential growth indefinitely. At a given moment the carrying capacity of the environment is reached (K) and mutual competition will impede further growth. The closer the number of individuals N approaches the carrying capacity, the less the growth per individual. From this follows the logistical equation: dN/dt = rN[(K - N)/K]. The concept of r-selection and K-selection should not be taken too far, since it is a simplification of reality.

Parasite

Trypanosoma cruzi in thick smear. Copyright ITM

Trypanosoma rangeli , a non-pathogenic blood parasite which can be mistaken for Trypanosoma cruzi. Copyright ITM

In stained blood preparations the parasites are C- or S-shaped with a prominent kinetoplast towards the rear (trypomastigotes). The nucleus is elongated and the undulating membrane is usually not clearly visible. After infection, multiplication of the parasite in the human is solely intracellular. They form microscopic pseudocysts in the tissues (similar to toxoplasmosis and sarcocytosis). This occurs mainly in the heart, muscle cells, some nerve cells and the lymphatic system. In the cell the parasite is small and rounded, with no flagellum (amastigote). When the infected cell ruptures, the parasites are released into the blood circulation where they become elongated and develop a flagellum. These forms can then infect other cells or be ingested by a bug.

An important protease for the parasite is lysosomal cruzipain (cruzain), a substance related to papain. This is being investigated as a possible therapeutic target. An unusual parasite-derived proline racemase was identified as a B-cell mitogen, resulting in polyclonal B-cell activation. The surface of the parasite is covered by mucin-type glycoproteins that attach to the membrane by glycosylphosphatidylinositol (GPI) anchors, similar to those of the variant surface glycoproteins in African trypanosomes. Mucins are recipients of the sialic acid transferred by surface trans-sialidase. T. cruzi has several hundreds of such mucin genes. The exposed N-terminal moiety of the molecules is hypervariable. It is postulated that this might play a role in immune evasion. Lateral DNA transfer from T. cruzi to the human genome, including the human germ line, has been demonstrated. In such interspecies DNA transfer, mitochondrial minicircle DNA was integrated into human chromosomes, leading to subsequent inheritance by the children.

Note: T. cruzi energy sources

The parasite appears to use the amino acid histidine as an energy source during its development in insect vectors, but uses fatty acids when residing in mammalian cells. Knowledge of stage-specific biochemical pathways may aid in selection of targets for drug intervention.

Note: Trypanosoma rangeli

Trypanosoma rangeli was first described in 1920 by Tejera, when examining the intestinal contents from R. prolixus. The first human infection was found in Guatemala. Its geographical range is from Central America to a large part of South America, in both humans and animals. There are no amastigotes in humans. The undulating membrane is prominent and the kinetoplast at the pointed tip is small. This parasite is transmitted via triatomine bugs. After the vector is infected during a blood meal, the parasite migrates from the intestine to the hemolymph and then to the salivary glands of the insect, where infective metacyclic stages will be formed. Transmission to humans is via the saliva injected during the bite (in this aspect it differs from T. cruzi , which is transmitted via the faeces of the insect). The parasite is apathogenic and in general does not survive in humans longer than one year (exceptions exist). There is no cross-reaction with the antigens currently used in Chagas' disease serology.

Clinical course

Clinical course, infection

Incubation period after exposure to vector-borne T. cruzi is 1 to 2 weeks, althogh longer incubation times are sometimes reported. If the parasites penetrate via the conjunctiva, there is unilateral redness and oedema of the upper and lower eyelids after 4 to 12 days. This is Romaña's sign, named after the Argentinean physician Cecilio Romaña, who described the oedema in 1935. This swelling may last for weeks. Sometimes there is also swelling of the ipsilateral lymph nodes (including the pre-auricular lymph nodes). Trypanosomes may be found in the tears at this stage. If inoculation is in the skin there is local oedema and redness (chagoma) in 75% of cases. This remains for 1 to 4 months. From these sites the infections spreads.

Romaña's sign. Early stage Chagas' disease. Photo Cochabamba, Bolivia

Romaña's sign. Early stage Chagas' disease. Photo Cochabamba, Bolivia

Romaña's sign. Early stage Chagas' disease. Photo Cochabamba, Bolivia

Clinical course, acute stage

The incubation period is followed by the acute phase, which lasts 4 to 8 weeks. Many infections are initially asymptomatic. Acute symptoms occur more frequently in children than in adults. Dissemination of the parasite from the inoculation site may go unnoticed but may also give rise to acute illness with muscle pain, local or generalised oedema, swollen liver, spleen and lymph nodes. Moderate fever is almost always present in symptomatic cases and may persist for a long time, two or even four months. Sometimes there is also acute inflammation of the heart (myocarditis) with arrythmias, decreased blood pressure, and heart failure. As with other forms of myocarditis the echocardiogram is frequently abnormal. There is low QRS-voltage, prolonged PR- and/or QT-interval, T-wave abnormalities. Rarely there are ventricular extrasystoles or atrial fibrillation (the prognosis is poor if this occurs). Acute inflammation of the brain and meninges (meningo-encephalitis) occurs, chiefly in young children. Inflammation of the heart and brain may be fatal. There is pronounced lymphocytosis and monocytosis. The acute-phase case fatality rate is estimated to be 0.25 to 0.50%.

Clinical course, latent period

If the patient survives the initial phase (which is usually the case), a latent period occurs of indeterminate duration. The patient is asymptomatic, seropositive and the parasitaemia is very low. Focal lesions are found in 60% of endomyocardial biopsies from patients in the latent phase. A positive xenodiagnosis can be obtained in 50% to 100% of these patients. For xenodiagnosis 10 to 40 non-infected bugs (e.g. Dipetalogaster maxima  or Triatoma infestans ) feed on blood from the patient. The faeces from these animals are investigated after 30, 60 and 90 days. In the event of immunosuppression there may be an acute flare-up, including meningo-encephalitis associated with AIDS or heart transplantation.

Clinical course, chronic phase

Gradually the patient develops symptoms. These vary greatly from region to region. Lesions of the heart, oesophagus and colon are the most common.

Chronic heart problems

Chronic damage to the heart muscle cells and the cardiac conduction system (including that caused by auto-immune mechanisms) leads to heart failure. There is dyspnoea during exertion, orthopnoea and sometimes paroxysmal nightly dyspnoea, oedema of the feet and ankles, congestion of the neck veins, enlarged liver, crepitations over the base of the lungs. Cheyne-Stokes respiration may occur in advanced heart failure. This phenomenon is characterised by periodic respiration in which apnoea episodes alternate with hyperventilation. It is assumed that the prolonged lung-to-brain circulation time plays a role in Cheyne-Stokes with a long cycle (e.g. 3 minutes) [Other causes of this phenomenon are for example brain stem lesions or compression]. Sometimes there is pulsus alternans: the peripheral arterial pulsations are alternately strong and weak. The precise physiopathological mechanism is not fully known. The apex of the heart, which is normally situated on the midclavicular line, is displaced to the left. The heart sometimes becomes enormous, which may lead to clot formation in the heart. If blood clots break loose, there may be embolic complications: CVA, ischaemia of limbs, renal infarction. Apical lesions in the left ventricle (wall thinning, intramural bleeding, aneurysms) are typical and occur in approximately 50% of patients. Unlike arteriosclerotic post-infarction aneurysms, in Chagas' disease the apical cardiac tissue does not consist of scar tissue, the wall is simply thinned. Right ventricular lesions occur in 10 to 20%. Cardiac arrhythmias may cause palpitations, dizziness, syncope and sudden death. On the electrocardiogram a right bundle branch block is often seen, together with a left anterior hemiblock, ventricular extrasystoles, abnormal Q-waves and/or AV-conduction disturbances. The coronary arteries are normal. A complete left bundle branch block is exceptional, unlike in idiopathic dilated cardiomyopathy. Sudden death is common in people with Chagas' disease. Probably this is due to ventricular tachycardia which changes suddenly into ventricular fibrillation.

In advanced heart failure, typical radiographic signs may be observed on a chest X-ray: cardiomegaly, prominent hili and distended pulmonary veins in the upper fields, pleural fluid, interstitial pulmonary oedema (fluid in the interlobular septa with Kerley B lines), peribronchial cuffing and finally alveolar pulmonary oedema ("butterfly oedema").

Megacolon secondary to chronic infection with Trypanosoma cruzi . Photo Cochabamba, Bolivia

The degree of heart failure is often indicated using the New York Heart Association classification:

Grade I             : asymptomatic

Grade II            : symptoms only during moderate to severe exertion

Grade III           : symptoms during mild exertion

Grade IV           : symptoms at rest. Patient generally confined to bed/chair.

Oesophagus and colon problems

Due to involvement of the small nerves in the oesophagus and colon, peristalsis is reduced and these organs are distended. This occurs in 5 to 10% of seropositive people south of the Amazon, and is virtually absent further north. Trypanosoma cruzi I  and II are both associated with cardiac lesions, but apparently intestinal lesions only occur in infection with T. cruzi II (the southern area).

Mega-oesophagus is characterised by difficulty in swallowing (dysphagia), choking, hiccups, nocturnal cough. This often leads to under-nourishment and loss of weight. A clinical aid for detecting delayed oesophageal emptying is to measure the time between swallowing a mouthful of water, and observing the abdominal noises (stethoscope on the epigastrium). Normally this is less than 10 seconds. A distended oesophagus may also be shown on X-ray. The parotid gland may hypertrophy and lead to so-called "cat's face".

Mega-colon can lead to pronounced constipation, meteorism, abdominal pain and functional intestinal obstruction (involvement of Auerbach's myenteric plexus). The abdomen is often distended. Faecaloma, volvulus and peritonitis are complications.

The nervous system

In no other infectious disease is the involvement of the autonomous nervous system so important as in Chagas' disease. Denervation of the parasympathetic nervous system is better documented and is much more pronounced than denervation of the orthosympathetic system. There can be sensorimotor polyneuritis. There is some hypoaesthesia and paresthesia, but chiefly a reduction or loss of tendon reflexes. The EMG is disturbed. In the central nervous system there is meningo-encephalitis in the acute phase, but the abnormalities in the chronic phase need to be better defined. In flare-up (e.g. AIDS) there may be intracranial hypertension, lesions of the cerebral nerves, paresis, plegia, stupor and convulsions. The cerebrospinal fluid exhibits a normal number of cells or pleocytosis with predominant lymphocytes and an elevated protein content. At times T. cruzi may even be detected in the cerebrospinal fluid. A CT scan of the brain shows one or more necrotising lesions which may or may not be ring-shaped, with haemorrhages, usually subcortical in the brain hemispheres and occasionally in the cerebellum or the brain stem. T. cruzi lesions rarely occur in the basal nuclei. These clinical pictures should be differentiated from cerebral toxoplasmosis, abscesses, mycoses, tuberculomata or other mycobacterial lesions, metastases or lymphoma. Of all cerebral vascular accidents leading to stroke, about 20% are secondary to embolism from a blood clot secondary to atrial fibrillation. If patients do not take oral anticoagulants, an average of 5% CVA's per year can be expected, which roughly translates to 50% of patients with CVA within 10 years after onset of atrial fibrillation. However, for several reasons (mostly hemorrhagic) 20-40% of patients cannot be treated with oral anticoagulants. Most of the clots (90%) originate when blood stagnates in the left atrial appendage, also known as the left atrial auriculum. At present, it is possible to insert via the femoral vein a so-called catheter-guided Watchman device. This device is inserted in the left auriculum and secludes it, so that no more clots can escape from this area. At present the price is still high and is only available in certain cardiological centres.

Child with hepatosplenomegaly due to congenital Chagas' disease. Photo Cochabamba, Bolivia

Child with hepatosplenomegaly due to congenital Chagas' disease. Photo Cochabamba, Bolivia

Congenital Chagas' disease in twins. Photo Cochabamba, Bolivia

Clinical course, congenital infection

About 1 to 2 % of babies born to seropositive mothers are infected. They may be asymptomatic (rarely) or may develop hepatosplenomegaly, neurological involvement, myocarditis, oedema and a bleeding tendency. The babies may be dysmature and/or premature. Fever is rare in these children. The mortality may be as high as 50% and they tend to die within a week. Those who survive will generally have permanent residual neurological damage.

Diagnosis

Trypanosoma cruzi in thick smear. Copyright ITM

Boxes with non-infected Dipetalogaster maxima or Triatoma infestans , used in xenodiagnosis of Chagas' disease. Copyright ITM

Boxes with non-infected Dipetalogaster maxima or Triatoma infestans, used in xenodiagnosis of Chagas' disease. Copyright ITM

In the acute stage the parasite may be found in the blood via a thin blood smear, thick smear or buffy coat. As a concentration technique an anion-exchange minicolumn may be used (Woo's technique similar to Lanham's column, but with a different buffer, see African sleeping sickness). Strout's concentration technique includes the double centrifugation of serum (from 10-20 ml of blood), after which the motile trypanosomes can be detected in the sediment. PCR techniques for T. cruzi   exist, but can only be carried out in better equipped laboratories. The serology is positive from the fourth week. To know whether the neonate from a seropositive mother is infected, PCR is performed and IgM antibodies in its blood are determined. A positive serology (IgG) 6 months after birth also indicates infection. In-vitro and in-vivo culture is possible, but usually not available. Biopsies of lymph nodes, heart and muscles sometimes show parasitic pseudocysts (amastigotes in the cells). This is quite an aggressive technique, however, and not very sensitive. Several chronic cases have been described where the parasites were seen on a blood smear, the PCR was positive, but the classical serological tests (ELISA, IFA, HAI) as well as anti-cruzipain antibodies were negative. How frequent this situation is, is not clear at present.

Note: Dipetalogaster maximus

Dipetalogaster maximus is a blood sucking bug which can take up to 4 ml of blood in one meal. It is best known for its use in xenodiagnosis of Chagas' disease. An original way in which the insects are being put to work is blood sampling of wild animals that are difficult to sample in any other way. The technique was already used to measure stress hormone levels in nesting terns without having to catch them (which is stressful in itself to the birds). The insects are put into artificial eggs with tiny holes big enough for their probosces. The loaded eggs are then slipped into the bird's nests. Blood samples can be removed from the insect by inserting a needle into their abdomen. Rabies survey in bats might also use a related technique.

Differential diagnosis

Broadened mediastinum due to mega-oesophagus in Chagas' disease. Copyright ITM

Mega-oesophagus in Chagas' disease can resemble achalasia. Copyright ITM

Chagas' cardiomyopathy. Differentiation from ischaemic, hypertensive or idiopathic cardiomyopathy is not always easy. The differential diagnosis includes high-output heart failure (anaemia, beriberi, hyperthyroidism, large AV-fistula and Paget's disease of the bone), postpartum heart failure, acute rheumatic fever, valvular disease, congenital abnormalities, pericardial disorders and the sequelae of acute myocarditis (e.g. Coxsackie virus). A cor pulmonale is usually obvious. It is useful to have an electrocardiogram available and if possible an ultrasound evaluation.

Mega-oesophagus: Achalasia of the oesophagus may be very similar to Chagas' oesophageal dilation. Strictures, benign and malignant tumours should be ruled out.

Megacolon may also occur in Hirschsprung's disease [congenital megacolon] [aganglionosis of a rectal segment, frequency 1/5000], diabetic neuropathy, psychogenic (mainly in psychosis, schizophrenia), after stricture (e.g. upon ischaemic insult of the colon or rectal stricture due to schistosomiasis or lymphogranuloma venereum), chronic severe laxative misuse (senna, cascara, aloe), neurological diseases (Parkinson's disease, myotonic dystrophy), chronic use of morphine analogues, chronic lead intoxication, Fabry's disease (glycolipid accumulation), systemic sclerosis (scleroderma), severe hypothyroidism and amyloidosis.

Prognosis

In an endemic region an asymptomatic person with positive serology is probably a carrier (xenodiagnosis positive in 50 to 100 % of cases). The percentage of seropositive persons who develop symptoms is highly dependent on the geographical region (e.g. 10 to 30%). Asymptomatic mega-organs occur. Chagas' disease variables associated with adverse outcome 2 points: Male 2 points: Low QRS voltage on ECG 3 points: Nonsustained ventricular tachycardia on 24-h Holter monitoring (run of 3 or more consecutive VES, with a frequency >100). 3 points: Left ventricular systolic dysfunction: segmental or global wall-motion abnormality on echocardio (quid apical aneurysms, intracavitary thrombus) 5 points: NYHA III or IV 5 points: Cardiomegaly present on CXR, defined as a cardiothoracic index > 0,5 Results : 0- 6: low risk 14% mortality rate in 10 years 7- 11: intermediate risk 44% mortality rate in 10 years 12-20: high risk 84% mortality rate in 10 years

 

Treatment

Treatment, clinical situations

 Acute phase The acute phase lasts up to 60 days. All patients who are in this phase should be treated.

 Congenital infection All infected children should be treated. The earlier therapy is begun, the better the results.

 Chronic phase Aetiological drug treatment is indicated for "recent" chronic infections (a few years). In practice all children younger than 10 years are treated. If mega-oesophagus is already present, the dysphagia should be treated (the passage and absorption of oral medication may be severely impeded). Aetiological treatment in these latter patients was not advised formerly, but more recent data have brought this into question. In a study in Argentina, 131 patients with chronic Chagas' disease were treated with benznidazole. After an average follow up of 8 years, 4.2% exhibited ECG changes compared to 30% in the untreated group. There was also considerably less clinical deterioration in the treated group (2.1% compared to 17%).

 Accidental infection This may occur, for example in laboratory staff. A serum specimen should be frozen before beginning treatment and a second blood sample taken 4 weeks later. Serology is performed on these paired sera. Benznidazole 7-10 mg/kg/day x 10 days is the usual treatment regimen in this situation.

 Transplant patients There are two possible situations: transplantation of an infected organ into a non-infected patient and transplantation of a healthy organ into an infected patient. A donor may be infected so that the recipient becomes infected. Normally the donor is tested beforehand, and positive donors are refused, but nevertheless these situations sometimes occur. Alternatively, a transplantation may be carried out on a patient who is a chronic carrier. The immune suppression that these patients undergo [steroids, azathioprine (Imuran®) and cyclosporin (Sandimmun®)], may lead to reactivation of Chagas' disease. In both cases treatment with benznidazole 5 mg/kg/day x 60 days, is indicated.

 HIV patients and Chagas Infection with HIV may lead to significant flare-up of Chagas' disease. In endemic regions all HIV patients should be monitored for Chagas' disease. If positive, benznidazole is recommended. There are insufficient data concerning chemoprophylaxis. Since the initial step is often serology, one would normally first try to confirm the diagnosis with a second serological test (ELISA-based preferably) and by looking for circulating parasites by microscope (QBC or buffy coat), PCR and perhaps xenodiagnosis. If the diagnosis is confirmed, the patients deserve to be treated as their risk of severe complications (cardiac, digestive or CNS) is high. Benznidazole is preferred to nifurtimox, since nifurtimox is a treatment that is really badly tolerated in adults (notably a lot of nasty allergic reactions). Benznidazole 5mg/kg (max 300mg) daily for 60 days is not an easy treatment to administer neither (beware of skin toxicity !).

 Pregnancy

Treatment during pregnancy is not recommended, although congenital Chagas has been well documented. It is clear that more understanding and better outcomes are sorely needed.

TREATMENT,  DRUGS

Radanil, benznidazole. This drug is used in the treatment of Chagas' disease. Copyright ITM

There are several problems. The drugs have an unsatisfactory cure rate. The chronic lesions may be caused by auto-immune mechanisms and might not be improved by eradicating parasites. [Nevertheless the role of auto-immune mechanisms should not be exaggerated: the disease worsens during immune suppression as in transplantation and in HIV]. The drugs should be given long term. Results vary from country to country, possibly due to a difference in sensitivity of the parasites. Side effects occur, more often in adults than in children. It is best to avoid steroids and possibly tetracyclines, since these may exacerbate the infection.

Nifurtimox  (Lampit®) 5 mg/kg/day orally, slowly increased to 15 mg/kg/day (divided over 3 doses) for 2 to 4 months. The drug is no longer produced. Side effects: neurotoxicity (insomnia, tremor, polyneuritis), nausea, leukopaenia, thrombocytopaenia or hypersensitivity. May cause haemolysis in G6PD deficiency [glucose-6-phosphate dehydrogenase]. In the acute phase the parasites disappear from the blood in 80 % to almost 100 % of cases. The actual cure rate is 50-60%.

Benznidazole  ( syn. benzonidazole: Radanil®, Ragonil®, Rochagan®) 5-10 mg/kg/day orally for 1 to 2 months. Administration (generally 100 mg tablets) is twice daily. The same side effects as nifurtimox, but less frequent and less pronounced, although skin rash occurs relatively frequently (up to 30% of patients), sometimes accompanied by swollen lymph nodes. The pharmaceutical company Roche has donated all commercial rights and the technology to manufacture benznidazole to the Brazilian government.

Allopurinol  (Zyloric®): 600 mg/day orally for 2 months (adult dose). The place of this therapy has not yet been determined, but it is currently used following flare-ups after heart transplantation.

Experimental:  posaconazole, squalene oxidase inhibitors such as terbinafine, proteasome inhibitors, trans-sialidase inhibitors, fexinidazole. Ravuconazole is a new triazole with in vitro activity against species of Candida , Cryptococcu s and Aspergillus , but also in vitro and in vivo (mice) activity against Trypanosoma cruzi . Ravuconazole has a long half-life in humans, which hopefully will facilitate compliance in patients. Clinical trials for its use in Chagas’ disease are planned for 2010, in Bolivia.

T. cruzi is auxotrophic for polyamine biosynthesis, since it does not have ornithine decarboxylase, and is thus naturally refractory to the effects of eflornithine.

In the chronic phase the usefulness of these drugs is doubtful, but the tendency is more and more towards treatment. Symptomatic therapy is indicated: oesophageal sphincter dilation, extramucosal cardiotomy (Heller's operation), colon surgery. An experimental treatment is the endoscopic injection of botulin toxin into the distal oesophageal sphincter (e.g. 20 U into each quadrant).

In heart failure, diuretics, ACE-inhibitors and antiarrhythmic drugs may be beneficial. Beta-blockers are best avoided in view of the AV-conduction problems and bradyarrhythmias. Aspirine or anticoagulants are indicated for patients with atrial fibrillation, previous embolic phenomena and apical aneurysms. Amiodarone (Cordarone®) is effective in more than 50% of patients who develop ventricular extrasystoles or ventricular tachycardia. A bifascicular or trifascicular conduction block, also a second or third degree AV-block are contra-indications. Amiodarone is an iodine-containing Vaughan-Williams class III antiarrhythmic drug. It may cause thyroid problems (either hypothyroidism or hyperthyroidism). Reversible deposits in the cornea, pulmonary toxicity, neuropathy, photosensitivity and grey discoloration of the skin may occur. The elimination half-life varies greatly from individual to individual (20 to 100 days). A high incidence of "torsades de pointes" has been observed during use of quinidine, disopyramide and other class I antiarrhythmic drugs. A beta-blocker can be used if amiodarone is contra-indicated. Patients with cardiac problems are generally very sensitive to digitalis (lower dose necessary). Pacemakers and cardiac surgery are reserved in practice for those with financial means and these persons have an inherently low risk of infection. Pacemakers can be used in cases of severe bradyarrhythmia. Implantation of an automatic defibrillator is indicated for patients with recurrent ventricular tachycardia or a history of cardiac arrest. Life-threatening dysrhythmia can be treated with an implanted pacemaker/defibrillator. For terminal heart failure, a heart transplant can be performed, but it is obvious that such costly procedures will be not within the financial means of the average Chagas' patient.

Prevention

Prevention, diverse methods

The animal reservoir of Trypanosoma cruzi  cannot be eradicated. There is no vaccine. Chagas' disease is typically a disease of poverty. Improvements in housing (brick or plaster walls, corrugated iron roofs, long-acting insecticides on house walls) diminish the insect population. A mosquito net has also proved its usefulness here. Serological testing of the blood used for transfusion is very helpful. In 1953 it was discovered that adding gentian violet kills the trypanosomes in 24 hours (0.25 g/litre of blood = 1/4000). This colours the blood purple, however. If 2 mg/ml of ascorbic acid (vitamin C) is also added and the blood is illuminated with a 75 Watt fluorescent lamp, the time necessary to sterilise the blood is greatly shortened (30'). To date the various biological methods of eradication of the vectors which have been tested (increasing natural enemies) have not been effective because a new ecological balance is very quickly achieved.

Prevention, National Chagas' Control Program of Brazil

This programme was begun in Brazil in 1975. The intention was to reach the whole country and the strategy included spraying houses with insecticides. In Sao Paulo the vector Triatoma infestans was eliminated in 1982. Between 1983 and 1993 reductions of house infestation, ranging between 100% (Mato Grosso) and 80% (Goias) were observed in 8 of the 11 endemic states. In the states Bahia, Tocantins and Rio Grande do Sul the results were less spectacular. Nevertheless further successes are expected in the coming years. The total incidence of seropositivity in children from 7-14 years fell by 96% between 1980 and 1994. In 1998 there was still one T. infestans per 10,000 houses. A much higher frequency would be required to maintain transmission of T. cruzi . In 2006, Brazil has been certified free of Chagas' disease transmission due to Triatoma infestans . It is reasonable to expect the transmission will be completely interrupted.

Map showing the distribution of Triatoma infestans before and during the Brazilian Chagas' disease control programma.

Prevention, Southern Cone Initiative

In 1991-92 the "Southern Cone Initiative" project was launched by Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, with the objective of stopping the transmission of Chagas' disease. In 1997 Peru joined the project. After an initial phase for preparation (charting the foci, programming the activities, calculating the costs), there was an attack phase with insecticides, repeated after 3 to 6 months. Insecticide-containing paint is cheaper than the traditional insecticides which are applied by spraying. Insecticides dispersed by fumigant canisters were also used. These are locally produced, e.g. in Argentina, are cheap, effective and also active against Aedes aegypti , the important dengue vector. At present there are effective colourless long acting insecticides. The fact that people see the bugs, cockroaches, etc. lying dead after spraying, is a bonus which makes it easier to accept the spraying procedure. In the Southern Cone Initiative, 1,800,000 houses were treated with pyrethroids (deltametrine, lambda-cyhalotrine, cyflutrine) by the year 2000.

Progress made in the fight against Chagas' disease. Southern Cone initiative. Adapted from WHO

Since then there has been further selective treatment of the houses which still exhibited infestation with triatomes. Simple "sensor boxes" of cardboard (traps for the bugs) were placed in the rooms and the occupants themselves could simply ascertain the presence of triatomes. The last phase is surveillance for the detection of residual foci. This is decentralised and involves the population. The effectiveness of the control programme has been demonstrated by the very pronounced drop in seropositivity among young children. The surveillance phase has been reached in 6 countries of the Southern Cone. At present there are several South American countries (Colombia, Ecuador, Venezuela) which have a national control programme. Similar programmes were begun in Central America in 1997: Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, Mexico and Panama. These programmes can only be successful if there is participation of the population and if they can be continued for long enough. The latter is a political decision. There have been investigations of the possibility to eliminate the pathogen from vector populations through paratransgenesis. This technique involves genetic modification of the vectors symbionts. Rhodnius prolixus carries the bacterial symbiont Rhodococcus rhodnii . These bacteria can be grown in vitro and can be genetically modified, e.g. through transformation with a plasmid containing a gene for a protein like cecropin A which is toxic for Trypanosoma cruzi . The modifed stable symbiont is then used to infect the vector, making the insect refractory to infection with T. cruzi .

Prevention, survey of control initiatives

The Southern Cone Initiative began in 1991-92. Many countries have now been declared free from transmission by the PAHO [Pan-American Health Organization].

The Central American Initiative was begun in 1997-98 in Guatemala, Honduras, Nicaragua, El Salvador, Panama.

The Andean Pact Initiative was set up in 1997-98 in Venezuela, Colombia, Ecuador. Northern Peru began surveys during the same period.

The Mexican Initiative was begun in 2000-1. In 2001 this was still at a very early stage.

In 2001 there was still no control or surveillance in the Guyanas, Surinam and Costa Rica. In July 2007 the WHO Global Network for Chagas Disease Elimination was launched in order to coordinate global efforts to eliminate this disease.

Exercises

Someone from Brazil has lived for 10 years in Belgium. One day he suddenly develops pain in his right leg. The leg is pale, cold and no arterial pulsations can be felt in the foot. Could this have something to do with Chagas' disease? Is urgent therapy with Lampit® advisable? Why is oesophageal sphincter dilation beneficial in Chagas' disease if the oesophagus is already distended? A mother who is seropositive (for Chagas' disease) gives birth to her first child. She asks you if you know what the chances are that the baby is infected, and how you can test for that. Is congenital infection serious? Belgium . A 50-year-old man consults you about palpitations. He lived for 7 years in a region endemic for Chagas' disease. Buffy coat, thin blood smear and thick smear are all negative. What do you think? Brazil . A 27-year-old woman suddenly develops fever and pain on the left side of her face. The skin is warm and red, and a few days later the left eyelid also swells up. Differential diagnosis? A colleague from southern Peru claims that Chagas' disease only causes cardiac problems. Another colleague from Nicaragua says that Chagas' disease is a highly exaggerated infection which, when it occurs, is almost always asymptomatic. A third person from southern Brazil disagrees totally and claims that swallowing disorders and heart problems are just as important and both are common. What is your contribution to this discussion? Brazil . Why should improvement in the housing in a region reduce the incidence of Chagas' disease? Brazil . A man complains of chronic constipation and cough at night. The previous year he suffered a severe pulmonary infection 4 times. You find no trypanosomes in his blood. What do you think and what do you do? A 23-year-old woman is brought to a hospital in Paraguay. She has fever, the spleen is enlarged and the blood pressure is 90/60. Should you begin Lampit® immediately? Brazil . A 68-year-old man is admitted due to acute discomfort of his whole left calf and foot. At physical examination the leg is colder than the right one. It is pale and when asked the patient reports paraesthesia. After a few hours there is loss of strength and he reports pain. Both femoral arteries are normal. The arteria dorsalis pedis is not palpable at the dorsum of the foot and the arteria tibealis posterior behind the inner ankle is also not pulsating. Do you suspect occlusion of the arteria femoralis superficialis? If so, where will the embolus probably be? What might be the underlying disease?

Summary

In humans obligatory intracellular parasite with replication in macrophages Cutaneous form: chronic painless ulcers or nodules, amastigotes in smear Visceral form: chronic fever, hepatosplenomegaly, pancytopenia, persistent inflammatory state. Lethal if not treated Diagnosis of kala azar: amastigotes in bone marrow and other sites, serology, antigen detection Mucocutaneous: chronic destructive lesions in mouth/nose, frequent clinical diagnosis Transmission via about 30 species of sandflies Zoonotic transmission: animal reservoir (especially dogs and rodents) Anthroponotic transmission: human reservoir, e.g. Indian kala azar and in cutaneous L. tropica Treatment with antimony derivatives, pentamidine, amphotericine B, miltefosine. Quid combination treatment?

General

Child with Leishmania tropica infection on the face (Marocco). Copyright ITM

Leishmania braziliensis ulcer on the wrist and spread via the lymphatics. Lesions occurred after a visit to rural Bolivia. Copyright ITM

Leishmania amastigotes, copyright ITM

Diffuse cutaneous leishmaniasis due to infection with Leishmania aethiopica . Copyright ITM

There are several species of Leishmania parasites and these can cause various clinical conditions. They can be responsible for chronic ulcers and skin nodules. Sometimes both skin and mucosae are affected (mucocutaneous leishmaniasis). When deep organs are affected, the condition is called visceral leishmaniasis. The Leishmania species that cause these various clinical conditions always have the same morphology under the microscope. However, there are differences in parasite DNA, proteins, enzymes and mode of development in the insect vector, etc. Leishmania parasites can in turn be infected with a RNA virus (the "leishmania virus"), though the significance of this is not yet known.

The classification, distribution and pathogenicity of the various Leishmania species is quite complicated. New data are regularly becoming available (for example, L. tropica was shown to be able to cause visceral leishmaniasis in rare cases). The whole taxonomy will probably change as more and more genetic information becomes available. At present there are some data suggesting that meiosis occurs in Leishmania , which is contrary to the classic view, which only recognises asexual multiplication. A distinction is made between zymodemes (iso-enzyme patterns), schizodemes (kDNA analyses with restriction enzymes), serodemes (via reactions with monoclonal antibodies) and rapdemes (using PCR with random primers). Some 30 different Leishmania species have been described (10 in the Old World and 20 in the New World). Many of these can infect humans. The genus Leishmania is frequently subdivided into the subgenera Leishmania and Viannia . This was originally based on the details of the development of the parasite in the sandfly intestine, but recent classification is based on rRNA analysis. Study of the chromosomes is difficult as, among other things, they do not condense during mitosis. Many genes of the parasites are present in the genome as multiple copies. This increases the risk of uneven crossing-over during division of the nucleus. The acquisition, rearrangement or loss of copies may play a major role in the evolution of the parasites (more important than point mutations). There are substantial geographical genetic variations. Hence in the dry western part of Peru L. peruviana causes the disease "uta", an ulcerative form without mucocutaneous lesions. This organism contains less DNA in some of the chromosomes than the virulent L. braziliensis, the pathogen causing espundia, a disease which occurs in the forests on the other side of the Andes in Eastern Peru. One of the differences is the number of copies of the leishmanolysin gene, which codes for an important surface antigen (gp63). This zinc protease has a role in adhesion to macrophages and survival in the phagolysosome. It is regarded as an important virulence factor. L. braziliensis contains more leishmanolysin genes than L. peruviana . The protein is being studied as, among other things, the basis for an experimental vaccine.

Classification

There is still no generally accepted internationally agreed definitive taxonomy. The following is for orientation : Leishmania species New World L. (Viannia) braziliensis LCL, mucosal  zoonotic  Latin America  L. (Viannia) panamensis LCL, mucosal zoonotic  Northern South America and southern Central America L. (Viannia) peruviana LCL zoonotic  Peru L. (Viannia) guyanensis LCL zoonotic  South America L. (Viannia) lainsoni LCL zoonotic  South America L. (Viannia) columbiensis LCL zoonotic  Northern South America L. (Leishmania) amazonensis LCL, DCL zoonotic  South America L. (Leishmania) mexicana LCL, DCL zoonotic  Central America, Mexico L. (Leishmania) pifanoi LCL zoonotic  South America L. (Leishmania) venezuelensis LCL zoonotic  Northern South America L. (Leishmania) garnhami LCL zoonotic  South America Old World L. (Leishmania) aethiopica LCL, DCL zoonotic  Ethiopia, Kenya L. (Leishmania) killicki LCL zoonotic  North Africa L. (Leishmania) major LCL zoonotic  North and East Africa, Middle East, Central Asia L. (Leishmania) tropica LCL anthroponotic  North Africa, Middle East, Central Asia L. (Leishmania) donovani LCL, visceral anthroponotic Central Asia, Africa Old and New World L. (Leishmania) infantum LCL, visceral zoonotic South Europe, North Africa, Central and South America LCL : localised cutaneous leishmaniasis DCL : diffuse cutaneous leishmaniasis

Visceral leishmaniasis is mainly caused by Leishmania donovani. There are several subspecies in this complex :

Leishmania donovani   (India, Pakistan, sub-Saharan Africa, East Africa) Leishmania infantum   (Mediterranean Basin, Middle East) Leishmania chagasi   (South America) Leishmania archibaldi (Africa)

In the Old World skin lesions are due to Leishmania tropica . This complex is subdivided into:

L. tropica (Mediterranean basin, Middle East). Frequently dry lesions L. major (Middle East, sub-Saharan Africa). Frequently moist lesions L. aethiopica   (Ethiopia, Kenya). Sometimes also affects mucosae L. killicki (North Africa)

In (mainly South and Central) America skin lesions are caused by L. mexicana and L. braziliensis . These parasites are subdivided into subspecies :

L. mexicana complex: L. mexicana, L. venezuelensis, L. amazonensis L. braziliensis complex: L. braziliensis, L. panamensis, L. guyanensis, L. peruviana

Mucosal lesions are common in infections with L. braziliensis . One should, however, always keep in mind that the clinical lesions of leishmaniasis are a consequence of the parasite species on the one hand and of the immunological resistance and reaction of the patient on the other.

Infections occur very rarely with other Leishmania species : L.(Viannia) naiff ,  L. (Viannia) shawi .

The genome of Leishmania major has been sequenced and published. Old World Leishmania (L. donovani and L. major groups) have 36 chromosome pairs, whereas New World species have 34 or 35 pairs. Chromosomes 8+29 and 20+36 are fused in the L. mexicana group and 20+34 in the L. braziliensis group.

Distribution

Map Leishmania infantum, L. aethiopica, L. tropica, L. major. Adapted from Color Atlas.

Map Leishmania mexicana and L.braziliensis . Adapted from Color Atlas.

Map of the areas endemic for Leishmania chagasi, L. infantum, L. donovani, pathogens leading to kala azar. Adapted from Color Atlas

Mucocutaneous leishmaniasis occurs in Central and South America and occasionally in East Africa.

Visceral leishmaniasis occurs from western China to the Mediterranean Basin, East Africa and Central and South America. It is very rare in Africa south of the equator. The majority of cases occur in 6 countries: Bangladesh, Nepal, India, Ethiopia, Sudan, Brazil

The cutaneous form is seen from India to the Mediterranean Basin, the northern half of the African continent and in Central and South America.

Leishmaniasis does not occur in Northern Europe, Canada, Uruguay, Chile, Southeast Asia, South Africa, Australia and Oceania. For additional information and geographical risk in Europe, see www.leishrisk.net

Vector

The parasite is transmitted by the bite of infected female sandflies: Phlebotomus in the Old World and Lutzomyia in Central and South America. These genera, together with the blood-sucking genus Sergentomyia  [little significance for man, as they suck blood from reptiles], belong to the Psychodidae family. Morphologically they very closely resemble each other. The name "sandfly" can be confusing as this name is sometimes used for other species as well. Sandflies are vectors of leishmaniasis, pappataci virus (an arbovirus) and Bartonella bacteria.

Phleblotomus freetownensis . Phlebotomes, also known as sandflies, are vectors of leishmaniasis in the Old World. Copyright ITM

Sandfly. Lutzomyia and Phlebotomus species are vectors of leishmaniasis in the New, resp. Old World. Photo Cochabamba, Bolivia

Dogs constitute a reservoir for certain Leishmania species, such as L. infantum and L. peruviana, but can also be infected with L. braziliensis and L. tropica . Photo Dr Van den Enden, copyright ITM

Only some 10% of the approximately 600 known species of sandflies are vectors, and only 30 of these are important. Epidemiological studies on leishmaniasis often begin with vector identification, though a number of problems arise here. Taxonomic identification of adult insects is difficult. Closely related species can sometimes be morphologically differentiated in one sex only. Morphologically identical species can sometimes be differentiated only with sophisticated techniques (e.g. analysis of the cuticle hydrocarbons, PCR, iso-enzymes, etc.). Understanding the genetic variability of the vectors is still in its infancy. A fly remains infected for life. In endemic areas, a minority of sandflies are infected, usually below one per cent.

The female insects need blood in order to lay their eggs. Most species bite at night and at dusk. There are exceptions to this, such as Lutzomyia wellcomei , the main vector of L. braziliensis , which bites mainly during daytime. They can suck blood both from animals (cats, dogs, various rodents, cattle, birds and lizards, etc.) and man. They are small, soundlessly flying insects (approximately 2 mm in length). Because of these small dimensions they can get through standard mosquito nets. Mosquito nets with a very fine mesh have the disadvantage that they make ventilation difficult, which is unpleasant in warm conditions. Impregnation with permetrine (cf. malaria) can help. Because of the very short mouthparts of the insects, they cannot bite through clothing. The body and the small wings are very hairy and when at rest the insects hold their wings upright in a V-shape above them. They are poor flyers. They will usually fly quite low and will remain in the vicinity of their breeding ground. They will also not fly when there is any wind. This knowledge can be used by having a fan or ventilator on at night in the bedroom to prevent sandflies from flying. They require high humidity and temperature for breeding, although they can be observed in dry regions provided there are sites with a favourable local microclimate (crevices, termite mounds, caves, hollows and holes in tree roots, etc) where 15 to 80 tiny eggs can be laid. The larvae cannot survive drying out. They will feed on organic waste and then pupate. Sandflies reproduce optimally at 23-28°C and at a relative humidity of 70-100%. Temperatures below 10°C or above 40°C are unfavourable for their survival.

Measures used to control adult sandflies include the use of insecticides for residual spraying of dwellings and animal shelters, space-spraying, insecticide-treated nets, impregnated dog-collars and personal protection through application of repellents/insecticides to skin or fabrics. Bednets will be most useful in areas with peridomestic vectors (e.g. P. argentipes in India), whereas in areas where the vector bites in the field (e.g. P. martini in Kenya and Uganda) this can be expected to be less effective. Whereas the legs of mosquitoes are used merely for slow walking, those of phlebotomine sandflies are used to run or hop across the skin of the host or other surfaces. As such the degree of contact sandflies may make with treated surfaces is greater and this should increase their susceptibility to contact insecticides.  Insecticides include products such as organochlorines (DDT and dieldrin), organophosphates (malathion), carbamates (propoxur) and synythetic pyrethroids (permethrin and deltamethrin). Because the breeding-sites of sandflies are generally unknown, control measures that act specifically against immatures are not feasible. Reports of insecticide-resistance refer to only three sandfly species ( P. papatasi , P. argentipes and S. shorttii ) against DDT in one country (India), although there are reports of DDT-tolerance in several countries.

Following the discovery that certain plants (e.g. Capparis spinosa , Ricinus communis , Solanum luteum ) used as sources of sugar by sandflies were toxic to Leishmania major , it was found that certain plant species were also able to kill sandflies. Planting these (e.g. Bougainvillea glabra , Ricinus communis , Solanum jasminoides ) in barrier zones might therefore provide a low-cost, sustainable alternative to insecticide use in the control of sandflies and leishmaniasis. Certain plant extracts used by Amazonian Indians to kill fish are highly toxic to Lu. longipalpis , such as dried leaf extracts of Antonia ovata (Loganiaceae) dissolved in water and Derris amazonica (Papillionaceae) killing 80% and 100% of females, respectively. These plants could therefore represent a readily available alternative to commercial insecticides for sandfly control.

Note: Hamsters infected with Leishmania emit odours that makes them more attractive to sandflies, and thus more likely to get bitten, promoting further transmission. The odours are highly volatile and gas chromatography can be used to analyze them. Such research might lead to the development of odour-baited sandfly traps.

Note: Immunomodulation by Leishmania parasites.

A gel produced by the Leishmania parasite in the gut of the sandfly prevents the insect from feeding properly. This causes more effort to feed, providing more chances for transmission of the parasite. The gel is injected into the human with the parasite, and increases the severity of the infection. The crucial molecule in the gel, called filamentous proteophosphoglycan, interferes with the human immune system.  The gel pushes the immune response to the non-protective T-helper2 arm. The parasite thus manipulates the sandfly to make it feed more, and it manipulates the host's immune system so that it can spread unchecked.

An important aspect of the immune system is the balance between two arms of the T-helper response. Broadly speaking, the T-helper1 (Th1) response is tailored to intracellular pathogens, such as viruses and some bacteria and parasites. Because these organisms live inside cells, they are not accessible to antibodies. The Th1 response therefore stimulates other defense mechanisms such as macrophages. The T-helper2 (Th2) system, by contrast, promotes a vigorous antibody response. The two arms are antagonistic, so a strong Th1 response means a weak Th2 response and vice versa. In leishmaniasis, where the parasites are intracellular, a strong Th1 response will kill the parasite, and a strong Th2 response will lead to uncontrolled disease.  Sandfly saliva is important for the establishment of infection and disease pathogenesis. The sandfly saliva contains the vasodilator maxadilan. Saliva proteins seems to influence the immune response, resulting in a shift from Th1l to Th2 response. It is possible that the age-related decrease of susceptability to leishmaniasis is due to anti-sandfy saliva antibodies. Note: biting midges In December 2010, Australian researchers announced that cutaneous leishmaniasis in kangaroos and wallabies is transmitted via biting midges ( Lasiohelea sp) and not sandflies. It would be important to confirm this, and to investigate if these insects could also be vectors in other parts of the world.

Life cycle, Leishmania sp.

The parasite's life cycle is quite simple. When an infected sandfly bites, the parasite (as a promastigote) is injected directly into the skin. This unicellular parasite then penetrates the cells of the reticuloendothelial system (macrophages), where it multiplies in the form of amastigotes (the non-flagellate form) ("a" = without; "mastix" = whip). It is this form that can be seen in a skin biopsy or bone marrow aspirate. Multiplication results in bursting of the host cell, whereupon other cells become infected.

Leishmania amastigotes. This is the form present in human tissue. Copyright ITM

Leishmania promastigotes. The parasite has this morphology when residing in the sandfly vector. Copyright ITM

Leishmania amastigotes. Copyright ITM

When another sandfly later bites, these infected cells can be ingested. The parasite is then still located in infected macrophages. The blood meal in the stomach is completely surrounded by a peritrophic membrane. The parasite transforms into a different form (promastigote with flagellum) in the insect and then multiplies. After 2-3 days the peritrophic membrane is digested and the parasites are released into the lumen of the stomach and intestine. They then attach to the microvilli of the intestine by means of their flagellae. They produce a chitinase which damages the chitin coating of oesophageal-gastric junction, so that the valve between stomach and oesophagus no longer functions adequately and leaks, resulting in a backflow of parasites to the mouthparts. The parasites accumulate 7 to 10 days later in the insect's proboscis and can be injected when the insect bites its next victim. The insect is infectious 7-10 days after an infected meal and has to survive for this time in order to be transmitted. Haemoglobin degradation products inhibit the secretion of chitinase and/or inhibit the enzyme itself making backflow of parasites to the mouthparts more difficult. Certain plant sugars do not have this effect. The insects also feed on plant juices. A balance between plant and animal feeding is required for successful transmission. A botanical description of the vector's environment (biotope) can be important in scientific studies.

Note: Sandfly gut

The intestine of the sandfly consists of three major sections: an anterior intestine (cibarium, pharynx, oesophagus or gullet and oesophageal crop or gizzard), a middle intestine (stomach; the cranial part is called the cardia) and a posterior or terminal intestine (ileum, rectum). The transition from anterior to middle intestine is formed by a small valve (stomodeum valve). The transition from middle intestine to terminal intestine is formed by the pylorus. The anterior and posterior intestines are coated with chitin. The middle intestine is not coated with chitin. Leishmania parasites that develop only in the stomach [the Leishmania (Leishmania) group] are sometimes known as the Suprapylaria. Those that develop on both sides of the pylorus are the Peripylaria [the Leishmania (Viannia) group].

Historical note

Historical note, visceral leishmaniasis - Kala Azar

Although our knowledge of the history of leishmaniasis is incomplete, the first recognized epidemic appears to have occurred in 1824, in Jessore, in what is now Bangladesh. In 1824 there were numerous deaths. People developed chronic fever with substantial loss of weight, emaciation and dark discoloration of the skin. They died of terminal dysentery or pneumonia. After a few years this "black disease" spread over the entire Ganges plain. In 1832 the infection reached West Bengal. In 1862 the infection was seen in Jageer, Dacca district, Bangladesh. There were enormous numbers of deaths, comparable to the plague epidemics (but proceeding more slowly). Four years later Jageer was as good as wiped out. In 1875 kala azar was noted in Assam, India. Over the next 25 years 25% of the population died of the disease. At around the turn of the century the epidemic diminished in the Ganges basin. Isolated cases still persisted, but in much lower numbers than during the epidemic years. The reason for this stopping of the epidemic is unknown. Between 1918 and 1923 a further total of more than 200,000 people died of kala azar in Assam and in the Brahmaputra valley. This was followed by another epidemic in 1944. Assam fever is an old term used for kala azar.

Historical note, discovery of the parasite

The search for the origin of kala azar initially proceeded with great difficulty. Many hypotheses were investigated: for example, hookworm infection (ancylostomiasis) or malaria were thought to be responsible for the clinical condition. In 1900 an Irish soldier developed kala azar, after a stay in Dum Dum, near Calcutta, India. He died in England. The Scottish physician Dr. William Boog Leishman, later Director-General of the medical service of the British Army, carried out the autopsy. In spleen tissue he discovered small particles within the macrophages. He suspected that these were a sort of partly digested trypanosomes. A previously used name for visceral leishmaniasis was "Dum Dum fever" and refers to this historical event. The Irish physician Dr. Charles Donovan investigated splenic aspirates (needle biopsies of the spleen) from kala azar patients and confirmed Leishman's discovery. The tiny particles were called Leishman-Donovan bodies.

Historical note, transmission

The manner in which the transmission takes place was for a long time a mystery. The first hint of an answer was found in 1904 by Leonard Rodgers. He put some spleen tissue from a patient into a flask with simple culture medium. The parasite appeared to multiply in vitro without much difficulty. In this culture medium the form of the parasite was, however, totally different. Instead of the spherical Leishman-Donovan bodies, such as were observed in man, elongated organisms (promastigotes) that had a flagellum were now seen. This implied that the Leishman-Donovan bodies that were found in man were but one of several stages in the life cycle of the parasite. The promastigote stage would thus occur somewhere in nature, outside of man. This information was crucial. After many years of investigation with numerous false leads, the Irishman John Sinton discovered that the distribution area of kala azar in India coincided with a map of the distribution of Phlebotomus argentipes , the "silverfoot sandfly". However, it took 25 years after the first description of the parasite before Knowles in Calcutta was able to show the presence of Leishmania parasites in the tiny Phlebotomus flies. He was however not able to prove transmission itself. It took a further 14 years to discover the reason for the failure of the transmission experiments. The parasite can only maintain and fully reproduce itself in female Phlebotomus flies if the vector has first had an infected blood meal, followed by further feeding with plant sap. If the insects suck blood again too soon, the development of the parasite is disturbed in a way which makes transmission impossible. If the fly continued to feed entirely on plant sap the parasites in the insect's intestine could multiply until they were present in such large numbers that they could almost clog up the insect's intestine such that the gastro-oesophageal valve would totally fail. When the fly tries to take its next blood meal, parasites are regurgitated and the host infected. This was formally demonstrated in 1940. Kala azar can be transmitted in other ways, but these are exceptional, namely shared use of needles among intravenous drug users, or infected blood transfusion. Very rare cases of congenital kala azar infection have been reported.

Historical note, DDT

In the same year, 1940, the Swiss chemist Paul Muller patented his discovery of DDT, the first cheap and very active insecticide. He received the Nobel Prize for this in 1948. The World Health Organization's Global Eradication of Malaria Plan was launched several years after a big successful trial of DDT for the control of malaria in the Tennessee River valley in the USA was completed in 1945. The basic strategy was, among other things, to spray all human and animal housing twice a year with DDT. An unforeseen effect of this campaign was the effect on kala azar. Phlebotomus is a poor flyer and in India it lives around the housing of domestic animals and man. This biotope is ideal for controlling them with a residual insecticide such as DDT. In India this stopped the transmission of kala azar and up to 1955 practically no further cases of kala azar occurred.

Chemical structure of DDT (dichlorodiphenyltrichloroethane). Insecticide used in malaria control. Copyright ITM

However, after the failure of the malaria eradication programme and the end of spraying in 1970 in India, kala azar returned. The first local epidemic was in Vaishali, a small town in Bihar. In 1975 West Bengal was also affected. Around 1980 the infection was widespread from Tamil Nadu in the south of India to the regions of Nepal and Bangladesh. About 80,000 cases were reported in north-east India in 1992. In November 2000 kala azar transmission was confirmed in Calcutta city. To make matters worse, resistance to the medication has developed in the meantime so that up to 10 % of the parasites are now resistant to the antimony drugs that form the basis of therapy. This percentage still increases every year. Vector control faces many difficulties, especially with respect to organization, long-term planning and execution, as well as financing. The threat of new major epidemics remains to this day, not only in Bihar, in the east of India, but also in many other areas, such as southern Sudan and in Gedaref (eastern Sudan). The interaction between AIDS and leishmaniasis is an additional problem. In parts of Spain up to 70% of the kala azar patients are infected with HIV and 9% of the HIV patients will develop kala azar. Intravenous drug users share non-sterile needles and can transmit not only HIV, but also leishmaniasis. Here transmission is dissociated from the traditional reservoir in dogs.

Visceral leishmaniasis - Kala Azar

Visceral leishmaniasis, distribution

At present 90% of all visceral leishmaniasis occurs in India, Bangladesh, Nepal, Ethiopia, Sudan and Brazil. Visceral leishmaniasis may be responsible for 500,000 new cases and > 50,000 deaths per year.

Visceral leishmaniasis, clinical features

After an initial multiplication in the skin, causing a transient small lesion, the parasites can further multiply in bone marrow, liver and spleen. This causes visceral leishmaniasis. The incubation period is usually 2 to 6 months. The pathogens are usually Leishmania donovani and L. infantum or sometimes Leishmania tropica . L. chagasi is possibly identical to L. infantum and was possibly introduced into the New World via infected dogs or rats at the time of the Spanish and Portuguese conquests, though there are doubts about this.

Visceral leishmaniasis in Southern Europe was initially considered to be a pediatric disease (hence the name L. infantum ). However, it is clear that all age groups can be infected. The disease is characterised by a persistent inflammatory state with chronic fever, enlarged liver and spleen and a low blood count (pancytopaenia = anaemia + leukopaenia + thrombocytopaenia). This must be distinguished from an aplastic bone marrow. The patient becomes very susceptible to other infections (pneumonia, tuberculosis, dysentery) which can sometimes prove fatal. Symptoms and signs of superimposed bacterial infections may confuse the clinical picture at the time of initial diagnosis. Low blood platelet counts result in a bleeding tendency (nosebleeds, bruising, etc.). Sometimes there are also other symptoms, such as swollen lymph nodes, more common in Sudan than in India. Weight loss and emaciation are frequent and substantial. The skin can turn a dark colour: kala azar (Hindi) means "black disease" and refers to this hyperpigmentation. This was mainly described from Indian cases. The reason of this hyperpigmentation is not clear. The infection can proceed atypically in HIV patients (for example, without fever or splenomegaly, or with negative serology). When immunosuppression is induced by chemotherapy, latent kala azar can become clinically apparent.

Visceral leishmaniasis (kala azar) with hepatosplenomegaly. Copyright ITM

Child with anaemia due to visceral leishmaniasis, kala azar. Photo Prof Eyckmans, copyright ITM

PKDL A skin condition, called post-kala azar dermal leishmaniasis (PKDL), can occur after a patient has suffered from kala azar. PKDL rarely occurs without being preceded by kala azar. This disease (PKDL) was originally described by Brachmachari in India. PKDL occurs on average 4 months after kala azar (range 0-3 years), though there are strong regional variations. This disease occurs mainly in India (up to 20% of kala azar patients), and to a much lesser extent in the Middle East. In Sudan the disease occurs regularly (56% of kala azar patients in one study). It is virtually unknown in the Mediterranean Basin or in South and Central America. It involves discoloured patches and painless nodules on the skin that usually contain few, but sometimes moderate numbers of amastigotes. Most of the lesions occur on the face (98%) and to a lesser extent on the thorax (80%), arms (70%), legs (40%), tongue (40%) and genitals (6%). Various degrees of severity can be clinically distinguished in PKDL. Grade 1 includes an extensive maculopapular to nodular rash, principally around the mouth, and possibly somewhat lesser lesions on the thorax and upper arms. Grade 2 is a similar but denser rash that covers the whole face and is also present on the chest, back, upper arms and legs, with fading of the lesions in more distal regions of the body. Grade 3 is a generalised dense rash with ulcers, scabs, cheilitis and possibly lesions of the palate (roof of the mouth). This disease has a very chronic course (years) and is therefore important for transmission. Parasites do not affect internal organs in PKDL. There is sometimes a concomitant neuritis, which can further contribute to the clinical resemblance to leprosy. Treatment with glucantime can be given 2 months, or longer (4 months in India, where resistance to antimony is higher). Amphotericine B is an alternative. The therapeutic place of miltefosine for PKDL is not clear at present. 

Visceral leishmaniasis, diagnosis

In endemic areas, fever lasting more than 2 weeks and accompanied by splenomegaly not responding to antimalarial therapy, strongly increases the suspicion of visceral leishmaniasis, but is in itself insufficient for diagnosis.

Leishmania amastigotes. Copyright ITM

Diagnosis of visceral leishmaniasis is not easy, since none of the tests have 100% sensitivity and 100% specificity. An ideal test would be affordable by those at risk of infection, sensitive (few false-negatives), specific (few false-positives), user-friendly (simple to perform and require minimal training), rapid (to enable treatment at first visit), robust (no requirment for refrigerated storage), equipment-free (such as micropipettes and well-plates) and delivered to those who need it (if it only exists in a research lab, it will not be useful). Clinical syndromic diagnosis lacks specificity, as malaria, hyperreactive malaria splenomegaly, trypanosomiasis, typhoid fever, disseminated tuberculosis, brucellosis, hematological disorders, splenic abscess or splenomegaly due to portal hypertension all can be accompanied by enlarged spleen, fever, wasting, anemia and/or lymphadenopathy. Because of the high cost and toxicity of current therapeutic options, empirical treatment is not defended. Therefore confirmatory diagnostic tests must be used. The leishmanin skin test is an indicator of past infection and is not used to diagnose visceral leishmaniasis. 1. Direct diagnosis is made by demonstrating the presence of amastigotes. The parasite is egg-shaped and measures 2-3 x 5 µm. With Giemsa staining, there is a pale blue cytoplasm, a well defined nucleus and a smaller kinetoplast. Microscopy requires considerable expertise and training. Usually bone marrow is obtained by sternum aspiration. Careful sterilisation of the large-bore needles is essential. Slide examination time is important and influences sensitivity. The technique of spleen aspiration is more sensitive (in some studies very nearly 100%, though in reality slightly lower) than bone marrow aspiration, but can be risky (spleen rupture, haemorrhage). If one wants to use a splenic aspirate, it is better to use the intercostal rather than the transabdominal route (safer, can be carried out more often). The 10th intercostal space between the anterior and the mid-axillary line is generally used. The procedure is safe when performed by an experienced physician, and when the prothrombin time is normal. The platelet count should be above 40 x 10 9 /liter. Active bleeding, severe anemia, jaundice, moribund state, pregnancy or lack of cooperation are contra-indications. Patients must lie in bed for several hours after the procedure. Vital signs must be checked frequently to allow early recognition of hemorrhage, Blood transfusion facilities must be available. One can use a 21-gauge needle and a 5 ml syringe. After penetration of the skin, the plunger is withdrawn, the needle is quickly inserted into the spleen while maintaining suction and withdrawn immediately (i.e. less than 1 second). Lymph node aspiration and/or liver biopsy are sometimes necessary. The parasites can rarely be detected in peripheral blood monocytes. Examination of a buffy coat using QBC can be performed. 2. Serology is positive in most cases of visceral leishmaniasis. Gel diffusions immunoelectrophoresis, complement fixation test, indirect hemagglutination, Western Blot and countercurrent immunoelectrophoresis have limited diagnostic accurary and/or feasibility in the field. Indirect fluorescence tests (IFA) are an alternative, but require a fluorescent microscope. The direct agglutination test (DAT) is often used, as this test has a high sensitivity and specificity. Both liquid and freeze-dried antigens can be used, although liquid antigen is associated with poor reproducibility in East Africa (most likely due to decay of liquid antigen during storage and transport). Note that freeze-dried antigen does not require refrigeration. The DAT is simpler than many other tests but requires equipment, such as microplates and micropipettes, training and regular quality control. A suggested cut-off value of 1/3200 is often used, but should be evaluated in each setting. An alternative is to consider titers < 1/1600 to be negative, borderline between 1/1600 - 1/12800, and positive > 1/12800. It can be defended that in a rural endemic area, a patient with more than two weeks fever and splenomegaly with strongly positive DAT values and no response to antimalarials doesn't necessitate formal demonstration of parasites. With borderline values tissue aspiration with search for amastigotes will be needed. A possibility in a small regional clinic is to absorb a drop of blood from a patient suspected to have kala azar on a small filter paper and then to punch out a standard size disk from the blood spot. In this way one obtains a well-defined, accurate aliquot of absorbed blood. This can be transported and used for DAT in a well equipped laboratory. Serology will remain positive after cure. The fast agglutination screening test (FAST) is a simplified (single serum dilution) and more rapid version of the DAT (2-3 hours versus 18h). Because DAT is not practical in many field conditions, alternatives are studied. ELISA is higly sensitive, but specificity depends upon the antigen used (amastigotes or promastigotes). Recombinant K39 antigen-based dipsticks using immunochromatography (ICT) are a promising alternative to DAT. K39 is a 39-amino acid repeat that is part of a kinesin-related protein of L. chagasi . This repeat is conserved within the L. donovani complex. The ICT tests are easy to perform, rapid and cheap. Twenty µl of serum are added on the dipstick, which is then placed vertically in a test tube. Two drops of chase buffer solution provided with the dipstick are then added. The results are read after 5 to 10 minutes. Even a weak band in the test region is considered positive. A control line has to be visible. It is the most promising tool for the diagnosis of visceral leishmaniasis in peripheral centres. The specific format (brand) of dipstick may play an important role (e.g. Opti-Leish TM , DiaMed IT Leish TM , DiaMed DUAL IT L/M TM versus Kalazar Detect TM ). 3. Formol-gel test. In kala azar there is a very high production of non-specific immunoglobulines (and a decrease in albumine), especially in advanced disease (i.e. more than 3 months). This can be demonstrated by serum protein electrophoresis, but this impractical in field conditions. The proteins can be precipitated as a gel by formaline. Twenty µl of 40% formaldehyde are added to 200 µl of serum in a glass tube. After twenty minutes, the gelificaton reaction is visualy assesed as positive or negative. The test is simple and cheap. The test can also be positive in patients with hyperreactive malaria splenomegaly.  4. A urinary antigen detection test using latex agglutination (KAtex) has been developed to circumvent the limitations of serological tests. It detects a heat-stable low molecular weight carbohydrate antigen. This will become negative upon successful treatment. It can therefore distinguish an active from a past infection.  A very high specificity and moderate to high sensitivity were reported.  The test requires the boiling of of 1 ml of urine for 5 minutes. About 50 µl of the treated urine sample is added onto a reaction zone on a glass slide and a drop of latex is added. The liquids are stirred to a completely homogenous mixture. Any agglutination reaction discerned when compared with a negative control is considered positive. The sensitivity varies with the parasite load. An alternative is the detection of sialic acid derivatives on erythrocytes which act as a biomarker for visceral leishmaniasis. These indirectly reflect the presence of parasites. Validation of this test is needed. 5. Culture can be done from peripheral blood, buffy coat or tissue aspirates. The microculture method improves sensitivity and decreases incubation periods. Cultures are expensive, time-consuming and require expertise. The parasites can be cultured in vitro, using Novy-Nicolle-McNeal (NNN-medium) Schneider's medium, Tobie rabbit blood agar (only Old World) or Nogushi soft agar with rabbit blood (Old and New World). In vivo culture in test animals (golden hamsters) is possible, though this is usually not feasible in clinical practice in developing countries. A Leishmania parasite can survive for 3 days at a temperature of 4° C, but for only 1 day at room temperature, in Locke transport medium (a buffered glucose-salt solution with antibiotics). 6. Genome assays. Lack of standardisation and quality control is a major concern of PCR and related assays. A multitude of gene targets, protocols and applications have been described. A PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species (it can be also be used in vector studies). The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species.The very high sensitivity of PCR-based assays may actually be a disadvantage by being a marker of infection (transient or permanent) instead of being a marker of disease, as it will pick up also asymptomatic carriers. Detailed genomic analysis of L. donovani showed that parasites can have two, three, four or even five sets of chromosomes in one organism. Further study of this ploidy-variation will investigate the possible clinical implications of this unexpected finding.

Montenegro test

Leishmanin is a compound obtained via in vitro culture of promastigotes. A skin test with leishmanin (Montenegro test) is negative during active kala azar, but later becomes positive (after 6 to 12 months). The Montenegro test reflects the suppressed cellular immunity during infection. There is a specific anergy for Leishmania parasites during active disease. This test is mainly of epidemiological value. To perform the test 0.1 ml is injected intradermally and the local reaction read after 48 hours (>5 mm induration = positive). A positive test eliminates the existence of active kala azar. Cutaneous leishmaniasis produces a positive Montenegro test.

Visceral leishmaniasis, treatment

Leishmaniasis, treatment with amphotericine B. Photo Cochabamba, Bolivia

Pentavalent antimonial compounds . One of the treatment option for visceral leishmaniasis are pentavalent antimony derivatives (antimony, chemical symbol Sb = Stibium). The derivative most frequently used is Glucantime® (meglumine antimonate , 85 mg Sb/ml) and, rarely, Pentostam® (sodium stibogluconate, 100 mg Sb/ml). The drugs can be administered IM (intramuscularly, painful) or by slow IV (intravenous) injection or infusion (diluted with 5% glucose solution, otherwise local thrombophlebitis occurs). The dose is always expressed as mg Sb: 2 x 10 mg/kg IM or slow IV infusion per day for at least 30 days. On an ampulle might be written 1500 mg/5 ml, which is 1500 mg calculated as the salt, not as stibium itself. This can lead to underdosing if one is not aware of this detail. As a dose is practically totally excreted and eliminated via the urine within 6 hours after administration, a twice daily administration would pharmacokinetically be more logical than an injection once daily. However, a single administration per day appears to suffice in practice. The dose should be reduced in patients with kidney failure. A maximum of 850 mg/day [10 ml Glucantime®] has been previously set due to the risk of cardiotoxicity with higher doses. This limit has been contested and higher doses are sometimes used. Negativation of T-waves and prolongation of the QT-time are indicative of threatening arrhythmia. The fever usually disappears after 1 week. The spleen begins to get smaller after 2 weeks but frequently requires 6 to 12 months to return to normal. Note: antimony . Antimony is just below arsenic in the periodic table. It mimics the toxic effects of arsenic, which result from binding to adjacent thiol groups on enzymes, thereby impairing their function. Antimony is found in trivalent and pentavalent forms. Inhalation of stibine gas (SbH 3 ) causes massive hemolysis. Medication which contains trivalent stibium such as antimony potassium tartrate (tartar emetic), stibophen and astiban are obsolete as antischistosomal agents. Tartar emetic should not be confounded with creme of tartar (potassium hydrogen tartrate), an innocent acidic cooking salt. Pentavalent antimonials (e.g. meglumine antimoniate, sodium stibogluconate) are used for treatment of leishmaniasis. One of their actions is to inhibit phosphofructokinase, the rate-limiting step in the parasites' glycolytic pathway.

Follow-up and response in the event of recurrence

Follow-up is necessary as a number of patients will relapse. This usually happens in the first 6 months after treatment. Cases of resistance are not infrequent. Splenectomy sometimes has to be carried out in cases of life-threatening anaemia or thrombocytopaenia. If possible a pneumococcal vaccination should be given before the operation, and lifelong antimalarial prophylaxis is indicated thereafter if the patient stays in an endemic area. Upon recurrence higher doses of Glucantime® can be used for a longer time (2-3 months), though if cost is no problem, amphotericin B can also be used.

Alternative treatments:

Amphotericin B was isolated in 1956 from Streptomyces nodosus from the Orinoco valley in Venezuela. It is a polyene and has a fairly complex structure with a hydrophilic and a lipophilic component. The name of the compound is derived from its amphipatic character: Greek: "amphi" = two-sided. The recommended dose of amphotericin B [Fungizone®] is 0.5-1 mg/kg/day IV, to be given over 6 hours; total dose max. 1-3 g. This drug is mainly used for the treatment of deep mycoses, though it is also active against Leishmania . It is a rather toxic medicament. Shivering, fever, nausea, vomiting, headache, anaemia, phlebitis at the site of the infusion, cardiotoxicity, kidney failure, hypokalamia and hypomagnesaemia are frequent side effects. Side effects occurring shortly after administration can be reduced by cortisone IV or meperidine (pethidine), a morphine analogue. Administration of 500-1,000 ml physiological isotonic saline solution before starting the IV-drip reduces the risk of nephrotoxicity. Amphotericin B is not eliminated via haemodialysis. The toxicity of the drug is reduced by pharmacological complexing with lipids prior to the administration. The drugs are then concentrated in the reticuloendothelial system and not in the kidneys so that a higher daily dose per kg of bodyweight can be administered and treatment time shortened (e.g. to 5 days). There are hopeful data which indicate that single-dose treatment (high dose; 10 mg/kg of the liposomal formulation) is useful. In 1990 AmBisome® was developed as a first-choice drug. Several lipid formulations of amphotericin B are now available. They differ from each other in the type of phospholipid and the ratio of lipid to amphotericin B. Good results have been obtained with these lipid formulations. The price of these medications (AmBisome®, Amphotec®, Abelcet®) has come down, but is still high for the average rural farmer in a developing country.

Formulations of Amphotericin B

Fungizone®: Amphotericin B deoxycholate. Contains no lipids. Emulsification of Fungizone® in Intralipid 20%: little reduction of toxicity AmBisome®: L-AmB: incorporation in liposomes (vesicles). Abelcet®: ABLC or Amphotericin B Lipid Complex. Microscopically small ribbon-like membranes formed by complexing with phospholipids. Amphotec®: ABCD (= Amphocil®) Amphotericin B Colloidal Dispersion: AmB-cholesteryl sulphate forms disc-shaped structures.

Pentamidine isethionate  (4 mg/kg every 48 hours IM for 4 months). Pentamidine (Pentacarinat®) should not be confused with Pentostam® (pentavalent antimony). The parasites are located intracellularly in phagolysosomial vesicles in phagocytic cells. These cells can ingest small carrier molecules. When pentamidine is bound to nanoparticles the intracellular concentration of the active compound increases greatly. One carrier that is being studied is polymethacrylate. The amine groups of pentamidine are ionically bound to the acid groups of the carrier. This binding is pH-dependent. In the acidic phagolysosome a substantial proportion of pentamidine is released and kills the parasite. By increasing the bioavailability of the drug in this way it should be possible to make these new formulations more effective and less toxic.

Combination therapy with gamma-interferon  (Immukine, 100-400 µg/m 2 body area /day SC for 10-30 days; 20.000 IU =1 microgram) was described with initially good results but its exact value in treatment has not been determined yet. Where does this idea of using gamma-interferon come from? [Its use is best known in the treatment of chronic granulomatous disease]. The immunological defence against Leishmania parasites is strongly dependent on the proper functioning of the reticuloendothelial system. Gamma-interferon (INFg) is secreted by stimulated lymphocytes and has the property of strongly activating monocytes/macrophages, which is essential for the defence against these parasites. Kala azar is associated with a specific anergy against the parasite, resulting in non-activated monocytes and macrophages. By giving INFg, one tries to activate these cells, increasing their killing capacity. This is the rationale for considering therapy INFg in some patients. In advanced laboratories, one can check activation of monocytes in vitro. After activation certain molecules show increased expression (FcR-1, HLA-DR, etc). If the receptor for INFg is deficient or defective, the activation status of monocytes is influenced very little if at all after addition of exogenous INFg. When INFg production itself is deficient, the INFg-dependent functions are also deficient even if normally functioning receptors are present on monocytes.

High-dose allopurinol  (Zyloric®), e.g. 3 x 7 mg/kg/day (that is, 20 mg/kg/day), for 4-12 weeks gave encouraging results.

Injectable aminosidine  (paromomycine) is now being studied. It is an aminoglycoside antibiotic. This compound is difficult to obtain at present. In 2007 the results of an Indian study showed that paromomycin IM, at a dose of 11 mg/kg/day x 21 days was noninferior to amphotericine B at a dose of 1 mg/kg IV every other day x 30 days. Pain at the injection site, liver toxicity and ototoxicity were reported as side effects. Paromomycin for IM administration is registered in India, and since 2012 also in Nepal.

Terbinafine  ( Lamisil®) is an antimycotic drug that is being evaluated.

Miltefosine   (Miltex®) was approved for use in India in 1992. It became more widely became available in subsequent years. Miltefosine or hexadecylphosphocholine is a lecithin analogue (=phosphatidyl-choline analogue). In the molecule phosphatidylcholine is bound to a carbohydrate component via an ether bridge instead of an ester. Miltefosine interferes with certain cellular signal cascades and with membrane synthesis, though its precise mode of action is still unknown. It was initially developed as an antineoplastic agent. Topical use in for example cutaneous metastases in breast cancer is being studied. In the 1990s it was also discovered that in vitro and in animal models it was active against Leishmania parasites. These organisms contain many ether lipids in the cell membrane. The main advantage of the compound is that it can be given orally, in contrast to the injectable antimony derivatives and amphotericin B. I cannot be given IV as this would lead to hemolysis. The molecule is fairly easy to produce and this should eventually bring down the price, which is very important in third world countries.  The price in the West is quite high. The daily dose for adults is 100-150 mg, and for children 2.5 mg/kg/day. It should be given for 4 weeks. Plasma concentrations are proportional to the oral dose. The half-life is about one week (6-8 days). The cure rate, according to preliminary studies, is excellent (97%). Dose-dependent gastrointestinal discomfort often occurs and reversible hepato- and nephrotoxicity sometimes occurs. It is teratogenic and so cannot be given to pregnant women or women who want to conceive in 6 months after treatment. How quickly resistance to miltefosine will develop when used as mono-therapy in the field is not yet clear. It is relatively easy to induce resistance in vitro.

The nitroimidazole fexinidazole has potential as a safe and effective oral drug therapy for treatment of visceral leishmaniasis (see also treatment of Human African Trypanosomiasis. Both metabolites of fexinidazole (sulfone and sulfoxine) were active against Leishmania donovani amastigotes. Reliance on a single enzyme for prodrug activation may leave fexinidazole vulnerable to the emergence of drug resistance. Pamidronate , a bisphosphonate drug typically used in the treatment of osteoporosis, is effective against experimental cutaneous leishmaniasis. Several bisphosphonates have significant activity against Leishmania donovani in vitro, and several are potent inhibitors of bone resorption and in clinical use for the treatment of osteoporosis and Paget's disease. Action on bone is based on binding of bisphosphonate moiety to the bone mineral and inhibition of the osteoclast's enzyme FPPS (farnesyl pyro-phosphate synthase). Expressed FPPS is also potently inhibited by bisphosphonates in the trypanosomatid parasite Trypanosoma cruzi , in which FPPS is postulated to be the major target of bisphosphonates. It is possible that currently approved clinical regimens of the drug are not high enough to cure human cutaneous leishmaniasis. Pamidronate could be a useful lead compound in the synthesis of new drugs against this disease. Sitamaquine. This is an oral 8-aminoquinoline drug leading to variable cure-rates (27-87%). Several cases of serious renal adverse effects have occured. Combination therapy. This is the suggested way forward to increase treatment efficacy, prevent the development of drug resistance, reduce treatment duration and possibly decrease cost. Pentavalent antimonials can be combined with paromomycin. Other combinations including liposomal amphotericine B and miltefosine are being studied.

Cutaneous leishmaniasis

Cutaneous leishmaniasis, distribution

Approximately 90% of all cases of cutaneous leishmaniasis now occurs in Iran, Syria, Saudi Arabia, Afghanistan, Algeria, Peru and Brazil.

Cutaneous leishmaniasis, clinical features

Various forms are clinically distinguished, the most important of which are :

Localised cutaneous leishmaniasis: skin ulcers that heal very slowly or nodular lesions, limited in extent and number. These chronic sores have regional names: clou de Biskra in Algeria and Aleppo boil in Syria. Diffuse cutaneous leishmaniasis: cutaneous nodules and plaques that do not ulcerate but sometimes spread over the entire body. Recurrent cutaneous leishmaniasis

"... After it is cicatrised, it leaves an ugly scar, which remains through life, and for many months has a livid colour. When they are not irritated, they seldom give much pain... It affects the natives when they are children and generally appears in the face, though they also have some on their extremities... In strangers, it commonly appears some months after their arrival. Very few escape having them, but they seldom affect the same person above more than once."

Early Leishmania braziliensis infection, with lesions on the nose. This differs from espundia. Copyright ITM

Skin ulcer due to cutaneous leishmaniasis.

Diffuse cutaneous leishmaniasis. Infection with Leishmania aethiopica . Copyright ITM

Infection with Leishmania aethiopica , resulting in diffuse cutaneous leishmaniasis. Patient is from Erithrea. Photo Dr Van den Enden Erwin. Copyright ITM

Localised cutaneous leishmaniasis

After a bite by a sandfly infected with L. tropica   (mainly urban infection), there is an incubation period of a few weeks or months, occasionally years. There is initially a small papula and usually only a single lesion, though sometimes there are several. This slowly spreads, can remain completely dry, become warty or nodular or develop into a painless, sharply delineated ulcer surrounded by a purplish raised border. Satellite lesions can occur. Spontaneous healing often occurs after 6 to 12 months, resulting in a depressed scar. Recurring cutaneous lesions - possibly with severe disfigurements - occasionally occur. There is usually immunity to any subsequent infection with the same organism. In infection with L. major   (mainly rural infections, particularly from a rodent reservoir) the lesions are usually larger and develop more quickly, hence the name. There is a greater tendency to local spreading via the lymphatics and have to be distinguished from sporotrichosis. The lesions will eventually spontaneously heal with scar formation. Clinical cure starts when macrophages become activated and start killing amastigotes. This is mediated via a T-helper cell type 1 (Th1) response. This immune reaction also prevent recrudescence of latent chronic infection. The Th1 response is accompanied by secretion of pro-inflammatory cytokines, such as interferon gamma and interleukin 12. If the immune response would be towards production of downregulating cytokines (interleukin 4, 10, 13, TGF beta), macrophages will not be capable of eliminating the parasites, but tissue destruction will be limited.

In South America the lesions often have their own local names and clinical expressions. Hence in Peru they are called "uta" (a solitary ulcer or a few restricted lesions brought about by L. peruviana , frequently on the face). In Guyana they are known as "bush yaws" or (French) "pian bois" ( L. guyanensis ) with rasberry-like lesions that resemble yaws. In Yucatan, Mexico an ulcer on the ear (usually caused by L. mexicana ) is know as "chiclero" ulcer.

Note: sapodilla trees

A "chiclero" is a man who collects chicle-latex in the forest. Chicle is a gum obtained from the bark of sapodilla trees ( Achras zapota  or Manilkara sapota ) and was originally used as a basis for chewing-gum. Numerous synthetic latex-like substances are now used as the base constituent. The latex protects the plant against insects, both mechanically (by virtue of its stickiness) and chemically. The chicleros have to cut into the bark to reach the milk ducts (laticiferae) of the secondary phloem but may not damage the cambium ("growth layer"). The sap is collected and processed. During their activity in the plantations the workers can get bitten by Lutzomyia olmeca  and as such are exposed to a high risk of contracting leishmaniasis, hence the term "chiclero ulcer".

Chiclero ulcer on an ear (leishmaniasis). Photo Cochabamba, Bolivia

Diffuse cutaneous leishmaniasis

Diffuse cutaneous leishmaniasis is a diffuse affection of the skin with extensive non-ulcerative nodules and is a very chronic disease. It is sometimes followed by chronic lymphoedema of an affected part of the body. This disease is poorly understood, but is probably caused by a diminished resistance to the parasite. This immunosuppression is possibly brought about by the parasite itself. One of the supposed mechanisms of escape of Leishmania parasites is downregulation of the expression of major histocompatibility complex (MHC) class II molecules on the macrophages they colonise. In East Africa diffuse cutaneous leishmaniasis is often caused by L. aethiopica  and in the New World frequently by L. mexicana .

If there are generalised cutaneous lesions the condition has to be differentiated from lepromatous leprosy, keloids, neurofibromatosis and post kala azar dermal leishmaniasis (PKDL). Due to the low resistance of the patient very numerous amastigotes are present and most skin smears are positive. Treatment is difficult, as the patient's immune system itself is functioning poorly.  DCL patients are anergic to leishmanial antigen. Patients with DCL have a predominantly Th2-type cytokine response. They have low concentrations of interferon gamma and interleukin 12. There is no tendency to self-cure. Differentiation from PKDL is important, as the latter can still be treated reasonably well. In Sudan 1 case of diffuse cutaneous leishmaniasis is found for every 100 cases of localised cutaneous leishmaniasis. The incidence varies greatly from district to district. It occurs frequently in South America, but in contrast to this it does not occur in India.

Recurring cutaneous leishmaniasis

Recurring cutaneous leishmaniasis seldom occurs (Iraq, Iran). This disease, also known as leishmaniasis recidivans leads to significant tissue damage. Parasites are very difficult to detect in these very chronic lesions. Differentiation from cutaneous tuberculosis is important.

Cutaneous leishmaniasis, diagnosis

Leishmania mexicana , ulcer on the arm. A skin biopsy is best taken from the edge of a lesion. Copyright ITM

Leishmaniasis. Material needed for skin punch biopsy. Copyright ITM

Attempts should be made to detect the parasite microscopically in a biopsy or smear from the edge of the wound. The biopsy will, if possible, be divided up for pathology (seldom available, not very sensitive, is principally used more for exclusion of another cause) and cultures (bacteria, mycobacteria, fungi, Leishmania ) and an impression preparation should also be made. Lesions on the face can be injected with 0.1 ml physiological saline and aspirated again while moving the small, thin needle back and forth in the skin. Serology is usually negative. Differential diagnosis includes ulcers due to mycobacteria, cutaneous diphtheria, tertiary syphilis, yaws, cutaneous carcinoma and deep or subcutaneous mycosis. Field sore (cutaneous diphtheria) and tropical ulcers are painful, particularly in the early phase. Differential diagnosis of disseminated nodular and ulcerated lesion includes leishmaniasis, sporotrichosis, atypical mycobacteria and nocardiosis.

Cutaneous leishmaniasis, treatment

The respons to treatment varies according to the species. Drugs for systemic and topical treatment can be used. There is an urgent need for better and cheaper drugs.

Indications for local treatment

lack of risk of developing mucosal lesions Old World cutaneous leishmaniasis small, single lesion absence of spread to lymph nodes

Indications for systemic treatment

presence of mucosal lesion or spread to lymph nodes New World cutaneous leishmaniasis, except localised Leishmania mexicana infection lesions unresponsive to local treatment

Overview topical treatment of cutaneous leishmaniasis

physical methods: cryotherapy (liquid nitrogen) for 15-20", repeated 2-3 times with an interval of e.g. 3 weeks. Blistering will occur. application of local heat via a CO 2 laser or an infrared lamp (40°C to 42°C for 12 hours) has been used, but heat-induced skin bullae are common. Application op topical 5-aminolaevulinic acid (a porphyrin-prcursor), followed by two laser irradiations, which photoactivates the compound. It is expected that very little scar tissue would form, so for esthetic important places, this might become first choice treatment, if the clinical studies confirm this expectation. ointment with 15% paromomycin and 12% methylbenzethonium chloride in soft white paraffin (e.g. Leishcutan® ointment). Urea can be added as a keratolytic. Twice daily application is advised, for a duration of 20-30 days. skin infiltration with pentavalent antimony with a fine gauge needle. Blanching of the lesions should be obtained. Treatment is repeated every 5-7 days, in general 2-5 times, sometimes more. imiquimod crème (Aldara®). This immunomodulator activates macrophage killing of Leishmania amastigotes, but is best used in combination with systemic meglumine antimonate. Experience with this drug is limited. Local application of imiquimod crème (250 mg, 5% weight/volume), i.e. one individual packet every other day x 20 days is possible. treatment with antimonium plus topical recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) has been described. GM-CSF (molgramostim = Leucomax®) was diluted for topical use to a concentration of 10 µg/ml. It was applied 3 times weekly for 3 weeks (1-2 µg/cm2/lesion). In vitro, GM-CSF has been shown to activate macrophages that kill Leishmania pathogens. Intralesional injection of GM-CSF (400 µg) has also been shown to reduce the healing time of leishmania ulcers.

Overview systemic treatment of cutaneous leishmaniasis

Pentavalent antimonials (meglumine antimoniate [85 mg Sb/ml, IM] or sodium stibogluconate [100 mg/ml, IM or filtered IV] can be given parenterally for extensive skin lesions. For unknown reasons, the incidence of herpes zoster is increase about 10 times during IV treatment with IV Pentostam relative to the incidence in the normal population. Cases of cutaneous leishmaniasis not treated with antimony do not have an increased incidence of herpes zoster. Pentamidine. First line against L. guyanensis (French Guyana). Check glycaemia. Several treatment schemes exist and the cure rate is dose-dependent. Some short-courses use 1200 mg as a total dose. In Guyana 3 mg/kg/day every other day is often used (4 injections). Imidazoles, triazoles. Infections caused by L. major can be successfully treated with oral fluconazole 200 mg/day for 6 weeks (cure rate of 80%). Ketoconazole 600 mg per day x 28 days is moderately effective for L. mexicana, but much lower against L. braziliensis . Treatment with ketoconazole is sometimes complicated by hepatotoxicity, abdominal pain and nausea. Itraconazole  (Sporanox®) gave good results in initial studies, but these were not confirmed later.  Miltefosine. Not yet widely available, but allows oral therapy.  Amphotericine B and its liposomal formulations (IV). Allopurinol. Not as monotherapy, but associated with e.g. pentavalent antimony for L. panamensis .

Treatment of diffuse cutaneous leishmaniasis ( L. aethiopica )

The treatment of diffuse cutaneous leishmaniasis caused by L. aethiopica is problematical, as this parasite is less sensitive to Glucantime®. Pentamidine  can be used against L. aethiopica . A dose of 4 mg/kg/week which has to be continued for at least 4 months after disappearance of the parasites from the skin is an acceptable guideline here. Parenteral aminosidine sulphate  is another therapeutic possibility. This is an antibiotic that is obtained from Streptomyces chrestomyceticus . It is an aminoglycoside and is thus potentially nephro- and ototoxic. It is chemically identical to paromomycin, which is obtained from a related Streptomyces strain. The compound is not resorbed from the intestine. Recurrences are frequently seen with aminosidine given as monotherapy. Aminosidine is, however, synergistic with stibogluconate and a permanent remission can be obtained with the combination of aminosidine with Glucantime® or Pentostam®. The dose is 14 mg/kg/day IM to be continued for up to 60 days after all parasites have been eliminated. The total treatment period takes 6 months or more. Good results were obtained with amphotericin B.

Mucocutaneous leishmaniasis

Mucocutaneous leishmaniasis, distribution

At present 90% of all mucocutaneous leishmaniasis occurs in Bolivia, Peru and Brazil. Illustrations of skin lesions and disfigurements suggestive of leishmaniasis are encountered on pre-Inca earthenware. These indicate that the disease was already in existence in Peru and Ecuador in the 1st century AD. Texts dating from the 15-16 th century Inca period and the Spanish conquest mention the risk of cutaneous ulcers in seasonal farmers. Espundia was also described as "white leprosy".

Mucocutaneous leishmaniasis, clinical features

When skin and mucosae are affected the disease is known as mucocutaneous leishmaniasis. This is very rare in East Africa but frequent in South America, where it is known as "espundia". After an initial skin lesion, that slowly but spontaneously heals, chronic ulcers appear after months or years on the skin, mouth and nose, with destruction of underlying tissue (nasal cartilage, for example). Tissue destruction with disfigurement can be very severe. Parasites are usually rare in the lesions. A substantial part of the disfigurement is possibly due to immunological mechanisms. One hypothesis is a relationship between the occurrence of mucocutaneous lesions and the presence of certain alleles of polymorphic tumour necrosis factor a and b genes.

Espundia or mucocutaneous leishmaniasis often results from infection with Leishmania brasiliensis. Photo Cochabamba, Bolivia

Espundia or mucocutaneous leishmaniasis often results from infection with Leishmania brasiliensis. Photo Cochabamba, Bolivia

Mucocutaneous leishmaniasis, diagnosis

The lesions often contain few parasites. Diagnosis is sometimes made solely on a clinical basis. Culture of the parasites is possible, but not really feasible in primitive rural conditions. Serology in espundia can be positive or negative (the quality of the antigen is of crucial importance). A practical problem in South America is whether a certain skin lesion with Leishmania amastigotes is caused by L. braziliensis  or not. The geographical origin of the lesion or PCR and/or zymodeme analyses may give an answer here, though these laboratory techniques are not available in rural areas.

Mucocutaneous leishmaniasis, differential diagnosis

Differential diagnosis includes skin cancer, tertiary syphilis and yaws, leprosy, rhinoscleroma (a very chronic granulomatous infection with Klebsiella rhinoscleromatis ), rhinosporidiosis, midline granuloma (a form of T-cell lymphoma), Wegener's granulomatosis, sarcoidosis, skin tuberculosis, infection with the free-living amoeba Balamuthia mandrillaris , chronic nasal cocaine abuse, noma, and fungal infections such cryptococcosis, histoplasmosis and South American blastomycosis (paracoccidioidomycosis). With this last disease, which is a very chronic infection, the lungs are frequently affected in a manner that can mimic tuberculosis. The yeast has typical oval cells with ectospores and can be detected in sputum.

Overview: Differential diagnosis of nasal ulcers :

Mucocutaneous leishmaniasis (espundia) Fungal infections, such as paracoccidioidomycosis (syn.  South American blastomycosis), histoplasmosis, cryptococcosis, coccidioidomycosis Actinomycosis Treponematoses (syphilis, yaws, bejel) Leprosy Tuberculosis Rhinosporidiosis Rhinoscleroma (chronic infection with Klebsiella rhinoscleromatis ) Balamuthiasis (infection with free-living amoeba) Non-infectious Wegener granulomatosis Midline granuloma (a form of T-cell lymphoma) Other non-Hodgkin lymphoma Squamous cell carcinoma Sarcoidosis Relapsing polychondritis Cocaine abusus

Exercises

Algeria . A patient has a skin ulcer that has already been present for 4 months. You find amastigotes in the scrapings from the edge of the wound. His wife's grandmother has advised him to eat more vegetables and fruits. Two months later the lesion has indeed healed. What do you think? A 29-year-old Dutch veterinary practitioner has lived in northern Kenya for 2 years and has had persistent malaise and general weakness for several months. You observe a pale man with a large spleen (6 cm below the left edge of the rib cage). He does not drink any alcoholic beverages. Before his arrival he was successfully vaccinated against hepatitis B (how can this be checked?). The edge of the liver is scarcely palpable and is smooth. What do you think and do? Ethiopia . A woman in a valley village has several nodules on her face. There is a painless, persistent wound on her right hand, possibly because she has repeatedly burned herself. What do you do? Algeria . In a village the headman of the village dies of visceral leishmaniasis. The traditional healer is called in and after some ritual he says that all stray dogs and all sick domestic dogs must be killed. What do you think? What would be your opinion if this happened in India? Would all goats also have to be slaughtered? In Mexico a 25-year-old "chiclero" has developed an ulcer on the left ear. It began small but became progressively larger and has now already destroyed half the earflap. You do not see any amastigotes in a smear. What now? In Oman  you see a local man with necrosis of the nose and palate. Could this be espundia? Justify your answer. India , Calcutta. 6 months ago a 30-year-old man left the village where he was born, went to the city and has ended up in the slums. He was picked up by the mission sisters. You see a very emaciated, pale person with fever, enlarged spleen and liver, nosebleed and heavy cough, sometimes with bloody sputum. The small laboratory at the dispensary finds a haemoglobin of 4 g/dl, 14,000 blood platelets/mm 3 and a white blood cell count of 2000/mm 3 . The bone marrow contains amastigotes. What do you think and do? Uganda . A bridge-builder develops a very painful, evil-smelling, progressive, deep ulcer in the calf of the right leg. He does not remember being injured at all. There are no undermined edges and no acid-fast bacilli are present. What now? Two years ago a farmer from Amazonia moved to Sao Paulo, Brazil and has had no contact with rural areas since then. He gets a chronic wound on his upper lip. Could this be leishmaniasis? Draw an amastigote and a white blood cell beside each other (correct proportions / size ratio!). A female patient comes to the clinic with numerous chronic skin lesions on her left arm, distributed according to the lymph drainage pattern ("sporotrichoid lesions"). Which organisms would be included in the differential diagnosis: Leishmania major, Leishmania braziliensis, Sporothrix schenckii, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Nocardia sp ., atypical mycobacteria, Streptococcus pyogenes, Staphylococcus aureus, Francisella tularensis, Pseudomonas pseudomallei? Is the geographical location of any significance?

Summary

  1. Entamoeba histolytica : trophozoites and cysts; faeco-oral transmission 2. Morphological identical but non-pathogenic Entamoeba dispar 3. Intestinal infection: asymptomatic - colitis - fulminant dysentery - amoeboma. 4. Amoebic colitis to be distinguished from bacillary dysentery, balantidiasis, colon tumor, Crohn's disease, ulcerative colitis 5. Liver infection: Liver abscess with fever, liver pain, pus upon aspiration, leukocytosis, positive serology, ultrasound. 6. Liver amoebiasis to be distinguished from pyogenic abscess, cholangitis, cholecystitis 7. Occasionally perianal skin ulcers and extra-intestinal locations 8.Treatment with nitro-imidazoles + contact amoebicides  

General

Entamoeba histolytica trophozoite in rectal mucosa. Copyright ITM

Entamoeba histolytica cysts. Cysts never contain red blood cells. Copyright ITM

Comparison of different gastro-intestinal amoebae and their nuclei: Entamoeba histolytica, Entamoeba coli, Entamoeba hartmanni . Adapted from the Atlas Human Protozoa,

By amoebiasis we mean here infection with Entamoeba histolytica . This is a unicellular cosmopolitan parasite. The first description of the parasite was in 1875 by Fedor Lösch in St Petersburg. This concerned an infection in a young Russian farmer in Arkhangelsk, 150 km from the Arctic circle. This illustrates the fact that the infection is not restricted to the tropics. Transmission depends on the level of sanitation and faecal hygiene in a country or region. 

Note:

There was considerable confusion concerning the nomenclature and pathogenic properties of Entamoeba histolytica . It is now recognised that there are morphologically identical amoebae, some of which are apathogenic (not able to cause illness) and some of which are pathogenic. This concept was introduced in 1925 by the French parasitologist Emile Brumpt. The non-pathogenic amoebae are called Entamoeba dispar . This should not be confused with the completely apathogenic Entamoeba hartmanni  (another amoeba species, previously sometimes called "small race" E. histolytica). In 1978 it was discovered in London that the two kinds of amoebae could be differentiated using isoenzymatic electrophoresis. Pathogenic amoebae always belong to one group and non-pathogenic amoebae always belong to the other group. In 1989 it was discovered that E. dispar always differs from Entamoeba histolytica by well-determined genetic (DNA) markers. Apathogenic Entamoeba dispar never change into pathogenic Entamoeba histolytica . Earlier reports of this appear to be due to laboratory errors: mixed cultures and/or contamination of cultures in the lab. In pathogenic Entamoeba histolytica (those which are able to cause disease), isolates with low virulence and with high virulence can be seen (virulence is a measure of the severity of illness which certain strains can cause in certain circumstances). The degree of virulence is variable, because this is determined by several parameters, including the environment (in contrast to properties which are genetically determined). Isolates with low virulence are non-invasive, while isolates with a high degree of virulence are invasive.

 E. histolytica trophozoites are highly motile. The fuel for this constant motion comes from the anaerobic conversion of glucose and pyruvate to ethanol. E. histolytica has no mitochondria (probably through secondary loss). Many of its metabolic enzymes seem to be of prokaryotic origin, possibly acquired from the lateral transfer of genes from bacteria.

Life Cycle and transmission

Infection is caused by ingestion of E. histolytica cysts. These cysts develop in the small intestine into motile trophozoites which then find their way into the colon. The trophozoites multiply by asexual reproduction and in turn produce cysts, which are then excreted with the faeces. The cyst is quite resistant and can survive for a long time in the outside world. Excreted trophozoites die quickly and therefore are not responsible for transmission. Cysts of E. histolytica are never found in tissues. The parasite is transmitted faeco-orally as a cyst, usually from person to person. Transmission via water also occurs. Dogs, cats, rats, pigs and monkeys may become infected but do not form a significant animal reservoir (Note: kittens were used by E. Brumpt as a very susceptible animal model to test the pathogenicity of amoebae). Flies and cockroaches may carry cysts. Their role in transmission has not been properly investigated but is probably of minor importance. The main source of infection is humans. Amoebiasis is thus not a zoonosis. Infection via sexual intercourse is rare (via anal contact). The latter method of transmission may result in severe and mutilating lesions of the genitals. Entamoeba histolytica is considered to be an asexual organism, but many mysteries persist. Some pieces of evidence don't fit with this asexual idea, such as the appearance of putative heterozygous populations after mixing homozygotic populations for certain isoenzym classes. Also,  E. histolytica has the full complement of meoisis genes, which one would expect to have decayed over time if the organism abandoned the sexual life cycle. E. histolytica shows heterogeneity in nuclear ploidy owing to various levels of endomitosis. Within a population individual trophozoits exhibit continuous variation from 4n to 40n. The consequences and importance of these findings are still poorly understood. Cyclical polyploidy might be another evasion strategy to avoid the impact of Muller's ratchet. Sex can be defined as meiotic reduction of the genome complement followed by karyogamy. Autogamy or self-fertilization occurs in hermaphroditic organisms where the two gametes fused in fertilization come from the same individual. Asexual organisms are argued to be subject to accumulation of deleterious mutations through a process known as Muller's ratchet. Sex is argued to be advantageous because it generates variablity via independent assortment of genetic material through recombination. It is possible that several solutions to Muller's ratchet have been identified by various organisms. Giardia has a parasexual system with haploidization by other means than meioisis followed by karyogamy. Bdelloid rotifers (which only has female organisms), acquire foreign DNA and reorganise genomic regions after rehydration following anhydrobiosis (suspended animation that allows these fascinating organisms to survive periods of extreme dehydration).

Prevention

Amoebic cysts are resistant to normal chlorination of drinking water. Boiling and filtering drinking water eliminates the parasite. Large scale prevention depends mainly on improved sanitation and hygiene. No vaccine is available. Amoebiasis is not an opportunistic infection in HIV patients.

Intestinal amoebiasis

Intestinal amoebiasis, clinical features

We can differentiate 4 different situations in intestinal amoebiasis :

asymptomatic carriers amoebic colitis fulminant colitis amoeboma

Asymptomatic carriers

Trophozoites can sometimes remain in the intestinal lumen for years without causing any damage: the patient is then an asymptomatic carrier. The majority (90%) of patients fall into this group. Asymptomatic carriers have by definition no symptoms of amoebiasis. These persons can be detected by faeces analyses. This may show cysts of non-pathogenic E. dispar or of potentially pathogenic E. histolytica , which for unknown reasons is not invasive. Differentiation with cysts of Entamoeba coli   (which are larger and have 8 nuclei), and others, is important. Entamoeba coli is not pathogenic.

Amoebic colitis

The incubation period of amoebic colitis varies greatly. When Entamoeba histolytica penetrates the intestinal mucosa (becomes invasive) it produces ulcerations of the colonic mucosa [Gr. histo-lytica, i.e. referring to breaking down tissues]. The ulcerations are sharply defined and have eroded undermined edges. This is expressed clinically as abdominal pain, diarrhoea with blood in the faeces, and only moderate or no fever, with good general condition. When the rectum is affected there is tenesmus (painful cramps in the anus). Peri-anal ulcers may occur via direct spread from rectal amoebiasis. The ulcers develop rapidly and are painful. After suffering from amoebic colitis there may be persistent intestinal problems, the aetiology of which is unclear.

Entamoeba histolytica rectitis, with spread to the perianal skin. Copyright prof Gigase, ITM

Entamoeba histolytica colitis. Notice the typical skipping lesions. Copyright ITM

Fulminant colitis

There is sometimes a fulminant course with high fever, a severely ill patient, intestinal bleeding or perforation of the colon. A slow seepage of intestinal content into the peritoneum is very likely in a severely ill patient whose condition deteriorates progressively, together with the formation of ileus (intestinal paralysis) and a distended abdomen. A fulminant course may occur if patients are treated with steroids (e.g. if amoebic colitis is wrongly thought to be Crohn's disease or haemorrhagic ulcerative colitis).

Amoeboma

In 1% of patients an inflammatory thickening of the intestinal wall occurs. A mass may then be palpated (amoeboma). The diagnosis may be made via biopsy. The inflammatory mass may mimick colon carcinoma. Countless trophozoites are found in the tissues (never cysts). Correct therapy produces a pronounced reduction in the volume in approximately 3 days.

Intestinal amoebiasis, diagnosis

When amoebic dysentery is suspected, a fresh faecal sample or a swab from a rectal ulcer should be examined under a microscope. If examined quickly (a fresh stool, still warm) the colourless motile trophozoites can be seen. Motility disappears when cooled, and the parasites are then difficult to recognise. They should be differentiated from actively motile macrophages. The trophozoite (motile form) has one nucleus. When colourless this nucleus is scarcely if at all visible. Once stained the nucleus is moderately visible. Lugol staining kills the parasite almost immediately (motility disappears). Stained Entamoeba histolytica   trophozoites have a transparent outer border (ectoplasm) and an opaque inner border (endoplasm). The border between endoplasm and ectoplasm is not distinct in Entamoeba coli . The trophozoite measures 20 to 40 µm and may contain red blood cells (unlike other amoebae). The last detail is probably pathognomonic for pathogenic Entamoeba histolytica, but is not always present, and this statement is contested by some. Ribosomes can be arranged in characteristically shaped elongate bars with rounded ends (=chromatoid bodies).

Entamoeba histolytica trophozoite. Morphologically, it is only possible to differentiate Entamoeba dispar from E. histolytica if the trophozoite contains engulfed red blood cells. Only E. histolytica is haematophagous, although this statement is contested. Copyright ITM

Entamoeba histolytica trophozoite. Copyright ITM

The cysts have 1, 2 or 4 nuclei and measure 8 to 15-20 µm. The nuclei are best revealed by means of an iodine stain. They have a dark circumference and a dark central point (karyosome). The karyosome of Entamoeba coli  is not centrally located, but eccentric. Iodine staining can also detect glycogen (brown) in young cysts. Fresh cysts of Entamoeba histolytica also contain what are called chromatoid bodies. These are squat, oval inclusions which can easily be detected (black) with an iron-haematoxylin stain (not with iodine stain). They are not present in Entamoeba coli or Endolimax nana  cysts. In active dysentery, often no cysts are found in the faeces, but if there is little diarrhoea, the parasites have time to encyst. Since excretion of the parasites is intermittent, it is best to carry out 3 different stool analyses before deciding upon a negative result. Sometimes it is easier to reveal the parasites in a stool obtained by means of a purgative.

Tests for tracing Entamoeba histolytica antigen in the faeces have been developed, but need to be further evaluated.

Intestinal amoebiasis, differential diagnosis

The intestines may contain several species of harmless commensal amoeba. Differentiation with these other, non-pathogenic amoebae is important; they include:

Iodamoeba butschlii : mononuclear cysts, big glycogen supply Entamoeba hartmanni : small cysts with four nuclei Endolimax nana : smaller round or oval cysts with 2-4 nuclei (measuring 6-12 µm) and slow-moving trophozoites (L.: limax =slug) Entamoeba coli : larger cysts containing 1, 2, 4 or 8 nuclei Entamoeba dispar is a special case (see above)

In dysentery it is important to distinguish between bacillary and amoebic dysentery since their treatment is completely different. A diagnosis may be made clinically, but it is best to confirm this by microscopy as there is partial clinical overlap of the two diseases. Balantidium coli  is a pathogenic ciliate which can cause severe colitis. This illness is very similar to intestinal amoebiasis and the diagnosis can only be made by coprologic examination. Treatment is with tetracyclines. Pseudomembranous colitis is caused by infection with toxicogenic Clostridium difficile . These bacteria can be selected out and can proliferate after administration of certain antibiotics. Metronidazole is a good treatment in this case. Vancomycin is equally effective, but will not be given in third world countries in view of its high cost. A related bacterium, Clostridium perfringens , can cause necrotising colitis (necrotic enteritis, Pigbel syndrome). This disorder has an acute course and is very severe. Sometimes gonococcal proctitis can be confused with amoebiasis. There are then no proximal intestinal lesions and culture of the mucus provides a diagnosis. Crohn's disease and ulcerative colitis are rare in the tropics. Radiology and biopsies are essential for their diagnosis.

Bacillary dysentery Amoebic dysentery Acute onset Gradual onset Poor general condition General condition normal High fever Little fever (adult) Severe tenesmus Moderate tenesmus Dehydration frequent Little dehydration (adult) Faeces: no trophozoites Trophozoites present Coproculture positive Coproculture negative

Intestinal amoebiasis, treatment

Asymptomatic carriers

Since high percentages of the population may be cyst carriers (e.g. 10%) there is little point in treating cyst carriers found by chance in an endemic region. In any case, 90-95% of these people are infected with the non-pathogenic Entamoeba dispar . If this is nevertheless desired (e.g. in people who prepare food) diloxanide furoate  (Furamide®) is indicated. Iodoquinol  (Intetrix®) and paromomycin  (Gabbroral®, Humatin®) can be used. In regions of low endemicity it may indeed be sensible to treat the patient to prevent transmission, and also to prevent possible development of later invasive amoebiasis.

Amoebic colitis

Parasites in the tissues (intestinal wall) can be treated with nitro-imidazoles, such as metronidazole, secnidazole or tinidazole. All nitro-imidazoles require reduction of the nitro group in order to kill susceptible organisms. Since oxygen is an excellent electron acceptor, it interferes with this reduction. The spectrum of action is mainly limited to those organisms with an anaerobic metabolism. Reduction in typical anaerobes is by the pyruvate:ferrodoxin oxidoreductase complex, in which the nitro-imidazole acts as an electron sink by capturing electrons from reduced ferrodoxin. Secnidazole has the longest serum half-life (17h), compared with 12-13h for tinidazole and 8h for metronidazole. The dose of metronidazole (Flagyl®) is 500 mg q.i.d. for 5 or more consecutive days (adults). Tinidazole (Fasigyn®) is more expensive but has fewer side effects. Two grams per day x 3 days is usually sufficient for amoebic colitis. Alcohol is forbidden during treatment due to antabuse effect with severe nausea. These drugs are rapidly absorbed in the proximal intestine. For this reason they are insufficiently active upon the parasites in the distal intestinal lumen.

Chemical structure of metronidazole. Copyright ITM

The latter are treated with diloxanide furoate (Furamide® =a contact amoebicide). This drug is not active, however, against parasites in the tissues. The two drugs thus complement each other. Dose: Furamide® 500 mg t.i.d. for 10 days (adults). Children: 30 mg/kg/day. Alternative contact amoebicides are iodoquinol and paromomycine (Gabbroral®, Humatin®) which have a somewhat higher relapse percentage, although this is not certain.

 

Hepatic amoebiasis

Hepatic amoebiasis, general

If amoebae are transported with the venous blood from the intestinal wall to the liver, an abscess in the liver may be formed: hepatic amoebiasis. If the abscess is located adjacent to the fibrous capsule of the liver, adhesions are formed. A subphrenic abscess is less frequent than direct perforation of the diaphragm with empyema or fistula formation to the bronchi. Perforation to the peritoneum is rare. Perforations of the intestine, biliary ducts or navel with secondary phagedenic ulceration of the skin are more frequent than generalised peritonitis. Abscesses of the left hepatic lobe may perforate the pericardium in a life-threatening manner. [The term "abscess" is not actually correct here in the strictest sense, because this is not a collection of pus cells (white blood cells). It is local cytolysis of liver tissue.]

Hepatic amoebiasis, clinical aspects

Liver amoebiasis with perforation of the abscess through the abdominal skin. Photo Prof. Gigase. Copyright ITM

Liver amoebiasis with perforation of the abscess through the abdominal skin. Photo Prof. Gigase. Copyright ITM

Upon physical examination there is fever and pain in the liver region (pain upon palpation or percussion). The pain increases during deep inspiration or coughing. If the volume of the abscess is significant, the liver will be enlarged and the diaphragm will be elevated (percussion, auscultation, chest X-ray). The patient may develop pain in the right shoulder (referred pain). Dullness upon percussion of the base of the right lung may be due to the elevation of the diaphragm, to reactive pleural fluid or breakthrough to the pleura, or to atelectasis of the lung. Jaundice occurs in a minority (6-29%) of patients and tends to be a very late symptom. Jaundice can result from biliovascular fistula (with backflow of the bile into the hepatic veins) or from compression of bile ducts. The abscess continues to spread until it breaks through to the surroundings: the pleura (empyema), the lung, the pericardium or the skin. If fistulisation to the skin occurs, there may be swift progression of a painful skin ulcer. Untreated amebic liver abscess is often fatal.

Note: right hemidiaphragm

The right diaphragm is usually about half an (rib) interspace higher than the left. This higher position of the right hemidiaphragm is generally and erroneously attributed to the bulk of the underlying liver. In fact, the left hemidiaphragm is depressed by the heart. In partial situs inversus with the heart on the right side, so that liver and heart are on the same side, the diaphragm leaflet on the right side is the one that is lower.

Hepatic amoebiasis, diagnosis

The diagnosis of a hepatic abscess may be suspected from clinical findings. Leukocytosis will be high. Ultrasound and serology (ELISA, Latex agglutination) can confirm the diagnosis, but are often not available. Antibodies will remain present for a long time -often years- after infection. An amoebic abscess of the liver will contain necrotic liver tissue at its centre. Upon aspiration this often has a dark brownish red colour called "anchovy " or "chocolate" pus, but the pus may also be yellow, grey or greenish. The pus has no offensive odour, unlike most bacterial (anaerobic) abscesses, which is an important difference. The wall of the abscess contains trophozoites, but the necrotic liver tissue itself does not. The presence of local oedema or bulging of the skin with or without fluctuation, indicates the proximity of the abscess and the site where a puncture can be carried out. In case of doubt a trial therapy quickly produces a spectacular improvement. Fewer than 20 % of people with a hepatic abscess have Entamoeba histolytica in the faeces. The absence of amoebae in the stools is therefore not a strong argument against the diagnosis.

Entamoeba histolytica . Echography liver showing an amoebic liver abscess. Copyright ITM

Liver abscess due to infection with Entamoeba histolytica . CT-scan of the liver shows a circular necrotic area. Copyright ITM

Hepatic amoebiasis, differential diagnosis

Pyogenic anaerobic hepatic abscess: stinking pus, poor general condition, often icterus, negative serology, sometimes portal-of-entry in the intestine (e.g. colon tumour). Subphrenic abscess: sometimes a history of peritonitis or surgery, possibly pleural fluid, negative serology. Hydatid cyst: slow development, no fever, no toxaemia, serology positive for Echinococcus , sometimes calcifications on abdominal X-ray, no leukocytosis. Ultrasound may show daughter cysts. Biliary cysts: ultrasound shows a thin wall and the content is anechoic, otherwise asymptomatic. Haemangioma: hyperreflective on ultrasound, otherwise asymptomatic. On CT scan with dynamic sequences there is a centripetal staining with a delayed isodense appearance to the surrounding liver tissue. On MRI a haemangioma is extremely hyperreflective on T2-weighted images (T2 = "water images"). Metastases: ultrasound shows generally (but not necessarily) irregular and hyperreflective structure, central necrosis may occur. Frequently peripheral oedema. Hepatoma: no fever or toxaemia, no response to trial therapy, elevated alpha-foeto protein, negative serology, biopsy is diagnostic.

Hepatic amoebiasis, treatment

An amoebic abscess of the liver is treated with metronidazole  for 10 days (often initially IV), followed by diloxanide furoate  for 10 days. The latter is to destroy any amoebae in the lumen of the intestines. Chloroquine is moderately active on liver abscesses and may in some cases be administered, but is now considered obsolete. If the diagnosis is known, aspiration is only carried out for very large abscesses or if there is a risk of breakthrough. Surgery is indicated if the abscess ruptures (e.g. into the peritoneum). If a relapse of the abscess occurs this will usually happen within two months.

Note: ipecahuanca or South American Vomiting Root Before metronidazole became available in the late 1950's, amoebiasis could be treated with ipecacuanha (= ipecac). It is prepared from the dried roots and rhizomes (underground part of the stem) of Cephaelis ipecacuanha and C. acuminata.  These plants belong to the vary large plant family Rubiaceae, which contain genera like Cinchona (cfr quinine), Coffea (coffee), Rubia (cfr madder as a source of red dye) and Psychotria (cfr ayahuasca poisoning). Western explorers came across the South American plant in the 1600's. The first mention of ipecac was in 1648, in the notes of naturalist Georg Markgraff and physician Willem Pison, who traveled to Brazil in 1638. A substantial amount was imported to France in 1672 by doctor Le Gras.  Ipecac in small doses was a diaphoretic (inducing sweating) and an expectorant. In larger doses the extract became emetic and cathartic (purging). The plant was used sporadically for the treatment of "flux" (dysenteria). In the 17th Century disease was still regarded as an imbalance of the humours. The rationale for using ipecac was that bad humours could be expelled by vomiting or purging, in a way similar to bleeding and cupping. Since it induced vomiting, ipecac was also used as an antidote against common poisons such as henbane (hyoscine) and arsenic. In 1682 the court doctor Jean-Adrien Helvetius succesfully treated the heir to the French throne, the dauphin, who was ill with dysenteria. As a reward for the cure of his son, King Louis XIV granted Helvetius a sole licence to sell ipecac root, which made the doctor a very rich man. Later on the French government decided to buy back the licence for the large sum of 1,000 Louis d'Or. Another use for ipecac was found by Thomas Dover (1660-1742), who was sea captain, privateer and physician. In 1709, as a second master of the sailing ship Duke,  he helped to rescue Alexander Selkirk from Mas a Tierra Island in the South Pacific archipelago of Juan Fernandez. This story became the basis for Robinson Crusoe by Daniel Defoe. Besides mercury (for syphilis and "cryptic venereal diseases", Thomas Dover was fond of using his mysterious "Dover powder" e.g. for gout, a disease which mainly struck the rich. It stood side by side with Dioscorides' older remedy with extracts of the autumn crocus Colchicum autumnale  (source of colchicine). At that time, the higher concentrations of colchicine if the spectacular beautiful creeping lily, Gloriosa superba , was not yet known. Dover powder contained ipecac, opium, saltpeter and cream of tartar. In the early years of the 19th Century, the French master chemists Francois Magendie, Pierre-Joseph Pelletier and Joseph-Bienaimé Caventou isolated a number of important plant alkaloids, including emetine (1817, from ipecac), brucine and strychnine (both in the seeds of the vomiting nut Strychnos nux-vomica ), quinine (from the bark of Cinchona , see chapter on malaria) and chlorophyll. The main alkaloids in the roots of ipecac were emetine (methylcephaeline) and cephaeline, both complex polycyclic molecules. Emetine inhibits protein synthesis by blocking elongation of polypeptide chains. It also blocks oxydative phosphorylation in mammalian mitochondria. Systemic effects include inhibition of adrenergic and cholinergic transmission in the autonomous nervous system and blocking the reuptake of noradrenaline in the heart. Emetine is lethal for Entamoeba histolytica trophozoites, but unfortunately, the therapeutic window of this substance is rather narrow. Adverse effects include vomiting, diarrhea, polyneuritis, cardiac arrhytmia, hypotension and sudden death. In case of suspected resistance to metronidazole, emetine could be administered by deep intramuscular or subcutaneous route: 1 mg/kg/day with maximum of 60 mg/day. Emetine has been used since 1912 in the treatment of amoebiasis. It was used in the study of the vomiting centrum located near the area post-rema in the medulla oblongata. At present, it is rarely used anymore.

Emetine induces nausea and vomiting, from which it has its name, and is only rarely administered orally. After parenteral administration absorption is rapid, but excretion is slow. Traces of emetine can still be recovered in tissues up to 60 days after a treatment.

Tachycardia and dizziness after a single administration are signs of hypersensitivity to the drug. Emetine is cytotoxic, including for the myocardium. Signs of intoxication are as follows:

- Nausea, vomiting and diarrhoea, although occurrence of these does not mean that the treatment has failed.

- Palpitations, tachycardia, retrosternal pain, hypotension, dyspnoea, ECG changes (ST depression, QT prolongation, T-wave inversion). The ECG returns to normal 1 to 2 weeks after discontinuation of the injections.

- Asthenia, tremor, myasthenia and paralysis including respiratory paralysis.

Treatment must not be repeated within six weeks following the initial treatment. Emetine must not be combined with other drugs which are toxic for the myocardium. The drug is preferably administered by deep subcutaneous injection and this is painful. IM administration may result in muscle necrosis which heals slowly and may lead to formation of fistulae and permanent scars. The drug is not administered IV. Contraindications are organic heart disease and conditions of advanced asthenia. The treatment is not without risk in children and the elderly, and in debilitated or undernourished patients. Admission and strict bed rest are necessary. Emetine is very effective in amoebic dysentery and amoebic abscess of the liver, but contact amoebicides should be used to supplement the treatment.

Chemical structure of dehydro-emetine. Used in second line treatment of amoebiasis ( Entamoeba histolytica ) and fasciolasis ( Fasciola hepatica ). Copyright ITM

Dehydroemetine  is better than emetine. Dehydroemetine is a semi-synthetic product with is more rapidly eliminated and has fewer side-effects. Recommended doses are between 1 mg/kg/day IM for 5 days and 2 mg/kg/day (maximum 90 mg/day) for not more than 15 days (two injections daily). Oral administration is sometimes recommended with tablets of 10 mg DHE which are slowly soluble. The same doses are used for oral and parenteral administration.

Amoebiasis of other organs

Amoebiasis of the lungs is generally the result of the spread of an amoebic abscess of the liver, which perforates through to the base of the lung. Breakthrough to a bronchus may occur. The prognosis is usually favourable. Amoebic pleuritis (empyema) is an unpleasant complication because of the need to drain the empyema. Other locations are rare and include:

Primary amoebiasis of the lung without prior hepatic amoebic abscess.

Abscesses in muscles, e.g. the thigh.

Ulceration of the skin of the lower limbs by amoebae, which could be the result of superinfections of skin wounds due to scratching with dirty nails.

Urogenital forms, either due to fistula formation of intestinal lesions to the bladder or even, in women, of peri-anal ulcers to the vagina and cervix of the uterus.

Location on the penis if the partner has ulcers of the vagina/cervix or anal ulcers.

Parasites may appear elsewhere and lead to abscesses in other organs, e.g. the brain.

Exercises

Tunisia . A 9-year-old child has diarrhoea. You find lots of cysts with 8 nuclei in the faeces. What do you think? Venezuela . A 34-year-old man has had fever for 3 weeks and pain at the lower right ribs. There is clearly pain upon percussion of the liver. On the lateral side a painful bulge is observed between the ribs. He has had diloxanide furoate for two weeks without improvement. The faeces contain no amoebic cysts, or trophozoites. What do you think, and what do you do? Peru . A 14-year-old child has bloody diarrhoea. He has fever (38.9°C). What enquiries do you make and what do you do? Iraq . A man exhibits a painful skin ulcer on his abdomen which is rapidly growing larger. There is a small lateral fistula at the level of the lower right ribs. His general condition is very poor. What do you do? Java . A shopkeeper has dysentery. Amoebiasis is suspected and he is started on metronidazole. After taking the first tablets he has severe nausea. What enquiries need to be made? Honduras . A patient receives surgery for appendicitis. Five days later he develops fever and pain over the liver region. His general condition is poor and becomes progressively worse. A liver aspiration produces 40 ml of stinking pus. What do you think? Ghana . A man has had loose, bloody stools for 10 days. Upon rectoscopy several ulcers are observed on the rectal mucosa. The ulcers have an eroded undermined edge. A rectal smear is taken and the material is placed on a slide. A number of other patients are still waiting to be seen. Afterwards all the material is taken to the lab. You eat lunch. In the afternoon you are told that the sample contained no amoebae. What now? Draw an Entamoeba histolytica trophozoite and cyst and indicate the important characteristics. Moçambique . You have 1000 tablets of diloxanide (500 mg) in your supplies. Each day 50 people consult you (6 days a week). 10% of these people have Entamoeba histolytica cysts in their faeces. If you are going to treat everyone (10 days therapy), how long will your supplies last? Indonesia . A man has progressive bloody diarrhoea. At another hospital, cysts of Entamoeba histolytica have never been found after repeated faecal analysis. Suggest some possibilities.

Summary

Giardia lamblia is an unicellular flagellate Faeco-oral transmission via cysts Sometimes asymptomatic infection Sometimes diarrhea, atypical abdominal discomfort, bloated abdomen First-line treatment with nitroimidazoles, by preference tinidazole    

General

Giardia lamblia trophozoite in faeces. Copyright ITM

Giardia lamblia cysts in faeces, copyright ITM

Giardia lamblia cyst. Copyright ITM

Giardia lamblia ( G. intestinalis, G. duodenalis ) is a unicellular parasite (flagellate) which causes intestinal infections. The infections are often asymptomatic and Giardia was for a long time thought to be apathogenic. Since 1981 it has been regarded as potentially pathogenic and as the cause of diarrhoea and various forms of abdominal discomfort. In developing countries the infection occurs often in children but its frequency diminishes as they grow older. Related species infect amphibians ( G. agilis ) and rodents ( G. microti ). G. psittaci and G. ardeae both infect birds. G. lamblia itself may infect various animals, including dogs, cats and beavers.

History

Giardia lamblia was described for the first time in 1681 by the founder of microscopy, the Dutchman Antony van Leeuwenhoek. [The microscope was invented by Zacharias Janssen, a Dutch spectacle maker]. The trophozoite was described in detail by Vilem Lambl and Alfred Giard in 1859. Subsequently Grassi described the cyst and its association with the trophozoite. In 1960 Karapetyan succeeded in culturing Giardia in-vitro using a complex medium of fibroblasts and Candida . In 1976 Meyer achieved the axenic culture (i.e. with no other micro-organisms) of human Giardia . Later the in-vitro excystation for starting cultures was achieved and in 1986 in-vitro encystation was achieved by Gillin. The whole cycle can thus now be maintained in the laboratory.

Taxonomy

Giardia was considered to occupy a unique primitive evolutionary position. In old phylogenetic trees, it branched off very early from the other eukaryotes, as determined according to certain 16S-rRNA analyses. This view is contested now, as more information becomes available. In some respects Giardia is closer to the prokaryotes than to the eukaryotes. This has implications for research into the origin and evolution of the cytoskeleton, nuclear membrane and so on. Giardia has lost its mitochondria during evolution, but each cell has several dozen highly reduced mitochondrial remnants, so-called mitosomes. These are surrounded by a double membrane, just as mitochondria and chloroplasts in aerobic organisms and plants. In the mitosomes, iron-sulfur (Fe-S) clusters are assembled. These are cofactors in electron-transfer reactions involved in ATP production. Among Giardia's proteins that contain Fe-S clusters are hydrogenases, ferrodoxins and pyruvate:ferrodoxine oxidoreductase, which is the target for anti- Giardia drugs. It is an anaerobic protozoon, which possibly explains its sensitivity to nitro-imidazoles (e.g. metronidazole). There is no de-novo synthesis of lipids, which means that the parasite is dependent on exogenous lipids and bile salts (hence its location in the duodenum).

As with many parasites which reproduce asexually, species definition within the genus is difficult. This touches on a fundamental problem: "How can a species be defined - as opposed to a strain - if an organism reproduces asexually?" Is each natural clone a strain? Giardia lamblia is possibly a complex of different species. Chemotaxonomy via determination of antigens using monoclonal antibodies shows that there is significant antigenic variation. DNA-analysis is promising, but Giardia has a complex genome. Using iso-enzymatic analysis, 13 zymodemes are known at present. The taxonomy of this intriguing species is still to be clarified. At present, seven distinct genetic groups based on protein and DNA polymorphisms can be distinguished in  Giardia . Each group has its own host range, with group A and B able to infect humans. Giardia contains two functionally equivalent and apparently identical nuclei. The two nuclei remain physically distinct during mitosis in the trophozoit. Both nuclei are diploid and transcriptionally active. The two daughters of a single nucleus segregate to different trophozoites. However, genome sequencing shows little heterozygosity between the two nuclei. If Giardia would be completely asexual, substantial allelic heterozygosity would be expected. Nuclear fusion (karyogamy, Etym: "nuclear marriage") occurs during encystation. If accompanied by homologous recombination, this would be a parasexual process (diplomixis), unique to Giardia , as far as is known. Certain genes which function only during meiosis in other eukaryotes, are present in Giardia . Recombination between different clones has been observed, but the data need confirmation. Other mechanisms for genetic exchanges within and between trophozoites could encompass genetic mobile elements and a Giardia -specific double stranded RNA virus. Plasmids can be transfected in trophozoits. They occur there as episomes or can be integrated into the genome. Episomes are found in only one of the nuclei. Plasmid transfer between the nuclei during encystation has been demonstrated in a minority. However, with all these data it is still not clear yet at present if Giardia is asexual (as tradionally assumed), parasexual or sexual.  Till present, Giardia has not been caught "in the act" however.

Life cycle of Giardia

Cysts are swallowed with water or food. In the duodenum excystation occurs, which releases the trophozoite. This measures 12-18 µm. It attaches itself to the duodenal and jejunal intestinal villi by means of a kind of ventral sucking disk. The parasite reproduces only by asexual division. The trophozoites may multiply until the whole surface of the intestine is coated with parasites. Possibly this mechanical screening off of the intestine contributes to malabsorption.

As trophozoites are carried to the more distal parts of the intestine, the parasite encapsulates. The cyst is resistant in the outside world, but trophozoites perish. Cysts remain viable in a wet, cool outside environment. They are not very resistant to drying out, however. The cysts measure 10 x 7 µm. Transmission is via direct faeco-oral contact, food or via water. There is an animal reservoir and this is sometimes involved in human infection (giardiasis is known in Canada as "beaver fever"). In industrial countries dogs and cats are frequently found to be infected, but almost always without symptoms. Research into host specificity has led to very complex studies, produces very variable results, but is of great importance in providing better knowledge of the epidemiology. The importance of animal reservoirs should be studied further.

Pathogenicity

In many cases infection is asymptomatic, but some patients develop symptoms. One hypothesis as to the pathogenicity is the mechanical covering of the intestinal epithelium (see above). This is not, however, the only way in which the parasite gives rise to symptoms. Giardia is cytopathogenic on cell monolayers in vitro. Probably there is also in-vivo enterocytic damage with secondary disaccharidase (lactase) deficiency. Indeed, villous atrophy is found in patients. Another way in which Giardia may be pathogenic, is the destruction of conjugated bile salts with secondary steatorrhoea. Yet another unanswered question is whether the immune response contributes to the pathogenesis. In vivo Giardia has frequent endosymbiotic bacteria up to 100 per trophozoite, or so it was thought. This may possibly influence pathogenicity. The same question arises as regards any ectosymbionts. Giardia itself can be infected with an RNA virus, of unknown clinical significance.

Clinical aspects

The disease is asymptomatic in approximately 80% of cases. The clinical spectrum ranges from silent carrier status to a malabsorption syndrome. The incubation time is 1 to 2 weeks. If symptomatic, an undifferentiated acute to subacute diarrhoea which lasts on average 1 to 6 weeks occurs. In some cases the diarrhoea is steatorrhoeic with malabsorption. This may be accompanied by mild fever, abdominal pain, ructus ("purple burps"), meteorism and anorexia, malaise and vomiting. The diarrhoea may be chronic and recurrent, chiefly in patients with an IgA deficiency, hypogammaglobulinemia or agammaglobulinemia. This reflects the fact that secretory immunity in the intestinal lumen is more important for clearance than cell-mediated immunity within the intestinal lumen.

Diagnosis

Giardia lamblia trophozoite in faeces. Copyright ITM

Giardia lamblia cysts in faeces, copyright ITM

Chilomastix mesnili in faeces. This nonpathogenic flagellated protozoon should be distinguished from Giardia . Copyright ITM

Trichomonas hominis in faeces. This nonpathogenic flagellated protozoon should be distinguished from Giardia . Copyright ITM

Diagnosis is quite difficult due to the intermittent character of the presence of Giardia in the faeces. The diagnosis is mainly based on fresh or enriched coprological preparations. Sometimes several analyses of faecal specimens are needed. One specimen gives a detection rate of approximately 70% while 3 specimens increase this rate to approximately 85%. Generally cysts are found, rarely trophozoites. Other techniques such as duodenal aspiration or the EnteroTest (the string test) are less practical. In rare cases infections have been recognised on jejunal biopsy material or mucus sampled during endoscopy. Recent techniques for detecting antigen in faeces have proved sensitive, specific and fast. One means by which antibodies to Giardia can be detected is immunofluorescence, but this is not specific or sensitive.

Microscopically a differentiation needs to be made with other flagellates such as the commensal Chilomastix mesnilii , Enteromonas hominis , Trichomonas hominis (= Pentatrichomonas hominis ) and Retortamonas intestinalis .

The histological intestinal lesions are not very pronounced: flattening of the intestinal villi, lymphocytic infiltration of the mucosa, no ulceration. Persons with hypogammaglobulinaemia have more pronounced histological lesions and the clinical consequences are more important, with steatorrhoea, persistent infection, resistance to treatment. Radiology of the small intestine is aspecific. If giardiasis is suspected, but cannot be proven, a trial therapy can sometimes be used.

Treatment

The drug of first choice are nitro-imidazoles, especially tinidazole (Fasigyn®), of which 2 grammes is to be taken in one dose (adult patient). This gives a cure rate of 90 to 95%. Metronidazole  may also be used, but produces more side effects. Ornidazole (Tiberal®) 500 mg b.i.d. is an alternative, but is best given for 5 days. Alcohol should be avoided since there may be an antabuse effect. Other nitro-imidazoles are also sometimes used: secnidazole (Flagentyl®), nimorazole (Naxogyn®). Resistance to nitro-imidazoles is increasing. Mepacrine (quinacrine, atebrine) is an old drug (3 x 100 mg/day orally for 5 days) which gives good results if tinidazole fails. It also kills cysts, as opposed to metronidazole. It is a yellow product and may cause a jaundice-like skin discoloration, which the patient should be warned about beforehand. It may also cause haemolysis if there is severe G6PD deficiency. Albendazole has also proved effective in vitro, but produces varying results in vivo. Nevertheless, it is a good second choice. Paromomycin (Humatin®, Gabbroral®) is an aminoglycoside which has very low absorption when taken orally and is thus active in the intestinal lumen. However, there is quite a high relapse rate (25%). Nitazoxanide is an alternative (500 mg BD x 3 days for an adult). Another alternative is furazolidone. Metronidazole is often available in tropical countries when tinidazole is unavailable. The molecule as such is inactive, but forms active metabolites when it enzymatically reduced. Selective toxicity is achieved because the drug is only reduced in an anaerobic environment (reduction is prevented by oxygen). It's action is limited to anaerobic protozoa ( Giardia, Entamoeba histolytica, Trichomonas vaginalis : all three lack mitochondria) and anaerobic bacteria.  In T. vaginalis resistance to metronidazole is correlated with a decrease is a certain enzym activity (PFOR), but changes in drug uptake and active efflux may also play a role in resistance. Side effects of metronidazole include a metallic taste in the mouth, gastrointestinal disturbances (vomiting, nausea, cramps, headache, and a disulfiram ("antabuse")-effect. Rarer are CNS toxicity, dizzyness, drowsiness, lassitude, paresthesias, pruritus and urticaria. It is mutagenic in Salmonella bacteria. Side effects of quinacrine include vomiting, nausea, bitter taste, blood dyscrasias, ocular toxicity including corneal oedema and retinal pigmentation, sweating, headache, dizzyness, pruritus, skin and urine discoloration, myalgias, toxic psychosis, insomnia, hemolysis in G6PD-deficient persons, disulfiram-effect, seizures and CNS stimulation. It is contra-indicated in pregnancy. Resistant cells exclude the drug. Furazolidone (Furoxone) is a synthetic nitrofuran, first used as an anti- Giardia drug in 1960. It is minimally absorbed from the intestines. It is selectively absorbed by the parasites, where it is activated. About 10% of patients have side-effects, including nausea and vomiting, malaise, pruritus, hypersensitivity and hemolysis in G6PD-deficient patients. It is also a monoamine oxidase inhibitor and can precipitate a hypertensive crisis on exposure to tyramine-containing foods. There is no cross-resistance between nitroimidazole and nitrofurans. There are a number of botanical products with anti- Giardia activity, including garlic. Allicin is the principal component of garlic, but is rather volatile and unstable. Diallyl trisulphide is a more stable degradation product and is commercially available in some countries (e.g. China) for amoebiasis and trichomoniasis.

In therapy-resistant giardiasis the questions should be considered as to whether (1) compliance is failing, (2) is there a possibility of counterfeit medication, a growing problem in many countries, (2) if there may be re-infection (e.g. via an asymptomatic cyst carrier), or (4) immunodeficiency, including IgA-deficiency, (5) possibly the presence of a duodenal diverticulum (mechanical reason for relapse, as the concentration of the therapeutic drug might be rather low) or (6) whether this is genuine problem of resistance. There is in-vitro cross-resistance between the different nitro-imidazoles. If symptoms persist, (7) long-term lactase deficiency or (8) bacterial overgrowth in the small intestine with possible inactivation of nitro-imidazoles by Gram-negative bacteria should be considered.

Prevention

Prophylaxis is difficult, both individually and in the community. Giardiasis is much more common than amoebiasis. The importance of giardiasis is underestimated according to some, and it is thought to be one of the ten most important parasitic diseases in humans. Nevertheless there is little connection between the prevalence and the pathology attributed to the infection. In general the treatment of asymptomatic infections in endemic regions is considered unnecessary. Treatment of large amounts of drinking water (flocculation, sedimentation, filtration and chlorination) is important. The activity of chlorine (Cl 2 , HOCl, NaOCl) upon Giardia cysts depends upon concentration, contact time,  temperature, pH of the water and the presence of debris and organic material. Chlorine compounds work best in water with a low pH and a high temperature, when the water contains little organic debris. Usual concentrations of chlorine in drinking water averages about 0.5 - 2 mg/L. For example : at 25°C, exposure to 1.5 mg/L for 10 minutes kills all cysts at pH 6, 7, and 8. at 15°C, exposure to 2.5 mg/L for 10 minutes killed all cysts at pH 6 at 15°C, exposure to 2.5 mg/L for 30 minutes, a small number of cysts remain viable at pH 7 and 8 (but not after 60 min.) at   5°C, exposure to    1 mg/L for 60 minutes failed to kill all the cysts at any pH tested. Alternatives for chlorine and hypochlorite compounds includes chlorine dioxide, ozonation and ultraviolet irradiation Boiling of large amounts of drinking water is too costly. The detection of cysts in a drinking water sample provides no information about their viability. Several methods have been developed to test for viability, such as exclusion of vital or fluorogenic dyes (e.g. propidium iodide, fluorescein diacetate), in-vitro excystation and animal infectivity. The detection of small numbers of cysts in large volumes of water demands specific techniques. 

Non- E. histolytica intestinal amoebae

Entamoeba coli cyst in faeces. Cysts can obtain up to 8 nuclei. Copyright ITM

Iodamoeba butschlii in faeces. The glycogen mass will stain brown with an iodine stain. Copyright ITM

At least 10 different amoeba species are found in the intestinal lumen or mouth. Some consider all amoebae apart from E. histolytica as non-pathogenic commensals, but it is clear that more investigation is needed to clarify some issues, especially regarding Blastocystis hominis and Dientamoeba fragilis . Genetic analysis indicate that D. fragilis is actually more closely related to Trichomonas than to amoebae. Entamoeba histolytica Entamoeba dispar Entamoeba moshkovskii Entamoeba hartmanni Entamoeba coli Entamoeba polecki Entaoeba chattoni Entamoeba gingivalis Endolimax nana Iodamoeba butschlii Blastocystis hominis Dientamoeba fragilis E. dispar and E. moshkovskii are morphological identical with E. histolytica . In order to distinguish between E. histolytica and E. dispar  molecular tools such as PCR technology are used. Most antigen-detection tests cannot distinguish the two organisms, although one test (Wampole E. histolytica test) uses reagents that differentiate between E. histolytica and E. dispar . If trophozoites in stool contain RBC, they are pathogenic E. histolytica , but if trophozoites do not contain RBC, no species identification can be reached. Limited research has been carried out on E. moshkovskii. At present there are no good practical tests to distinguish this organisms from the two other look-alikes. Its presence is suspected especially in people who have E. histolytica / E. dispar -like cysts in the stools, but who test negative for E. histolytica/E. dispar antigen. E. moshkovskii is   highly resistant to the current amoebicidal drugs. It has been isolated from riverine sediments and brakish coastal pools, and it is considered a free-living amoeba. It is osmotolerant and can be cultured at room temperature. More study is needed on this organism. The existence of these non-pathogenic look-alikes, often results in clinical doubt and leads to overtreatment. Infections with non-pathogenic amoebae are much more frequent than infections with pathogenic E. histolytica . E. hartmanni is a non-pathogenic intraluminal parasite which can only be distinguished from E. histolytica forms by its smaller dimensions, and which was previously regarded as a non-pathogenic zymodeme of E. histolytica , the so-called small-race E. histolytica . At present it is considered a separate non-pathogenic species. Entamoeba coli is a non-pathogenic organism that is commonly mistaken for a pathogenic E. histolytica . Trophozoites move slowly and never contain red blood cells, unlike the swift and haematophagous E. histolytica . E. coli cysts are larger (10-30 µm) and may contain up to eight nuclei. The cysts contain no chromatoid bodies. E. polecki was originally found in the intestines of monkeys and pigs. Later, it was also detected in humans. In Papua New Guinea, it is not uncommon. Trophozoites are nonprogressive or sluggish. They measure 10 to 12 µm. The cytoplasm is granular and contains bacteria. If only trophozoites are present (without cysts), it is very difficult to identify this species with certainty. Most cysts have a single nucleus, and contain an inclusion body and chromatoidal bars. Cysts measure 5-11 µm. It is considered non-pathogenic. E. chatonni infects apes and monkeys, and can occasionally infect people. These infections are asymptomatic. E. gingivalis was the first human amoebic parasite to be described. Trophozoites are found in the mouth, tonsillar crypts and in the gingival sulci. Sometimes they are present in cervical smears (role of intrauterine devices?).  No cyst stage is known and it does not occur in the intestine. The parasite is transmitted by kissing and also by orogenital contact. It is usually regarded as a non-pathogenic commensal, but should be noted that some doubt the absence of its pathogenic capacity. It is sometimes recovered from patients with gingivitis and pyorrhea alveolaris, but an etiological connection is doubted. Once it has been recovered from a neck nodule by fine-needle aspiration in a patient who had received radiation treatment. It is important to distinguish it from E. histolytica, for example from a pulmonary abscess. It can contain ingested white cells and nuclear fragments of the ingested cells can be seen within the food vacuoles. No other amoeba ingests leukocytes. Endolimax nana is apathogenic. The trophozoites are small (up to 10 µm), move slowly with blunt hyaline pseudopods. The cysts are oval, small (6-12 µm) and contain two to four nuclei. Chromatoid bodies are very rare. Iodamoeba butschlii also has small cysts, about 9 µm. These have only one nucleus and a glycogen mass which stains with iodine (Lugol), from which it gets its name. Occasionally trophozoites can be seen in stool samples.  Dientamoeba fragilis is an amoeboflagellate. The fact that it can develop flagella puts it in a different taxonomic group from the above mentioned amoebae (more closely related to Trichomonas sp than to Entamoeba histolytica ). It is a non-invasive intestinal parasite. Many infections are asymptomatic, but it has been associated with aspecific diarrhea. There are two genetically different forms, but it is unclear if these correlate with virulence. It is very difficult to demonstrate with the microscope because the vegetative form is easily damaged (fragilis = breakable). No cyst stage is known and it is unclear if transmission via trophozoites can take place. One hypothesis as to how transmission of such a fragile microorganism is possible, is that Enterobius vermicularis (pinworms) could function as vectors, but solid evidence is lacking. If the faeces cannot be brought quickly to the laboratory (ideally < 10'), they should be fixed in PVA (polyvinyl alcohol) or SAF (sodium acetate formalin), otherwise the parasite will most likely not be detected. There seems to be  wide genetic variability between isolates, e.g. as demonstrated by differences in DNA melting temperature or variability of certain DNA markers. As more information will become available in the future, it is possible we will encounter a scenario like the one with Entamoeba histolytica (being pathogenic) and Entamoeba dispar (non-pathogenic): i.e. a heterogeneous species with genetic variants that have similar morphologies but different pathogenicities. It is clear that more study is needed. Dientamoeba fragilis infections can be treated with a 5 day course of metronidazole, but a single 2 gram dose (adult patient) of ornidazole it is easier and gives less side-effects. Paromomycin and iodoquinol can also be used and actually give higher cure rates. For Blastocystis hominis , see below (separate chapter).

Free-living amoebae

Saprophytic amoebae from water, silt and wet soil which belong to the genera Naegleria, Acanthamoeba, Balamuthia  and Sappinia  are cosmopolitan and potentially pathogenic. They seldom cause infection, although underreporting is probable. In industrialised countries with a moderate climate these amoebae prefer fresh water with a temperature higher than average, such as public swimming pools and warm waste water from factories or power stations. This suggests that these amoebae must be widely distributed in a tropical environment. The protozoa can be cultured on agar on which bacteria such as Escherichia coli or Enterobacter aerogenes are growing. Species identification is the work of a specialist.

Free-living amoebae, Naegleria fowleri

Naegleria fowleri trophozoite, one of the free-living amoebae.

Naegleria fowleri amoebae. Copyright ITM

Acanthamoeba sp. Notice the typical thorn-like projections (acanthopoda). Copyright ITM

Naegleria fowleri . Culberston et al in 1958 were the first to launch the concept that free-living soil and water amoebae could cause disease in humans. There are both trophozoite and cyst stages in the life cycle. The trophozoites occur in two forms, amoeboid and flagellate. The amoeboid form is the only one present in human tissue. The amoeba tends to be elongate, rather pear-shaped. It measures 7-35 µm. Round forms are about 15 µm in diameter. The pseudopodia are lobate (thick and blunt). The nucleus is large and round, with a big central karyosoma and no peripheral nuclear chromatin. The cytoplasm is somewhat granular. In order to feed, trophozoites form a kind of "cell-mouth", called a amoebostome. This is quite spectacular in electronmicroscopic pictures. In addition to phagocytosis via these food cups, contact-mediated cytolysis occurs. When flagella develop, e.g. after transfer from culture or from tissue to water, they sprout at the broad blunt end. This change to the flagellate form can take 2-20 hours. The flagellate form does not divide, but is motile. When the flagella are lost, the amoeboid form is regained and the parasite resumes asexual reproduction. Cysts measure 7-15 µm, but are absent from human tissue, in contrast with Acanthamoeba infections. They have a thick double wall and contain a single nucleus. Infection with Naegleria fowleri is the consequence of bathing or swimming in contaminated freshwater ponds or lakes at quite high temperatures, such as fresh water lakes in the summer (e.g. southern USA), ponds, rivers and hot springs. Sampling of such warm water has indicated that N. fowleri is commonly present in such environments. The location and number of amoebae in the water can vary over time. Inhalation or aspiration of contaminated dust, water or aerosols brings the parasite in contact with human tissue. The infection follows penetration of water into the nasal cavities. From there the lamina cribriforma of the ethmoid bone is penetrated, probably through phagocytosis of the olfactory epithelium. Via the first cranial nerve, the infection spreads to the lowermost part of the frontal cerebral lobes. Extensive tissue damage follows. The amoebae reproduce rapidly in the cerebrospinal fluid. There is virtually no inflammatory reaction. Haemorrhagic necrosis of the base of the brain, cerebral cortex and the olfactory lobes develops. The incubation time is 2 to 15 days. Early in the infection, upper respiratory distress, severe headache, sore throat, runny or stuffy nose, altered smell and taste occur. Fever, vomiting and neckstiffness follow. Mental confusion and coma occur after 3 to 6 days. Most infections are lethal. The CSF tends to show cloudy or haemorrhagic liquor, with neutrophilic pleiocytosis, increased protein concentrations and low glucose levels, very similar to the findings in acute bacterial meningitis. A high index of suspicion is needed for diagnosis. A history of bathing in surface water during the previous two weeks is significant. The disease closely resembles acute bacterial meningitis and is known as PAM (primary amoebic encephalitis). It is rapidly fatal in humans (there have been rare exceptions), and death usually occurs in 3-7 days. On CT-scan basal arachnoiditis with obliteration of basal cisterns in the precontrast scan and enhancement with contrast material is suggestive of amoebic encephalitis. It is best to notify the lab when sending the specimen. Motile trophozoites can be seen in a fresh preparation on a microscopic slide with coverslip, but the microorganisms need to be distinguished from leukocytes. Optimal temperature for observation is 35°C, in order to stimulate trophozite motility. Refrigeration or freezing should be avoided, and if the CSF needs centrifugation, low speeds (150-250 x g ) should be used in order not to damage the trophozoites. Giemsa or modified trichrome stains can be used.  N. fowleri trophozoites can be visualised by indirect fluorescent antibody in tissue slide sections of either hematoxyline-eosine-stained unfixed/frozen brain tissue or H&E fixed brain tissue. Amoebic DNA can be demonstrated by PCR from spinal fluid or unfixed brain tissue samples. In vitro culture requires an agar plate seeded with living bacteria or cell culture. N. fowleri does not grow in salt concentrations higher than NaCl > 0.4%.  In clinical practice, most cases will be diagnosed only at autopsy (immunofluorescense and immunoperoxidase techniques). Serology is not helpful for diagnosis. Immediate chemotherapy is required for survival. N. fowleri is rather sensitive to amphotericine B. Combination treatment with ampho B, miconazole, rifampicin, azithromycin, chloramphenicol an/or ketoconazole has been used. There have been rare documented cures of N. fowleri meningo-encephalitis after treatment with amphotericine B (Fungizone) 1-1,5 mg/kg/day administered IV and intrathecal, and associated with rifamipicine 10 mg/kg PO/IV (max 600 mg/day). Intensive supportive care is required. The mortality is high. The only certain way to prevent N. fowleri infections is to refrain from water-related activities, especially those in warm freshwater during periods of high temperature and low water volume. Nose clips should be used during activities in warm fresh water. During such activities, one should avoid digging in or stirring up sediment. The genome of Naegleria gruberi was sequenced and reveals the presence of meiosis-specific genes, suporrting the occurence of sex in this species (although there has no formal confirmation of a sexual life cycle).

Free-living amoebae, Balamuthia mandrillaris

Balamuthia mandrillaris infection with important skin lesion. Copyright Alexander von Humboldt Institute, Peru

Infection with Balamuthia mandrillaris , a free-living pathogenic amoeba. Copyright Alexander von Humbolt, Peru

Balamuthia mandrillaris infection with important skin lesion. Copyright Alexander von Humboldt Institute, Peru

Balamuthia mandrillaris amoeba in biopsy. Copyright Alexander von Humboldt Institute, Peru.

Balamuthia mandrillaris amoeba in biopsy. Copyright Alexander von Humboldt Institute, Peru.

In the past, Balamuthia mandrillaris was regarded as a harmless soil inhabitant. Infection with this free-living leptomyxid amoeba was first described in a pregnant mandrill at the San Diego Wild Animal Park. The animal died of meningoencephalitis. Later, it was found that several other mammals can be infected with this protozoon (gorillas, baboons, monkeys, sheep, horse). Genotyping studies have shown that lethal infections are due to a single species with global distribution. It is not clear how humans get infected, but transmission via swimming in contaminated surface water is one possibility. The pathogen has also been isolated from a potted plant in a home. Trophozoites of B. mandrillaris measure 12 to 60 µm in diameter. In tissue culture, broad pseudopodia form, although on cell monolayers, the pseudopodia are thin and fingerlike. Cysts are spherical and measure 6-30 µm. The cyst has a three-layered structure: endocyst, mesocyst and an outer wrinkled ectocyst. Infection with this amoeba causes peri-orbital swelling and ulceration, followed by symptoms of granulomatous meningo-encephalitis, in both immunocompetent and immunocompromised persons. Other tissues can also be infected. Symptoms include headache, nausea, vomiting, fever, visual disturbances, dysphagia, seizures and hemiparesis. Both trophozoites and cysts are found in CNS tissues. Differentiation with Acanthamoeba is difficult when using only simple light microscopy. Electron microscopy, immunofluorescence testing and histochemistry are needed for definite species identification.  B. mandrillaris does not grow well on Esch. coli -seeded nonutrient agar plates. It can be cultured on certain cell monolayers. A cell-free medium has been developed. In vitro studies show that B. mandrillaris is suspectible to pentamidine. Ketoconazole, propamidine, clotrimazole, sulfadiazine, fluconazole and clarithromycine have all been used in treatment of patients. Treatment is not standarised yet. Experience in Peru showed that prolonged administration of itraconazole 400 mg/day (adults) can be useful.

Free-living amoeba, Sappindia diploidea

A newly (2003) recognised pathogenic free-living amoeba is Sappindia diploidea . Infection results in amoebic encephalitis with hemorrhagic inflammation which presents as a tumor-like cerebral mass. Trophozoites measure 40 to 70 µm. They have a distinctive double nucleus. Both nuclei are connected to each other by perpendicular filaments. Trophozoites ingest host blood cells. They can be demonstrated with Giemsa and periodic acid-Schiff stains. Cysts have not yet been detected in human tissue.

Free-living amoebae, Acanthamoeba sp.

In the past, there was some taxonomic confusion between the genera Acanthamoeba and Hartmanella , but at present, no true Hartmanella species has been shown to be pathogenic to humans. Acanthamoeba sp are free-living protozoa which occur in numerous places (water, dust, waste).  Several species have been described: A. castellani, A. culbertsoni, A. polyphaga, A. healyi, A. astronyxis, A. hatchetti, A. rhysodes, A. griffini, A. quina, A. lugdunensis. The trophozoite measures 15-45 µm and exhibits fine thorn-like protrusions, acanthapodia. The name of the genus is derived from this feature (Gr. acanth = thorn). In the environment, they live from bacteria, algae and yeasts. However, they can also exist on liquid nutrients taken up by pinocytosis. Cysts form under adverse environmental conditions. The cysts have a double wall. The outermost wall is wrinkled. They are rather resistant to a variety of biocides, chlorination and antibiotics. They easily survive low temperatures (24 years at 4°C has been described). In 2003, an extreme large virus (mimivirus) was isolated from Acanthamoeba polyphaga , present in water of a cooling tower. This remarkable virus, just visible to the naked eye, remains mysterious. In 2008 an even bigger virus ("mamavirus") was discovered. It was found that this large virus itself can be infected with a small virus, a so-called virophage. Human seroconversion against this virophage has been observed, but the significance and relevance of this finding is still unclear. Acanthamoeba species are responsable for several clinical problems: (1) granulomatous amoebic encephalitis, (2) keratitis, (3) disseminated lesions, including skin ulcers, but also lesions in adrenals, kidneys, liver, spleen, thyroid.... 1. Granulomatous amoebic encephalitis (GAE): incubation period for is not known. Unlike with Naegleria , infection of the central nervous system progresses slowly and occurs where there is immunesuppression or in the course of a severe general illness. Infections are more common in AIDS patients with a low CD4-count. Generally it presents as a subacute meningo-encephalitis with signs of a brain abscess, and develops in two to three weeks (range 7 days - 5 months). An acute onset of disease, with fever, headache and lethargy has also been described. Confusion, dizziness, headache, drowsiness, neckstiffness, seizures, nausea with or without vomiting, hemiparesis. The cerebral hemispheres tend to be involved, with an inflammatory exudate covering the cortex, granulomatous necrosis of the brain parenchym and thrombosed blood vessels. Multinucleated giant cells are easily seen in immunecompentent individuals, but are scarce in immunesuppression. Such infections can mimick malignancies, fungal infections or abscesses. In AIDS patients, the differential diagnosis with cerebral toxoplasmosis can be very difficult. 2. Keratitis. This is more common than cerebral inflammation. This was first described in 1974. The amoebae may infect small wounds of the cornea and then trigger a dangerous ulcerative keratitis which may develop into painful uveitis with hypopyon, scleritis and panophthalmitis. Levels of amoeba-specific IgA in tears were significantly lower in patients with keratitis. Acanthamoeba keratitis should be considered in the differential diagnosis of uveitis in AIDS patients. It is possible that bacterial endosymbionts contribute to the pathogenic potential of these protozoa. Initially this diagnosis is often missed and the lesion is considered to be a herpetic or fungal keratitis. Acanthamoeba infection may follow upon a herpetic keratitis. It is possible that decreased sensation plays a role in these cases. Infection can follow corneal trauma (e.g. corpus alienum). The number of cases has grown in recent years as the result of increased use of contact lenses and the practice of rinsing these with tap water. It is likely that bacteria in the biofilm on dirty contact lenses constitute a good source of nutrition for the amoebae. If there is a superficial epithelial defect, whether or not caused by the contact lens, the amoebae may become invasive in the corneal stroma. Since keratitis and cornea ulceration can follow direct exposure of the eyes to contaminated material or solutions, proper desinfection of contact lenses is imperative. Commercial contact lens disinfectant solutions differ in their ability to kill Acanthamoeba species. Those containing hydrogen peroxide (H 2 O 2 ) or chlorhexidine-thiomersal were effective against cysts and trophozoites. Benzylalkonium chloride-based solutions are only active against trophozoites, and polyquaternium or polyaminopropyl are ineffective. Of course, the cyst is more resistant than the trophozoite. Exposure times need to be sufficient. Cysticidals must be able to gain access to the interior via pores in the cyst wall. The pores (ostioles) are plugged with mucopolysaccharides. These plugs need to be disrupted in order to kill the parasite inside the cyst wall. The amoebae are often scarce in corneal smears. Culture is possible on nonnutrient agar plates with an overlay growth of Esch. coli or Pseudomonas aeruginosa bacteria on which the trophozoites feed, but there are also several other culture methods, including cell monolayers. Filtration of clinical samples through micropore filters gives higher yields than centrifugation. Sometimes the diagnosis is made purely on anatomopathological grounds, e.g. during a cornea transplantation.  Identification however, is difficult. Tissue stains are effective. Cysts stain with Gomori's silver methenamine, periodic acid-Schiff and calcofluor white. CSF or bronchoalveolar lavage fluid cytospin preparations can be used to search for the amoebae. So far, serology is not useful for diagnosis.   3. Other locations . Abscesses in other locations, and granulomatous skin lesions in which histological investigations show amoebae, have also been observed. Skin lesions are more common in AIDS patients. Hard erythematous papulonodular lesions or non-healing indurated ulcers may be the first sign. It is not known yet if the skin lesions represent a primary focus (direct inoculation) or result from hematogenous spread from elsewhere in the body. Treatment is very difficult. Combination treatment with penicillin, chloramphenicol, rifampicin and pentamidine has been used in systemic infections. Treatment of Acanthamoeba keratitis employs a combination of propamidine isethionate eye drops (Brolene®), topical neomycin, polyhexamethylene biguanide collyre (Lavasept®) and/or topical chlorhexidine (Hibitane®). Brolene®, available in Great Britain, is an antiseptic which is moderately toxic for the corneal epithelium. The use of topical steroids  is controversial but probably beneficial. Oral itraconazole is probably also active. Topical miconazole  is sometimes also used. Pentamidine (a diamidine related to propamidine) is being evaluated.

                   COMPARISON  Naegleria fowleri   Acanthamoeba sp  Amoebic form with lobate pseudopodia;   Flagellate form (two flagella)  No flagella, filiform sharp pseudopodia  Cysts not present in tissue; they are small and smooth  Cysts can be found in tissues;  large and wrinkled with a double wall   Culture requires living cells (bacteria or cell culture)  No growth if NaCl concentration > 0.4%  May grow without bacteria; not affected by NaCl 0.85%  Smaller than Acanthamoeba ; dense endoplasm; less distinct nuclear staining   Large round, less endoplasm; more distinct nucleus

Free-living amoebae and endo-amoebic bacteria

Many amoebae feed on bacteria. Sometimes, the bacteria are killed and digested by the amoebae, sometimes the bacteria kill the amoebae, and sometimes, a kind of more or less stable symbiotic relationship is achieved. This can be temperature-dependent. E.g. certain Parachlamydia bacteria are endosymbiotic in amoebae at 30°C and lytic at 37°C. This suggests that bacteria present in amoebae in the cooler upper respiratory tract (nose) might be liberated in the warmer lower tract, for example during an aspiration event. Co-culture of amoebae (e.g. Acantamoeba polyphaga ) with bacteria recovered from different places (mouth, nose swabs, …) could become an interesting tool for new discoveries in this field. Free-living amoeba have been shown to be a reservoir of pathogens such as certain Legionella species. The pathogenic role -if any- of certain other amoeba-associated bacteria remains to be established (e.g. some amoebae may be natural hosts for pathogens such as Campylobacter jejuni, Listeria monocytogenes, Vibrio cholerae, Mycobacterium leprae, Burkholderia sp., Cryptococcus neoformans and Pseudomonas aeruginosa, as well as microorganisms of unclear status such as Bosea massiliensis , Legionella anisa, Parachlamydia sp., Rasbo bacterium, Bradyrhizobium sp.). The clinical relevance of these findings are not clear yet .  

Background on Rhizopoda

The amoebae belong to the Rhizopoda (Gr. rhizo = root). These animals are related to a number of other protozoa, including the Actinopoda (Gr. actis = ray) and Foraminifera (L. foramen = opening). The taxonomic classification varies greatly from author to author. Some are being studied for their unique genetics. The genome of Amoeba dubia (syn. Polychaos dubia ) contains over 600 Gigabase pairs, which is 200 times more DNA than in humans. Acanthamoeba castellanii contains mitochondria in which a special form of transfer-RNA editing takes place. This forms a subject of intensive research. The class of Lobosea includes the orders of naked amoebae (e.g. Entamoeba and Naegleria ) and the armoured amoebae (Arcellinida, amoebae with an armour of grains of sand or excreted silica).  The class of Actinopoda contains almost exclusively sea plankton. These organisms have no direct medical importance but include some pretty micro-organisms, which by itself is already a good reason to study them. The Actinopoda are also known as Radiolaria (radiating creatures) and Heliozoa (sun creatures). They often have an exoskeleton of opal (SiO 2 ×n.H 2 O) and have radial or bilateral symmetry. The cytoplasm forms radiating pseudopodia (axopodia) which penetrate through small holes in the exoskeleton, and which give this group its name. The accumulation of countless silica skeletons from dead Radiolaria has lead to geological sediments. In this respect they are similar to diatoms which are algae belonging to a completely different taxonomic group. Diatoms have a siliceous skeleton consisting of two parts, like a pill box; see also "amnesiac shellfish poisoning". The Acantaria are radially symmetrical Actinopoda with a skeleton of strontium sulphate, also known as celestite. This mineral dissolves relatively quickly in sea water after their death when they sink to the depth of the ocean. The Foraminifera are exclusively marine protozoa. At present the majority are benthic (living at the bottom of the sea), and only some species belong to the plankton. Their exoskeleton consists of crystallised calcium carbonate (calcite or the rather more dense aragonite, sometimes even dolomite, i.e. Mg/CaCO3). They have the shape of linear vessels or complex spiral chambers. Some Foraminifera are large (up to a few cm) and are then called nummulites (Lat. nummus = coin). The cytoplasm penetrates through pores in the skeleton and forms pseudopodia which may anastomose (reticulopodia). In the course of time the exoskeletons from the small dead animals accumulate in thick layers on the seabed provided the water is no more than 3700-4400 m deep. Below this depth they dissolve because the deep cold water can contain more CO 2 than warmer water. These may later be converted into chalk cliffs, which can also contain dead coral, the shells of molluscs and the microscopically small calcium shells of a certain group of very small algae (Haptophyta, Prymnesiophyta or Coccolithophoridae). The Egyptian pyramids, for example, were built of this material.

Balantidium coli

Balantidium coli trophozoite. Notice the numerous cilia. Copyright ITM

Balantidium coli cyst. Cysts do not have cilia. Copyright ITM

Ultrastructure of flagellum, showing the classic 9 and 2 structure. Copyright ITM

Balantidium coli is a large protozoon. The trophozoite measures 30-200 µm x 40-60 µm. The whole surface of the trophozoite is dotted with countless cilia. These are very characteristic and because of this, it is classified as a ciliate (compare with Paramecium ). Balantidium coli is the only ciliate pathogenic to humans. The cysts are oval and measure approximately 45 x 60 µm. As yet there are insufficient data concerning this organism. It is a commensal of pigs. Transmission occurs from pigs to humans and from human to human in poor hygiene situations, also via water or food contaminated with cysts, including poorly cooked pork sausages. The infection is cosmopolitan, rare, yet locally prevalent, e.g. in Papua New Guinea and in tropical Africa. As with amoebiasis the infection may be intraluminal and latent or invasive in the intestinal wall and symptomatic. In the invasive forms ulcerations of the intestinal wall are found, quite similar to those of amoebiasis, with the same complications and the same clinical forms. Liver abscesses caused by B. coli have been observed but are extremely rare. Diagnosis is parasitological by direct stool microscopy or enrichment techniques. Treatment is not always simple. Tetracyclines (10 days) have been used as well as imidazoles in high doses.

Note: Flagella

In a fresh preparation, B. coli can be very quickly recognised due to its swift manner of propulsion. Under the microscope, the creature is difficult to keep in the field of vision due to its relatively high speed. How in fact do such organisms move? One of the problems which micro-organisms face is propulsion. The protozoa are in an aqueous environment and have to overcome enormous resistance during movement. Due to their small dimensions it is as if they were moving in thick syrup (they cannot "freewheel"). One important element in this context is Reynold's number. This number is relevant not only for micro-organisms, but also for insects, fish, aeroplanes, etc. This dimensionless number indicates the relationship between powers of inertia and powers of viscosity [Reynold's number = density of liquid x speed x length / viscosity]. If Reynold's number is high, there is turbulent flow and the effect of the viscosity is negligible. For small organisms in water, and therefore associated with small Reynold's number, there is laminar flow and the influence of the viscosity is dominant. For this reason many micro-organisms have special organelles with which to move. One important organelle is the flagellum. A certain group of micro-organisms (flagellates) take their name from the fact that they possess flagella. The term flagellum (L. flagellum = whip) is used, however, for two totally different organelles. Some micro-organisms are dotted with myriads of these organelles which work in a coordinated way and which are then called "cilia".

Flagella in Eukaryotes : In eukaryotes the flagellum is a long, thread-like structure, formed by the external plasma membrane with a central axoneme. This axoneme consists of what is called a 9 + 2 structure, formed by contractile microtubuli. The main proteins are tubulins (the building proteins of microtubuli), nexins (forming connections between microtubuli) and dyneine (ATPase). Cilia have the same structure as flagella, but are rather shorter and they are always present in large numbers on one cell. Their movement is co-ordinated. The flagellum will cause a sinusoidal wave, for which ATP is used as an energy source. The base of the flagellum is formed by a basal body, situated just next to a kinetoplast (the grouped DNA of a special mitochondrion). The position of the kinetoplast as regards the nucleus is important (cf. promastigote, epimastigote). Note the direction of the movement: e.g. in a human sperm cell the movement will begin at the base of the flagellum, the sinusoidal wave moves towards the tip of the flagellum and the cell is driven forwards (direction away from the tip of the flagellum). The movement of the cell is similar to that of a motorboat with a screw propeller. In protozoa such as trypanosomes the movement is reversed. The sinusoidal wave moves towards the cell and pulls the cell in the direction of the flagellum. The movement of the cell can be compared to that of an ordinary propeller aircraft.

Flagella in Prokaryotes : In prokaryotes the term flagellum is used to indicate a different, somewhat shorter organelle, consisting of the protein flagellin (see chapter on Cholera).

Note: hydrogenosomes and mitochondria

Balantidium coli is the only pathogenic ciliate for humans, but other ciliates are certainly worth studying. In aerobic cells, most ATP is produced by mitochondria. Normal mitochondria, which consume oxygen, do not function in anaerobic circumstances. Several alternative ways of ATP production are possible, including the biochemical reactions in hydrogenosomes. These are cell organelles that produce ATP and hydrogen. They are found in various eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. The evolutionary origin of this organelle is unclear. In 2005, it was shown that hydrogenosomes of Nyctotheris ovalis , a ciliate living in the hindgut of cockroaches, have retained a rudimentary DNA genome which encodes for some components of the mitochondrial electron transport chain. At first, this finding was baffling. However, it suggests that the N. ovalis cell organelle might be a missing link between mitochondria and the "usual" hydrogenosomes.

Cryptosporidiosis

Cryptosporidia are coccidia and belong to the Apicomplexa phylum. Coccidia form an order of unicellular eukaryotic micro-organisms, which includes the following human pathogens: Toxoplasma gondii, Sarcocystis sp., Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli . Microsporidia do not belong to the Coccidia and form a totally different taxonomic group. DNA analysis of Cryptosporidium suggests that there could be more than twenty different species. C. parvum is the most common parasite in this group in human infections, but C. meleagridis, C. canis, C. muris  and C. felis  are also found in immune-compromised persons with acute diarrhoea. C. parvum has two genotypes: genotype I is a purely human (anthroponotic) parasite while genotype II is zoonotic and can infect various animals as well as humans. The zoonotic genotype retained the name C. parvum , whereas the anthroponotic genotype was renamed Cryptosporidium hominis . The complete genome of Cryptosporidium parvum type II has been sequenced in 2004. Analysis shows that this parasite has very streamlined metabolic pathways and depends upon the host for certain nutrients. It lacks an apicoplast and possesses a degenerate mitochondrion without its its genome. Gene loss in nothing new for mitochondria, but complete absence of mitochondrial DNA is very unusual. In quasi all other mitochondriate organisms, usually a small set of core genes (those closely involved in electron transfer reactions) are retained. The extreme reduction of the mitochondrion means that it has dispensed with most of the biochemical processes usually performed by this organelle. Consequently, C. parvum is resistant to inhibitors of the classic respiratory chain, such as cyanide, azide and atovaquone.

Cryptosporidium parvum , Ziehl stain. Copyright ITM

Cryptosporidia are very small parasites of the intestinal mucosa (2.5-5 µm). They infect many vertebrates (various mammals [calves], birds, fish, and so on). They have been known since 1907, but were only described in humans in 1976. Several species can be differentiated according to the type of host, but the host specificity does not appear very precise. Transmission to humans occurs from calves, dogs and cats. Transmission via drinking water or via insufficiently chlorinated water in swimming pools happens frequently. This species is resistant to standard chlorination. In 1993 a huge epidemic took place in Milwaukee in which 403,000 persons were infected via drinking water. The parasite was first observed in humans in cases of persistent diarrhoea in patients with immunosuppression, and since 1981 in cases of AIDS. Since 1983 the infection has frequently been recognised as a cause of benign and brief diarrhoea, both in children and adults, and it is one of the more common aetiologies of travellers' diarrhoea.

The complete cycle of the parasite, sporogony and schizogony, takes place in the same host. People become infected by swallowing thick-walled, resistant oocysts. Once in the intestine the parasites excyst and release sporozoites. They penetrate epithelial cells via the apical membrane. Then follows a reorganisation of the actin cytoskeleton of the host cell. The parasites are intracellular but extracytoplasmic. After maturation of the sporozoite there is asexual reproduction via schizogony with the formation of merozoites. These may either penetrate a new epithelial cell to repeat the cycle (type 1 merozoites) or undergo further intracellular changes (type 2 merozoites) to the sexual form of the parasite. The macrogamont is the female form, the microgamont the male form. The microgamont releases microgametes. After fertilisation and the formation of zygotes, thin-walled oocysts are produced, which after meiosis release sporozoites in their turn which amplifies the infection (auto-infection). Thick-walled oocysts are released into the lumen of the intestine, and are directly infectious via the faeco-oral route. C. parvum induces apoptosis in epithelial cells. It is assumed that an enterotoxin is produced, but this has not yet been proven.

The parasites may be found throughout the entire digestive tract and even in the mucosa of the respiratory tract, but are usually limited to the duodenum and jejunum. The incubation period is 4 to 12 days (usually 7-10 days) and is followed by moderately severe diarrhoea without fever and with little abdominal pain but no particular characteristics. Asymptomatic infections may occur. If there is no underlying immunosuppression, spontaneous recovery occurs within a few weeks. It is estimated that 4 to 10% of all commonplace cases of diarrhoea in children in tropical environments can be attributed to Cryptosporidium . In patients who have a deficiency in cellular immunity (such as in HIV infection), the diarrhoea is more pronounced, chronic for several months, and recurrent. This is accompanied by painful abdominal cramps, nausea, dehydration, loss of weight and mild fever. Fulminant infection with cholera-like diarrhoea may occur in patients with fewer than 50 CD4 T-cells/mm 3 . Sometimes the protozoa enter the biliary tract, resulting in sclerosing cholangitis, strictures and papillary stenosis. Diagnosis is difficult and requires invasive procedures such as retrograde cholangiography (ERCP [endoscopic retrograde cholangiography]). A biliary tract reservoir may contribute to the chronic course of infection. Other extra-intestinal manifestations include infections of the respiratory tract, pancreas and middle ear.

Diagnosis is based on looking for the parasite in the faeces on smears stained with modified Ziehl-Neelsen or Kinyoun staining. The small dimensions of the parasites and their similarity to yeast cells, were responsible for the fact that infection in humans was only recognised in 1976. The parasites can easily be recognised on intestinal biopsy material obtained by endoscopy. There is villous atrophy, hyperplasia of crypts and an inflammatory cellular infiltrate in the lamina propria. There are other diagnostic techniques, such as immunofluorescence, antigen-capture ELISA [enzyme-linked immunosorbent assay] and PCR [polymerase chain reaction], but these are not available in most tropical settings. Serology is of little benefit. It is possible to detect thickening of the wall of the biliary tract and/or dilation of the gall bladder by ultrasound.

Treatment is mainly symptomatic and can be quite difficult in AIDS patients. The best practical method in these patients is via HAART (highly active antiretroviral therapy). Good results have been described using colostrum from hyperimmune cows (Lactobin-R®). Paromomycin  (Humatin®, Gabbroral®) is a non-absorbed aminoglycoside and is of limited use. A swift cure has been reported in infections with C. felis . Azithromycin and letrazuril are being investigated. At present the drug of choice is nitazoxanide (Alinia®, Cryptaz®, 500 mg tablets or syrup). If papillary stenosis exists, papillotomy may be carried out to achieve decompression of the biliary tract. Prevention is based on the prevention of contamination of the water supply, e.g. by run off from cattle farms. Cryptospodirium cysts are very resistant to chlorination (much more so than Giardia cysts, although even those have a certain resistance to standard concentrations in drinking water). The exact sensitivity depends upon several factors, such as water temperature, acidity, contact time wiht chlorine, turbidity, presence of organic material, and the of course details of the chemical (chlorine, hypochlorite, chlorine dioxide ...). The cysts are more sensitive to ozone.

Isosporosis

Isospora belli mature oocyst containing two sporocysts. Copyright ITM

Chemical structure of pyrimethamine. Copyright ITM

Isospora belli  was first described by Virchow in 1860 but not named until 1923. It is a coccidian parasite of the duodenum and proximal small intestine (jejunum) in humans. It is cosmopolitan, but more frequent in a tropical environment. No reservoir hosts other than man are known. The oocysts are very resistant to environmental conditions and may remain viable for months if kept cool and moist. The sexual and asexual cycles occur in the same host. The parasites are located intracytoplasmic, unlike Cryptosporidium . There is a prepatent period of about 9-10 days. Infection may be latent or lead to diarrhoea for one to two weeks, occasionally with mild fever, headache, malaise and abdominal pain. The stools tend to be soft, watery or foamy, with an offensive smell, suggesting malabsorption. In immunosuppressed people, the infection can become chronic. In such cases, oocyst shedding can continue for years. In AIDS patients, the parasites can occasionally be found in lymph nodes and walls of large and small intestine, mesenterium and even liver and spleen. Diagnosis is difficult and is based on coprological examination, duodenal tubage and biopsy of the duodeno-jejunal mucosa, in which the parasites are not very numerous. The oocysts are rather large and measure 20-33 µm by 10-19 µm. The oocysts are very pale, transparent and are easily overlooked, especially in a concentrated sediment of a polyvinyl alcohol-preserved stool sample. For this reason, it is best to diminish the light intensity of the microscope and additional contrast should be obtained for optimal examination conditions. Wet preparations are generally preferred. Charcot-Leydig crystals (derived from eosinophils) are occasionally found in isosporiasis cases. The oocysts are acid-fast and can also be detected with auramine-rhodamine staining.  Usually the oocyst contains only one immature sporont, but two may be present. Continued development occurs outside the human host with the development of two mature sporocysts, each containing four sporozoites. Normally this takes about 48 hours. The sporulated oocyst is the infective stage which will excyst in the duodenum. The condition can be treated with cotrimoxazole  (e.g. Bactrim forte® 4 x 1 tablets/day for 10 days). If there is diminished sensitivity or resistance, either pyrimethamine  (Daraprim®) 25 mg/day x 20 weeks or the combination ornidazole (e.g. 2 gram on day 1, 15, 30) with albendazole (400 mg BD x 30 days) is used. Ciprofloxacin  is also moderately effective (70% cure rate). Prevention is based on improved personal hygiene measures and improvement of the sanitary conditions.

Eimeria in faeces. To be distinguished from Isospora and Sarcocystis . Copyright ITM

Note: Eimeria

Do not confuse with Isospora hominis (pathogenic) or Eimeria , a genus not pathogenic for humans. Eimeria sp. form the largest and the most economically important genus of Coccidia and includes species which can infect various mammals and birds. How long the protozoa survive, the period of sporulation under various circumstances, the infectious dose (probably low), the existence of alternative routes of transmission than faeco-oral transmission are all insufficiently clear.

Sarcocystosis

Sarcocystosis, general

Sarcocystis species are parasites of mammals, birds and reptiles. Human sarcocystosis (syn. sarcosporidiosis) is rarely diagnosed. The parasites almost always have a life cycle involving 2 hosts. Sexual reproduction (gametogony followed by sporogony) takes place in the intestine of a carnivore or omnivore (the predator). Asexual reproduction (schizogony) takes place in the muscles of another host (the prey).

In 1843 Miescher described the parasite for the first time as white threads in the muscles of a mouse. In 1869 Lindemann erroneously described 3 human cases. In 1893, Kartulis described the first authentic human case. In 1972 it was discovered that the intestinal parasite Isospora hominis and the muscle parasite Sarcocystis hominis  were 2 stages of the same organism (do not confuse with Isospora belli ).

For some species, humans are the definitive host i.e. the host in which sexual reproduction is completed. In this case there is intestinal sarcocystosis. Humans may also act as accidental dead-end intermediate hosts for several other species and in these cases there is muscular sarcocystosis. The natural final and intermediate hosts of many Sarcocystis species, which infect human muscle are still unknown. The Sarcocystis species themselves are also still unknown. Histologically the tissue cysts are often similar to those found in local monkeys. All in all, little is known about these parasites.

Sarcocystosis, intestinal sarcocystosis

Isospora belli Copyright ITM  

Sarcocystis sp. Copyright ITM

Sarcocystis, pseudocyst in muscle. Copyright ITM

Sarcocystis, pseudocyst in muscle. Notice the typical perpendicular striations of the cyst wall. Copyright ITM

Sarcocystis bovihominis and Sarcocystis suihominis are parasites of humans. Infection occurs due to eating raw or insufficiently cooked meat from cattle or pigs containing tissue cysts (intestinal infection cannot be triggered by the ingestion of sporocysts). The sexual cycle takes place within the cytoplasm in the cells of the human intestinal mucosa. The sporocysts which are released with the faeces are infectious for the intermediate host. These infections are cosmopolitan and generally asymptomatic. They can nevertheless trigger enteritis with peripheral hypereosinophilia. The diagnosis is based on faecal examination. Sometimes the parasites will be detected in surgical resected intestinal specimens.

Sarcocystosis, muscular sarcocystosis

Muscle infection is caused after swallowing sporocysts (faeces of an infected predator). Each sporocyst releases 4 sporozoites. These penetrate the intestinal wall. Reproduction begins in the vascular endothelium. After dissemination of merozoites, there is invasion of skeletal and cardiac muscle tissue and possibly the central nervous system (in animals). The merozoites develop first to metrozoites and then to cystozoites. These tissue cysts remain dormant until the host is eaten by a predator, after which the intestinal cycle begins. The tissue cysts gave the genus its name (Gr. sarx = flesh).

Most human infections are apparently asymptomatic. It is also possible that the diagnosis is systematically missed (data from investigation of routine autopsies). No cases of neurological involvement in human patients are known. Some patients with muscular sarcocystosis develop an eosinophilic myositis. The myositis is characterised by muscle pain, painful mild muscular swelling, mild fever, general weakness, bronchospasms and eosinophilia. This should be differentiated from trichinosis ( Trichinella spiralis ). Eosinophilic fasciitis, toxoplasmosis, polymyositis, dermatomyositis and polymyalgia rheumatica may lead to similar clinical pictures. During the short blood phase, the parasites can be mistaken for Plasmodium falciparum gametocytes, or even a rare blood form of Toxoplasma gondii .

Diagnosis is made via muscle biopsy. The intact cysts in the muscle generally do not trigger a local inflammatory reaction. Dead and ruptured cysts, however, may cause inflammation. It is necessary to differentiate from tissue cysts of Toxoplasma gondii ( Toxoplasma generally has smaller cysts with a thin non-striated capsule) or Trypanosoma cruzi . Not much is known about treatment. At present there is no known and agreed upon therapy. For many years, intestinal sarcocystis forms were named Isospora hominis , reflecting their morphological similarity. Since  Isospora belli is treated with cotrimoxazole, this product appears to be a logical -but unproven-choice for intestinal sarcocystosis. The activity of primaquine on parasites in muscle is in unclear.

Cyclospora cayetanensis

Cyclospora cayetanensis in faeces, unstained. The parasite is about double the size of Cryptosporidium parvum . Copyright ITM

Cyclospora cayetanensis  is a protozoon which belongs to to the Coccidia. Several related protozoa were described in moles and myriapods by Eimer (1870) and Schaudin (1901). It is closely related to Eimeria . It was named in 1994 by Ortega and collegues. The name is derived from the morphology (the sporocysts are spherical) and from a Peruvian university (most of the epidemiological and taxonomic work has been carried out at the Universidad Peruana Cayetano Heredia, Lima, Peru). Before 1992 the organism was wrongly regarded as a cyanobacterium. Distribution is probably cosmopolitan, but the species is only common in regions with poor hygiene.

After swallowing mature (i.e. sporulated) oocysts, there is excystation after contact with bile salts. The released sporozoites penetrate the jejunal enterocytes. In 1997 it was demonstrated that during infection of humans two different asexual stages occur as well as sexual stages. The full cycle can be completed in one host. Infected persons eliminate non-sporulated oocysts in their faeces. Until they sporulate, which takes days or weeks, these parasites cannot infect a new host. This delay makes direct human to human transmission improbable. After sporulation the oocyst contains 2 sporocysts each with 2 sporozoites. A reminder: In Isospora one oocyst has 2 sporocysts each with 4 sporozoites. Cryptosporidium oocysts contain no sporocysts, only 4 naked sporozoites.

Oocysts are spherical, measure 8-9 micron in diameter and contain granular material when excreted. At that point they are not yet sporulated (i.e. no sporozoits yet), and are not infectious. The oocysts to mature for a minimum of one week in the outside environment before becoming infectious. This implies that person-to-person transmssion would be very unlikely. The oocysts stain with varying degrees of acid fastness, but they are recognisable even without staining. They can be differentiated from Cryptosporidium oocysts because the latter are smaller (half the diameter). Isospora cysts are also acid-fast but are much larger (20-33 x 10-19 µm). Under UV light Cyclospora displays autofluorescence (blue with 355 nm dichroic filter and green with 450-490 nm filter). Isospora and Cryptosporidium do not autofluoresce. Fluorescence with auramine shows bright yellow disks in Cryptosporidium , but is weak in Cyclospora . Nevertheless in practice these more expensive techniques do not have to be used since diagnosis can be made using standard light microscopy.

The protozoa are present in the duodenum and jejunum and cause persistent watery diarrhoea, often accompanied by significant abdominal discomfort, nausea, tiredness and anorexia. The symptoms may last several weeks. In particular, non-immune persons such as travellers or small children, will be symptomatic. Cotrimoxazole is used in treatment. This protozoon also causes persistent diarrhoea in HIV-positive persons. If these cannot tolerate cotrimoxazole, the rather less effective ciprofloxacin may be used.

Knowledge about this parasite is insufficient. Transmission via water is possible and food may be contaminated by rinsing vegetables and fruit. Protozoa can be detected in surface water with special techniques. No reservoir is known to date. Similar parasites are found in primates and Cyclospora -like microorganisms have been found in ducks, chickens and dogs, but morphological similarity does not imply genetic identity.

Microsporidiosis

Microsporidiosis, general

Microsporidia in muscle of AIDS-patient. Ziehl-stain. 

Microsporidium sp. in faeces. Species identification requires PCR or electron microscopy. Copyright ITM

Species belonging to the phylum Microspora are called microsporidia. At present, more than 140 genera are recognized and 1200 species have been described. These obligate intracellular organisms appear to have separated very early from the eukaryotic family tree. They have true nuclei, but no mitochondria or peroxisomes. Their ribosomes are prokaryote-like (70S). Since the spore wall contains chitin, some researchers regard them as aberrant fungi. They are obligate intracellular parasites, and are recovered in countless widely varying host groups (insects, fish, rodents, and so on). They have long been know to be pathogenic and are the cause of significant damage in, for example, silk worms (pébrine [see Pasteur and the ravages of the French sericulture in the 19 th Century]), apiculture and fish farms. Species which can parasitise humans are very small (1-2 µm). Encephalitozoon cuniculi  holds the record at present for the smallest eukaryotic genome (<2.9 Mb). Other species known to infect humans are Brachiola vesicularum, Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis (previously Septata intestinalis), Enterocytozoon bieneusi, Microsporidium africanum, Microsporidium ceylonensis, Nosema algerae, Nosema connori, Nosema ocularum, Pleistophora sp, Trachipleistophora hominis, Trachipleistophora anthropophthera  and Vittaforma corneae (previously Nosema corneum ). These organisms have mainly been described in immunodeficient persons. Note: Pasteur and silkworms In 187O the French scientist Louis Pasteur was called in to help to save the French silk industry. A disease, known as pébrine, was destroying the insects.  It became so severe that the only method of getting uninfected eggs was to import them from Japan. Infection interferred with pupation of silkworm moths and caused affected larvae to produce coccoons of poor quality. Infected larvae displayed black or brown spots, due to infection with Nosema bombycis . He also identified a second (viral) disease called "flacherie", where dark spots were absent. Pasteur found parasites in all tissues of insects with pébrine, particullary in gut epithelium. Each produced a single small spore, only 3-4 µm in length. This spore was passed in the stools. Larvae became infected when they fed on mulberry leaves contaminated by these spores. Another mechanism of infection was found to be transovarial transmission. Parasites developing in reproductive tissues incorporated in eggs, so that newly hatched larvae were already congenitally infected. When this knowledge was acquired, Pasteur advised good animal husbandry methods to the sericulture industry. Care was taken to isolate infected moths, eggs, and contaminated mulberyy bushes.  The French silk industry revived.

Microsporidiosis, ultrastructure

The parasites have a very characteristic ultrastructure. The organism forms oval-shaped spores with an external exospore (glycoproteins) and an internal endospore (chitin). Within the spore is a coiled spiral tube (polar tube). After it is ingested, the spore is stimulated to protrude this polar tube which then penetrates a host cell. The sporoplasm is then injected via this tube into the cytoplasm of the host cell. Subsequently there is reproduction of the parasite (merogony and sporogony). New spores may infect other neighbouring cells or be passed to the outside world to infect a new host.

Microsporidiosis, transmission

Transmission is chiefly via the faeco-oral route, but much is still uncertain. Transplacental transmission occurs in carnivores, but has not yet been described in humans. Possibly transmission is via aerosol for those protozoa which cause corneal lesions. Transmission via infected water is being investigated.

Microsporidiosis, clinical aspects

Symptoms will be determined by the anatomical location of the parasites. Disseminated infections, corneal infections (keratitis), intestinal locations etc. all occur. In HIV patients with low resistance (CD4 < 100/µL), there is often persistent diarrhoea, abdominal pain, loss of weight and sometimes sclerosing cholangiopathy.

Microsporidiosis, diagnosis

Diagnosis by light microscope (faeces, biopsies, corneal scraping) is often difficult due to the small dimensions of the parasites and the labour-intensive staining techniques. New staining techniques, such as the fluorochromes calcofluor, Fungifluor or Uvitex 2B and modified trichrome staining (modification according to Weber or Kokoshin), are now available. Experience is essential and the parasites must be properly differentiated from fungal spores and bacteria. With the optical brightening agent Calcofluor White 2MR and a wavelength of 395 to 415 nm (obeservation light 455 nm) the spores appear as bluish-white or turquoise oval halos. This stain is nonspecific. Small fungi and some artifact material present in stool may also fluoresce. However, for specimens other than stools, the microsporidial spores tend to be much easier to detect. Cytocentrifugation (duodenal aspirate, bile, urine, BAL fluid, CSF) followed by Giemsa or Gram staining can be used. The spores are often Gram variable (immature spores tend to be red; mature spores are violet to purple). Electron microscopy is a good technique for species identification, together with PCR [polymerase chain reaction], but of course this can only be carried out in specialized centres. The organisms can be detected in routine formail-fixed and paraffin-embedded tissues. Birefringence results from the presence of chitin in the endospore layer. This property can be observed in sections stained with Gram and modified Warthin-Starry stains and is used to differentiate the spores form other intracytoplasmic granules and mucin droplets.

For a good review of laboratory diagnosis, see: http://jcm.asm.org/cgi/content/full/40/6/1892

Microsporidiosis, cell culture

The microsporidia cannot be grown axenically, but several species have been grown in cell culture. However, these in vitro techniques are not relevant for routine diagnosis.

Microsporidiosis, treatment

There is still too little known about treatment. Fumagillin  is a product originating from Aspergillus fumigatus which is used in microsporidiosis of honey bees. It has been used topically in keratitis with good results. Other drugs have been used with varying success. Albendazole  is effective in infections with Encephalitozoon intestinalis and to a lesser extent in Enterocytozoon bieneusi . Nitazoxamide  possibly has a place in treatment. Improving immunity in HIV patients, e.g. by combination antiretroviral therapy, often leads to remission of the infection. For symptomatic treatment (e.g. in persistent diarrhoea without knowing its cause), loperamide  (Imodium®), opioids (laudanum) or even somatostatin analogues may be used. The latter is of course not easily available in developing countries.

Blastocystosis

Blastocystis hominis . Copyright ITM

Blastocystis hominis was first described in 1912. It is a rather common enteric unicellular protozoan. Although previously regarded as a yeast, has the morphological and biological characteristics of an anaerobic protozoon. At present it is classified is a taxonomic class by itself (Blastocystea; some classify it among the Stramenophiles or heterokonts, a rather heterogenous group). The parasite colonises chiefly the caecum and to a lesser extent the distal colon. Very little is known of the basic biology of this organism, including the life cycle. Several morphological forms have been recognized: ameboid, vacuolar, avacuolar, multivacuolar, granular, cyst.  Which of the forms is responsable for transmission is not known. The vacuolar stage divides, while the amoeboid stage might be invasive and is capable of budding. The granular or cystic form is thought to give rise to numerous new organisms when conditions become unfavorable. B. hominis forms pseudopods, and ingests bacteria and debris. It reproduces by binary fission or sporulation. There are thin-walled and thick-walled cysts. Transmission is faeco-oral through contaminated food or water. There seems to be a large animal reservoir. B. hominis has been isolated form numerous animals, but more study of the zoonotic potential of this organism is needed. Until about 1930 it was regarded as a cause of diarrhoea, but thereafter it was generally considered to be an apathogenic commensal.  It infects a variety of vertebrates. It's pathogenicity is controversial. Several studies using different methods and examining different patient groups have reported very variable results, from asymptomatic infection, acute symptomatic infection, chronic symptomatic infection, with abdominal pain, diarrhea, constipation, irritable bowel, fatigue, skin rash, and other symptoms. The variation in results led to disagreements concerning a possible pathogenic role of Blastocystis in humans. Maybe Blastocystis has several variants which differ in their pathogenicity or virulence. The pathogenicity might depend on the parasitic load (more than 5 Blastocystis per 40x field, but different pathogenic properties of different strains will likely also play a role). Specimens vary enormously in size, from 6 to 40 µm. Molecular typing has revealed extensive genetic diversity in morphological identical strains. It is possible that additional studies will show that what we call Blastocytis hominis , will turn out to be a mixture of different microorganisms, a situation similar to the past confusion about the morphological identical Entamoeba histolytica, E. moshkovskii  and E. dispar. According to current PCR-based genotype analysis, there may be 12 different species which are lumped together under one name. Confirmation of the existence of separate species, and determination of their pathogenic properties will help to clarify the clinical relevance. If considered necessary, metronidazol, trimethoprim/sulfamethoxazole or clioquinol are used for treatment. Blastocystis is an anaerobic protozoon, and as such has no use for fully functional mitochondria, but contains "incomplete" mitochondrion-like organelles with cristae, a transmembrane potential and DNA. Other anaerobic protozoa tend to have hydrogenosomes (making ATP and hydrogen; most hydrogenosomes contain no DNA) and/or mitosomes (viewed by many as a kind of relict mitochondrium without DNA, but essential for biosynthesis of crucial cytosolic Fe–S proteins). The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. It is a kind of transition form. These findings blur the boundaries between mitochondria, hydrogenosomes and mitosomes. There is an ongoing discussion over the hypothesis if these cell organelles have a common ancestor.  It is clear that more study is needed of this fascinating enigmatic organism. Beware: don't confuse Blastocystis with Blastomyces .

Myxozoan parasites

Not much is known about myxozoan parasites. Myxozoans are commonly found as parasites in aquatic vertebrates (esp. fish) as well as some invertebrates. The phylum of Myxozoa contains two classes: Myxosporea (infect fish, amphibians, reptiles) and Actinosporea (infect mainly oligochaetes and sipunculids). More than 1,000 species have been described so far. Many of the spores contain chitin (which cannot be digested by humans) and can pass intact through the gastrointestinal tract. There is at present little evidence that these organisms can infect people. However, spores have been recovered from stools of patients with gastrointestinal problems. Of course this is no proof of pathogenicity. Spores of Henneguya salminicola have been detected in stool samples of human patients. Since they morphologically resemble spermatozoa, misidentification can occur. The teardrop-shaped spore measures about 10 µm in diameter and has a tail of about 40 µm. Most spores have two pyriform polar capsules situated in the anterior region of the spore body. They stain with modified trichrome stain. Iodine preparations and acid-fast stains can be employed, but at present there is insufficient information on these organisms.

Rhinosporidiosis

Rhinosporidiosis is an infectious disease which occurs in the New Wold, Europe, Africa and Asia, but is most common in the tropics (India and Sri Lanka). The disease was first described in Argentina around 1890 by Malbran and Guillermo Seeber (the subject of his thesis). The disease is characterised by slow-growing, painless polyps or tumour-like masses, which are usually found on the nasal mucosa, lachrymal sac, conjunctivae, palate, larynx or penis. Chronic rhinitis and/or epistaxis may occur. Disseminated infections with lesions of skin, liver, spleen and kidneys, are very rare. Treatment consists of surgical excision, but recurrence can be expected in approximately 10% of patients. Biopsy shows a characteristic picture of large (up to 300 microns) spherical thick-walled cysts, containing hundreds or thousands of endospores measuring 7-9 µm. Around the cysts there is an inflammatory infiltrate with neutrophils and small abscesses. The protozoon may be detected by staining with silver (methenamine), PAS [periodic acid-Schiff] or mucicarmine after KOH digestion of the specimen. Rhinosporidium seeberi has not to date been cultured in vitro. This species is a eukaryote and is difficult to classify, having the properties of both fungi and protozoa. A few years ago the species was classified under Mesomycetozoa (a cladistic group). Recently, it has been shown, using PCR [polymerase chain reaction], that the protozoon is not closely related to the Eumycota. It also became apparent that R. seeberi is genetically close to the genus Dermocystidium as well as the "rosette agent" (pathogen of fish). The name Ichthyosporea was proposed for a new taxonomic group which would include these organisms. The group would be placed in a taxonomic tree close to the division of fungi and animals. No natural reservoir is known. It is also assumed that people become infected by swimming in fresh water lakes or rivers. It is likely that fish or other water creatures are the normal hosts. Swans can be infected. A related species, R. equi , can infect horses and cattle. More recent studies found arguments to consider cyanobacteria of the genus Microcystis aeruginosa are causative agent of this condition.

Protothecosis

Protothecosis is a rare infection in humans. Infection is more common in cases of immunodepression (AIDS, leukaemia). The disease was reported originally from Sierra Leone, but cases were later identified in the New World (Panama, USA), South Africa, China, Vietnam and New Zealand. The disease is caused by Prototheca wickerhamii and P. zopfii (segbwema ). These are aerobic unicellular round ( P. wickerhamii ) to oval ( P. zophii ) algae which activity belong to the Chlorococcales [Chlorophyta or green algae]. However, they contain no chlorophyl and are colourless. They are typically 8-16 µm in diameter, but their size may vary from 3 to 30 µm. These algae reproduce by internal division of a mother cell, which produces two to fifty small endospores enclosed in a sheath (theca). This ruptures and the endospores are released, after which the cycle is repeated. No sexual reproductive stages are known. The protozoa occur in still water, sewage sludge, mud and slime on trees. Various animals may be infected (cattle, dogs, rabbits, mice, rats, pigs, deer). Humans are infected via traumatic inoculation of the germ into the skin or via infection of an open wound. Infection is usually limited to the skin, where local painless granulomatous hyperkeratotic dermatitis results. Bursitis and tenosynovitis have been described. Indolent olecranon bursitis can be tender. Sometimes there is systemic involvement, including cholangitis, chronic meningitis and retinitis. There have been cases of peritonitis after peritoneal dialysis. Diagnosis is made by biopsy. The pathogens are morphologically similar to mulberries. Confusion with yeasts is possible. For tissue sections a PAS [periodic acid-Schiff] or a Gomori methenamine silver stain are used. In tissue the sporangia are usually larger (30 µm) than in culture ( P. wickerhamii  7-13 µm; P. zophii 14-16 µm). The algae can be cultured in vitro on glucose-containing media. Generally a blood agar and a Sabouraud medium are used. Growth is optimal at 30°C and is inhibited at 37°C. Treatment is surgical with or without amphotericin B. Ketoconazole has frequently been used with success, but requires long-term administration. The possible therapeutic roles of itraconazole and fluconazole need to be better determined.

Babesiosis

Babesiosis, general

Babesiosis is a zoonotic disease which is triggered by infection with a protozoon of the genus Babesia . The disease is also known as piroplasmosis. The order of Piroplasmida belongs to the Apicomplexa (cf. malaria). There are more than 110 species in the genus Babesia . Some infect fish, birds, reptiles or mammals. The rodent parasite Babesia microti (USA) and the bovine parasites B. divergens and B. bovis (Europe) cause most infections in humans. Occasionally other species may be responsible for human infections (e.g. the WA1 strain). The species was first described in febrile cattle by Victor Babes in 1888. In 1957 the first case in humans was described in Yugoslavia by Skrabalo.

The protozoa are closely related to Theileria , a very important pathogenic genus in veterinary medicine. East Coast fever is triggered by Theileria parva , a disease transmitted by Rhipicephalus ticks.  It was proposed that Babesia microti would be renamed Theileria microti , and that the human disease would be named human theileriosis. Note: Theobald Smith Theobald Smith (1859-1934) was a pioneer microbiologist, the son of a German immigrant to the USA. He is best know for his work of Texas cattle fever. He discovered a new species of Salmonella , S. enterica (formerly S. choleraesuis ). His chief, Daniel Salmon, claimed credit for this discovery, which is why we now speak of salmonella infections instead of smithella infections. Texas cattle fever was a devastating disease, which could destroy up to 90% of a herd. After talking to and listening to cattle herders, he was intrigued by the possibility that ticks might have a pathogenic role in this disease. Smith and his collegues Fred Kilbourne and Cooper Curtice started to investigate. In 1889 Smith saw small intraerythocytic bodies in the blood of sick animals. They are now recognised as  Babesia microti;  initially named Piroplasma bigeminum . Transmission experiments proved without doubt that this microorganism was responsable for the disease and that ticks ( Boophilus ) were responsable for transmission. Delineation of the tick's life cycle paved the way for control of the disease by dipping cattle in a chemical which destroyed the arthropods. The discovery by Smith that arthropods could transmit pathogens was a fundamental step forward in medical scientific thinking. It was partly due to his work that the hypothesis of malaria transmission by mosquitoes was taken seriously. A couple of years later, insect transmission of African trypanosomiasis was discovered by David Bruce, malaria transmission via mosquitoes was identified by Ronald Ross, yellow fever transmission by (a different species of mosquitoe) was proven by Walter Reed and his team and the spread of epidemic typhus through body lice was identified by Charles Nicolle. Smith also discovered the cause of turkey blackhead, a devastating enterohepatitis of these birds. It is caused by a protozoon called Amoeba meleagridis (now Histomonas meleagridis ). He also found that embryonated eggs of the intestinal roundworm Heterakis papillosa (later renamed H. gallinae ) could  transmit the amoebas. (Cfr the hypothesis of transmission of Dientamoeba fragilis , still not resolved to this day.)

Babesiosis, transmission

Voles form the reservoir. Transmission is via the bite of hard ticks such as Ixodes scapularis   and Ixodes ricinus . The transmission of B. bovis is via Boophilus microplus . In the USA larval nymphs of Ixodes scapularis feed chiefly on Peromyscus maniculatus ("the white-footed deer mouse"). The adult ticks suck blood from deer (cf. Lyme disease). Strangely enough the deer are not infected with B. microti . In ticks transstadial transmission occurs. The parasite passes from larva to nymph to adult tick. There is no transovarian transmission. Infections in humans are accidental occurrences. After injection of saliva of the tick, the micro-organisms penetrate red blood cells and mature. Babesia microti trophozoites undergo asexual reproduction in human blood and divide into two or four merozoites. To date no exo-erythrocytic cycle, as exists in Plasmodium species, has been described. Infected red blood cells undergo haemolysis. This releases the protozoa which can then penetrate new red blood cells. Infections via blood transfusions have been described. Transplacental infection may occur. When a tick sucks blood, the parasites are in the blood meal. This blood meal is enclosed in a peritrophic membrane in the tick's gut. The parasite changes shape and it is assumed that it produces gametes. Once fertilisation is complete the zygote penetrates the peritrophic membrane and passes through the intestinal epithelium by endocytosis. Once in the haemolymph it forms an ookinete, which penetrates the salivary glands. There, the parasite forms sporoblasts, a stage which can overwinter. Thousands of sporozoites are formed. At the time of the tick's next feeding, the parasite is mature and ready to be injected with the saliva. The part of the cycle between sporozoites and intra-erythrocytic merozoites is as yet unclear.

Babesiosis, geographical distribution

Endemic regions in the USA include Massachusetts and New York State with Nantucket Island, Long Island, the coast of Connecticut as well as foci in Georgia, California and Wisconsin. Cases have also been reported from various European countries such as Ireland, Scotland, Sweden, former Yugoslavia, France and Russia. There have been isolated case reports from Africa, Asia and Latin America.

Babesiosis, clinical aspects

Asymptomatic infection may persist for months or years. If symptomatic, the first symptoms occur after an incubation period of one to two weeks. Malaise, tiredness, fever, headache, nausea and abdominal pain, myalgia and joint pain are early but aspecific symptoms. The body temperature may rise to 40°C. Hepatosplenomegaly with haemolysis and jaundice, haemoglobinuria, mild neutropenia and thrombocytopenia follow. There is no lymphadenopathy. In severe cases ARDS [acute respiratory distress syndrome] with shock may develop. Infections may have a dramatic course in asplenic persons, chiefly in the European forms. Here the infection is similar to P. falciparum malaria. Splenectomy or immunosuppression during asymptomatic infection may lead to a full blown infection.

Babesiosis, diagnosis

Babesia microti . Copyright ITM

Babesia microti . Copyright ITM

Babesia microti in thin bloodsmear. Copyright ITM.

Babesia microti . Copyright ITM

Diagnosis is made from a blood smear stained with Giemsa. The parasitaemia is generally 1 to 10%. Haemoglobinuria and proteinuria occur. Sometimes the mature parasite is in the form of a clover leaf: a so-called tetrad or Maltese cross. The intra-erythrocytic dimension of the merozoite is 1 to 2.5 µm. It is pear-shaped, oval or round. The circular appearance means that Babesia is often confused with Plasmodium falciparum , but malaria pigment cannot be detected. There are also no gametocytes or malaria schizonts. In Babesia infections, large parasites may contain a central white vacuole, which is not present in malaria. Serological tests and DNA analysis may help in diagnosis. Hamsters and gerbils can be inoculated to cultivate the protozoa, but this can only be done in specialised centres. It is possible that some of the "malaria" in Africa is in fact babesiosis.

Babesiosis, treatment

Quinine  is the drug of choice, 650 mg TDS  plus clindamycin  600 mg TDS or 1.2g BD IV for 7 to 10 days. Children receive 25 mg quinine/kg/day. Atovaquone  (750 mg BD) and azithromycin  (500 mg on day 1, then 250 mg daily) are also used and this combination is better tolerated. Exchange transfusion may be considered if there is life-threatening parasitaemia. A blood transfusion may be life-saving. Remember that ticks can be infected with more than one pathogen. In endemic regions co-infection with Borrelia burgdorferi , certain Ricketssia , Anaplasma,   Ehrlichia or viral pathogen must be considered.

Babesiosis, prevention

Asplenic persons should avoid endemic regions and pay extra attention to tick prevention (proper clothes, repellent containing at least 30% DEET, permetrine, physical inspection after walking).

Summary

Toxin from intraluminal intestinal bacteria, Vibrio cholerae O1 and O139 Acute profuse to catastrophic watery diarrhoea with severe dehydration and ion loss Low fever and limited abdominal cramps Rehydration essential; preferably Ringer's lactate Antibiotics are of secondary importance

General

Cholera is an acute infectious disease, characterised by profuse watery diarrhoea. It is caused by a Gram-negative bacterium: Vibrio cholerae O1 (the characters O1 indicate the serogroup). It is a very small, motile, curved bacterium (vibrio is the Greek word for comma). There is a single polar flagellum. Various subtypes exist, with classification according to biological and biochemical behaviour (biotypes) and serological characteristics (serotypes). Analysis of rRNA-genes (ribotyping), electrophoretic typing of multiple enzymes (zymovars) and bacteriophage typing are used in epidemiological research. Until 1992 it was thought that bacteria causing cholera must belong to V. cholerae , serogroup O1 and that they must be toxicogenic (must possess and express the genes for toxins). It was known that non-O1 Vibrio cholerae could sometimes cause mild gastro-enteritis or even septicaemia in immunodepression, but not cholera. In October 1992 in Madras (India), a mutated pathogenic bacterium (a new serogroup) was discovered. This new bacterium also causes cholera. The isolate was given the name Vibrio cholerae O139, nicknamed Bengalen. After a short bloom, the traditional strains (O1) became again more common. A few years later, V. cholerae O139 Calcutta was identified. Hybrid El Tor strains that encode classical cholera toxin have been identified. In August 2000 the whole genome (2 circular chromosomes) of V. cholerae O1, biotype El Tor was charted and published. Antibiotic resistance genes in V. cholerae are often positioned on plasmids and can be transmitted to vibrios from non-pathogenic intestinal flora. There are arguments to suggest that V. cholerae recently isolated from the intestine of a patient is much more infectious than bacteria which have been in the outside world for a long time.

Map. Cholera spread during the seventh pandemic.

Biotypes

There are 2 biotypes: classic Vibrio cholerae and V. cholerae biotype El Tor. Biotype El Tor agglutinates chicken erythrocytes and causes lysis of sheep erythrocytes, unlike the classic biotype. The name El Tor originates from the Egyptian town and quarantine camp El Tor in the Southern Sinai dessert, where the bacterium was isolated for the first time in 1905 (during the 6 th pandemic) from an asymptomatic Hajj pilgrim from Mecca. The importance of this germ was long disputed (until 1961). At present El Tor has replaced the classic variant in most places, except in the Ganges and Brahmaputra delta. El Tor may also survive longer in the environment, is less dependent on transmission via water and produces more asymptomatic infections (symptomatic/asymptomatic infections = 7/100).

The El Tor and classical biotypes can be distinguished with several techniques. For example, El Tor will be resistant to polymyxin B, agglutinates chicken erythrocytes, yields a positive Voges-Proskauer reaction (a type of glucose fermentation leading to a color change), is positive for the El Tor hemolysin and will be sensitive to group IV El Tor phage but resistant to the classical group V phage.

Genetic hybrids between El Tor and classical biotypes of V. cholerae O1 were reported among sporadic isolates and were named Matlab variants after the place where they were first isolated.

Serotypes

V. cholerae O1 of both biotypes can be subdivided into serotypes according to the structure of the O antigen. If only O antigen A and C are present, the bacterium is known as serotype Inaba. If only A and B are present, the bacterium is known as serotype Ogawa. If A, B and C are present, the name Hikojima is given. Serotype shift seldom occurs (from Ogawa to Inaba and vice versa). The difference between these serotypes is only of importance for epidemiological studies. For example: in 1991 all cases of cholera in South America were caused by toxin-producing Vibrio cholerae , serogroup O1, biotype El Tor, serotype Inaba. The cholera epidemic in the Rwandan Hutu refugees in Zaire (July and August 1994) was caused by El Tor, serotype Ogawa. These bacteria were resistant to tetracyclines, cotrimoxazole, chloramphenicol and ampicillin. The outbreak in Haiti in October 2010, 9 months after the earthquake in January, was due to V. cholerae O1, Biotype El tor.

Note: flagella, pili and fimbriae

Most motile bacteria move about with structures called flagella (spirochaetes move with the help of axial filaments). Do not confuse active bacterial movement with random Brownian movement. Do not confuse a bacterial flagellum with the flagellum of a eukaryote such as Giardia (cf. also the remark concerning cilia in Balantidium coli ). The flagella are too thin (0.2 µm) to be observed with a standard light microscope. The bacterial flagellum carries out a rotating movement. Some bacteria have several flagella. When the flagella rotate anti-clockwise, they form a coherent bundle, so that the bacterium moves in a straight line. On the other hand when the flagella turn clockwise, there is no longer any co-ordination and the bacteria move randomly.  By timing the duration of clockwise and anti-clockwise spinning, this mechanism can be used in chemotaxis. The motor is in the membrane and the immediate driving power is not ATP, but a proton gradient. The bacterial flagella must not be confused with fimbriae , thread-like appendages which have no function in movement, but play a part in adherence to cells or tissues (important for virulence). Flagella rotate, fimbriae do not. Pili (singular pilum) are important in conjugation, the bacterial equivalent of sex. These hollow rigid tubes permit DNA transfer between bacteria. F-pili [ F ertility] are important in the spread of resistance to antibiotics. Pili may also act as receptors for bacteriophages.

V. cholerae can only cause disease if there are pili [Lat.: "hairs"] present. Pili are shorter and thinner than flagella. The pili adhere to the intestinal mucosa. In 1996 it was discovered that cholera toxin can be transmitted from a toxicogenic V. cholerae strain to a non-toxicogenic strain via filamentous bacteriophages. These phages were given the name "cholera toxin phage" or shortened CTXPhi. The genetic material of the phage can integrate into the genome of the bacterium and become lysogenic. The bacteriophages adhere to the pili (called toxin-coregulated pili) prior to penetration. Many bacteria carry in their genome a segment of DNA which is known as a pathogenicity island. This piece of DNA codes for a number of virulence factors. The genes which code for the toxin coregulated pili are in a pathogenicity island, called VPI ( Vibrio cholerae Pathogenicity Island). In 1999 it was discovered that the VPI itself codes for a bacteriophage. This was given the name VPIF. We thus have the situation that the first bacteriophage (VPIF) codes for the receptor of another bacteriophage (CTXPhi). This may be of importance for the development of a live V. cholerae vaccine.

Epidemiology

Epidemics, Pandemics

Cholera has always been endemic in India and Bangladesh, in the huge delta formed by the confluence of the Ganges, Brahmaputra, Jamuna and Meghna rivers. Probably there was no cholera in Europe or America before the 19 th century. Between 1817 and 1923 there were various great pandemics, probably caused by the classic V. cholerae (there is no certainty as to the exact strain). The first pandemic which started in 1817 did not reach Western Europe. In 1829 the bacterium was introduced into the countries around the Persian Gulf via a British army unit stationed in India. From Iran the infection spread to Iraq, Syria, Georgia and Astrakhan (north of the Black Sea). It then travelled towards Odessa, Moscow, Vienna, Warsaw and Hamburg reaching England via the port of Sunderland. The first cases in London were seen in February 1832. The third pandemic merged with the second and was amplified by the miserable conditions during the Crimean war. The pathogen was discovered in 1884 by Robert Koch during the fifth pandemic (first work in 1883 in Alexandria, Egypt, confirmation followed by research in India in 1884, with isolation of the bacterium in culture). In fact the bacterium had already been described in 1849 by Pouchet and in 1854 by Filippo Pacini, an Italian physician. However, the latter's work on this was not known outside Italy (he was known abroad due to his description of Pacini's corpuscules, the pressure receptors in the skin). The germ theory and in particular the work of Koch were attacked by Pettenkofer. Pettenkofer was a proponent of the "ground water theory", believing that the fermentation of organic matter in the subsoil ("miasma") released cholera into the air (no transmission from person-to-person) which then infected the most susceptible e.g. those with poor diet, constitution, etc. Both Pettenkorfer and his loyal student Emmerich drank a vial filled with cholera bacteria as proof against Kochs type transmission of V. cholerae . Amazingly, Pettenkofer did not then get cholera, but Emmerich suffered severe diarrhoea for 48 hours. When each pandemic began and ended is rather unclear. There was cholera in Belgium in 1832, 1848, 1854, 1859, 1866 and 1892. In 1866, 1 Belgian in 100 died of cholera. Since cholera was present in Belgium in 1892, this means that cholera was present when my grandparents were young.

Cholera pandemics since 1817 Number Years Origin Pathogen 1 1817-1823 India ? 2 1829-1851 India ? 3 1852-1859 India ? 4 1863-1879 India ? 5 1881-1896 India V. cholerae O1, classic 6 1899 -1923 India V. cholerae O1, classic 7 1961 to present Sulawesi V. cholerae O1, El Tor 8? 1992 to present Madras, India V. cholerae O139

After the sixth pandemic there was a strange silence for about 40 years, for which no good explanation exists. The seventh pandemic was caused by El Tor. It started in 1961 in Celebes (Sulawesi), Indonesia, reached India in 1964 and Africa in 1970. In 2 years the infection passed through 29 African countries. In 1973 it arrived in the Gulf of Mexico. Early in 1991 the infection spread rapidly in Peru. In 3 weeks there were 30,000 cases. The bacterium then spread further into South America, causing 360,000 cases within the year. In the summer of 1992 a second, less severe outbreak occurred. Nevertheless by August 1992 "only" 5,000 deaths had been reported (from an estimated total of 600,000 cases), thanks to the wide-spread use of rehydration therapies. The case-fatality ratio varied depending on the region. After 1993 the disease assumed an endemic character in several countries, sometimes with local outbreaks. At the end of 1993 the cumulative total amounted to 900,000 cases in three years (1991-1993), with a cumulative mortality of 8,000. According to one hypothesis cholera bacteria infected the marine plankton off the Peruvian coast via the ballast water from a Chinese freighter. The possible role of changes in the nutrient-rich von Humboldt current is still unclear.   Basic Reproduction Number In epidemiology of infectious diseases, a central and important concept is the basic reproduction number (syn. basic reproductive rate, basic reproductive ratio) or R° (R zero or R nougth). This reflects the number of cases one case generates on average over the course of its infectious period. This metric is useful because it helps determine how an infectious disease can spread through a population. In general, the larger the value of R 0 , the harder it is to control the epidemic. For simple models, the proportion of the population that needs to be vaccinated to prevent sustained spread of the infection is given by 1 − 1/R 0 . This method is an approximation, and becomes more complicated with diseases where e.g. intermediate hosts or vectors are involved, such as malaria. In malaria, the estimated R 0 can vary by several order of magnitude, depending upon local conditions. The formula for calculating the number is  R 0 = beta.C.D, with beta = virulence (degree of infectiousness, probability of transmission between infected and non-infected person) C     = average number of contacts per infected persons per day D     = duration (number of days that an infected person stays infectious) If R 0 < 1 : the spread of disease will eventually cease If R 0 = 1 : the disease becomes endemic If R 0 > 1 : the epidemic will increase   Values of R 0 of some infectious diseases        Disease                        Transmission               R 0 Measles                      Airborne                      12–18 Pertussis                     Airborne droplet          12–17 Diphtheria                   Saliva                             6–7 Smallpox                     Social contact                5–7 Polio                            Fecal-oral route             5–7 Rubella                        Airborne droplet            5–7 Mumps                        Airborne droplet            4–7 HIV/AIDS                    Sexual contact                2–5 SARS                          Airborne droplet             2–5 Influenza 1918            Airborne droplet             2–3 Cholera Haiti 2008      Fecal-oral route             2-2.7

Cholera epidemic in Congo. The mortality was high. Photo courtesy Els De Temmerman

Cholera epidemic in Congo. Photo courtesy Els De Temmerman

Cholera epidemic in Congo. Photo courtesy Els De Temmerman

Cholera epidemic in Congo. The mortality during this epidemic was very high. Photo courtesy Els De Temmerman

Cholera in Bangladesh, Copyright Alexander von Humboldt Institute, Peru

Cholera epidemic in Congo. Refugees live in very poor conditions. Photo courtesy Els De Temmerman

About 80% of the cholera in 1997 occurred in Africa, chiefly in the horn of Africa (118,000 cases were reported officially). The increase in cholera in this region followed heavy rains and flooding (possibly associated with the El Niño weather phenomenon).

Since 1992 V. cholerae O139 is recognised as a cause of a disease which is clinically identical to classic cholera, but which also occurs frequently in adults. Classic cholera in India, on the other hand, is common in children. There is no cross immunity with V. cholerae O1. Bacteria of the 0139 serogroup have a polysaccharide capsule (unlike V. cholerae O1), which may explain the increased risk of septicaemia. In the following years this new serogroup spread across Bangladesh, India, Pakistan and Southeast Asia. By the end of March 1993 more than 100,000 cases had been reported in Bangladesh. Further spread continued, but somehow diminished again, as the classic form and El Tor took over, reducing the incidence of the new serogroup. The reason is unknown.  Therefore it is difficult to make then new Bengalen serogroup responsable for an 8th pandemic. It was observed in India that, after the first spread of V. cholerae O139, new variants (clones) of V. cholerae O1 El Tor once more gained the upper hand. Cholera also surfaces regularly in Madagascar. From the beginning of December 1999 until the end of February 2000 more than 12,400 cases were reported. The disease can thus certainly not be regarded as an entity which only existed "in the past". At the end of 2008 a large cholera outbreak appeared in Zimbabwe. By February 2009, this led to more than 60,000 cases with a mortality of more than 5%, reflecting the general degradation of the nation's basic infrastructure and the crumbling Zimbabwean health care system. By mid-April 2009 the official count was 96,591 cases with 4,201 deaths. In 2010, more than 38,000 cases of cholera were identified in Nigeria. In January 2010, a devastating earthquake with magnitude of 7 on the Moment Magnitude scale (compare with Richter scale) hit Haiti, with its epicentre 25 km from Port-au-Prince, the capital. A couple of weeks before Nepalese United Nations peacekeepers arrived in Haiti, a cholera outbreak occured in Kathmandu, the Nepalese capital. The forces were stationed in Mirebalais, 60 km north-east of Port-au-Prince. Late October 2010, patients with cholera were recognized in some Haitian rural areas. In less than 6 weeks, more than 10,000 cholera cases were identified. The disease quickly spread to the capital, where many people were still living in temporary shelters and tents, without access to safe drinking water or proper sanitary facilities. By January 1, 2011, the Ministry reported 171,304, with a cumulative mortality of 3651. The hospital case fatality rate was too high, and a target of hospital CFR of < 1% should be achievable. A possible epidemiological connection with the Nepalese forces was suspected and created tension between the local population and the UN forces. The current Haitian strain of cholera was identified as a virulent hybrid of the El Tor O1 biotype and the classic type, serotype Ogawa.

Historical note: John Snow and contaminated water

In the first half of the nineteenth century a cholera epidemic occurred in London. In 1848, a cholera outbreak started which would kill more than 14,000 people in London. Another outbreak in 1853 killed more than 10,000 people. The physician John Snow, already well known in 1853 as anaesthetist to Queen Victoria. Chloroform had become available in 1831 by using a process based on acetone or ethanol and calcium hypochlorite. As an anesthetic, it could be used during during childbirth, and Dr Snow was the Royal obstretrician. Dr Snow had also a special interest in cholera. In 1854 he examined the various families presenting cases and calculated that the mortality in the houses that were supplied with water by the Southwark and Vauxhall Water Company was 31/1000 houses. This was 8.5 times higher than in houses supplied by the rival Lambeth Company. Although neither of the two companies offered purified water, the first company took its water from the Thames near London Bridge, downstream from the city sewage outlets while the second company pumped its water upstream from the city at Thames Ditton. In 1849 there had been no difference in mortality between the families that received water from Lambeth or Southwark. Before 1851 the Lambeth Company drew its water from a highly contaminated stretch of the Thames near Hungerford Market. It was this spectacular change (1849 compared to 1854) which made Snow conclude that contaminated water had a causal connection with cholera. Although both companies delivered water in the same streets, the water used in any particular house could be identified by its salt content (London Bridge is closer to the sea and its water is saltier than that at Thames Ditton). Adding silver nitrate leads to precipitation of silver chloride, proportionate to the amount of salt in the water. This was the basis of a simple test that could be carried out in every house.  Similar findings were made in Hamburg in 1890. The incidence of cholera was 34/1000 in Hamburg, where the drinking water was drawn from the river Elbe, and 3.9/1000 for the surrounding areas where other sources were used. In Altona, to the west and downstream from Hamburg, contaminated water was also taken from the Elbe, but there was less cholera. How could this be explained? If anything, more cholera would be expected in Altona. The difference was that in Altona the water was first filtered slowly through sand before being supplied for consumption. These observations led to attempts to provide cities with clean drinking water and to construct adequate sewers. Cholera was the first disease for which surveillance was set up and because of this the disease still has code number 001 in the international classification list of diseases. It was quite a new concept to use maps for epidemiological purposes. A street map (or any surface area) with a number of dots on it can be divided into a number of neighbouring polygons. Points within a polygon lie closer to the central dot than to other dots. The sides of the polygons are equidistant between the dots of the polygons. A cartographic presentation such as this is called a Voronoi diagram. The scientific technique was discussed by Johann Dirichlet in the mid-19 th century. Dirichlet who would later succeed the legendary Carl Gauss. The diagrams take their current name from Mr Voronoi, after his elaborate work on the subject in 1908. Historical note: Bazalgette and The Great London Stink of 1858. Cholera had an enormous effect on the development of the principles of public health and was the basis for the so-called "sanitary revolution" at the end of the 19 th century. The Great London Stink of 1858 was also a catalysor for action. In the late 19th century, flush toilets with a water lock (WCs) began to replace chamber pots. Since flush toilets are not designed to handle waste on site, they need a drain pipe to cesspits. These toilets greatly increased the volume of human waste discharged into cesspits, which regularly overflowed into the streets. To remedy the problem, a law was passed in 1815 that allowed the discharge of sewage into the Thames River through a primitive sewer network then under construction. By the mid-nineteenth century, the Thames had become choked with sewage and refuse that sloshed back and forth with every high and low tide. The river became anoxic and turned into a stinking sewer in the heart of London. In the very hot and dry summer of 1858 the stench became so bad that the summer sittings of Parliament were cancelled. Heavy rains finally brought the great stink to an end, but a long term solution was called for. In 1859 the chief civil engineer of the Metropolitan Board of Works Joseph Bazalgette suggested to eliminate the stench which was believed to cause cholera ("miasma"  theory). Bazalgette's solution was a 6-year-project to construct a sewer system for Central London. It was a massive enterprize for which 132 km main underground sewers were constructed, together with 1800 km of enclosed street sewers, to intercept the raw sewage. The sewers would be lined by special  heat-hardened bricks to make them more resistant to corrosion by waste water. High-quality Portland cement was needed, as Portland cement hardens by exposure to water. However, quality varied a lot and each batch had to be tested. The sewer drains had a slope of 75 cm for each km, using gravity to displace the sewage. Bazalgette calculated the diameters of the sewer pipes, allowing for a very generous production of liquid waste, and then doubled to diameter, for "just in case and unexpected events". This allowed the sewers to be operational till present times (if he had not doubled their diameter, the sewers would have reached their limit in 1960). The outflows were diverted downstream where four massive pumping stations outside the city dumped the untreated waste into the Thames. The construction of the great London Victorian sewers started in 1859 and was completed in 1865. As an unintended consequence of the fight against stench, the water supply ceased to be contaminated. This contributed to the decline of the number of cholera cases. Downstream sewage treatment facilities to clean the raw waste were built only decades later.

       

Transmission

Cholera is spread by the faeco-oral route, via contaminated water and food. The infectious dose of bacteria required to cause clinical disease varies according to the mode of transmission and varies according to bacterial strain, with hyperinfective strains occuring immediatly after gut passage. In people with normal gastric function and if ingested with water, the infectious dose is one thousand to one million vibrios. When ingested with food, is is lower about one hundred to ten thousand vibrios. The low pH of stomach acid kills most vibrios. When a person uses antacids, proton pump inhibitors or ranitidine, a lower infectious dose is required to trigger infection. The same applies to chronic atrophic gastritis and status post-gastrectomy. Asymptomatic infections are common, especially in case of El Tor. People excrete bacteria for about 10 days. This is sufficient time to ensure continued contamination of the environment. Chronic carriers are very rare, but occur, sometimes with vibrios lodging in in the biliary tract. In third world countries many people have no chlorinated, filtered, treated, pure drinking water. The lack of good toilets and sewers leads to contamination of the surface or ground water. Too often untreated sewage water is still poured into surface water. Sometimes sewage pipes and drinking water pipes are laid in the same trench, which may result in contamination if there are leaks or greatly varying water pressures in the pipes. If drinking water is contaminated in this way, bacteriological checks of the drinking water when it leaves the pumping station will not show anything amiss. In houses, drinking water containers with a wide opening often become contaminated, because people are inclined to scoop up water in their (dirty) hands. Containers with a small spout, from which water must be poured, are safer.

'Cholera faeces which look like ''rice water''. Copyright Alexander von Humboldt Institute, Peru'

There is also direct transmission from person to person, but it is rare. The number of bacteria on dirty hands is usually lower than the minimum infectious dose necessary for direct transmission. Health workers who respect basic hygiene are at extremely low risk. Filter feeders such as mussels or oysters (especially in estuaria) concentrate the bacteria in their bodies. When the organisms adhere to food particles (e.g. the chitin of crustaceans) and in the case of hypochlorhydria, lack of gastric acid due to gastric surgery, antacids, anti-ulcer drugs or atrophic gastritis, the number of organisms needed to trigger infection is much smaller. Food may be infected by dirty hands during or after preparation. The bacteria can survive and reproduce in food such as cereals, rice or lentils and crustaceans. This intermediate replication step is very important. If someone dies of cholera and a meal is made for the mourners at the funeral by the persons who have washed the corpse, the risk of further transmission is very real. The bacteria are very sensitive to drying out, sunlight and acid. Meals which contain acid, e.g. tomatoes and/or lemon, are much less dangerous than neutral or alkaline meals. Vegetables and fruit on the market are often sprayed with water to make them appear fresher and more attractive. If this is done with contaminated water, transmission may occur.

Reservoir

Humans are the only vertebrate hosts. Vibrio cholerae can survive long-term and probably permanently in brackish water, especially if there is a a neutral or slightly alkaline pH and the water contains some minerals and organic material. The bacteria are concentrated in phytoplankton (certain algae) and zooplankton which live in this water. Among the latter, copepods, a group of crustaceans, are important. V. cholerae O1 produces chitinase, an enzyme which can break down chitin. This is a structural protein of the exoskeletons of invertebrates. The full importance of this chitinase has still to become clear.

Vibrio cholerae is also found in association with a cyanobacterium ( Anabaena variabilis ). Vibrio cholerae has similar interactions with marine diatoms ( Skeletonema costatum ), phyaeophytes ( Ascophyllum nodosum ) and copepods. The bacteria also flourish in fresh water where plants such as water hyacinth are growing. The impact of specific ecological circumstances which might lead to a sudden increase in plankton (algal bloom) needs to be studied further. Cholera is clearly seasonal. A chronic aquatic reservoir is likely and this might be independent of continuous human faecal pollution. V. cholerae excreted by humans can be cultured in the laboratory. These bacteria however may assume a living form which cannot be cultured in vitro and which multiply in the environment. However, those bacteria are not dead since they multiply when instilled in a rabbit's ileum (rabbit ileal loop model). It may revert to a replicating form in its natural environment when there are favourable environmental factors and this has important epidemiological implications. The living, but non-reproducing form of V. cholerae can probably cause disease. Traditional culture methods for tracing V. cholerae in water miss these "dormant" bacteria. Tests based on fluorescent antibodies may offer a practical solution, since they stain dormant and active bacteria the same.

Half empty cargo ships take water in their ballast tanks in order to lie deeper and with more stability in sea water. This ballast water of course contains micro-organisms. In the past potentially contaminated ballast water was pumped out again when the ship came into harbour at its destination. In view of the importance of an aquatic cholera reservoir, freight ships have to empty and refill their ballast water tanks while in open sea, with clean oceanic water and not when they come into harbour, using dirty coastal water.

Note: dormant bacteria

The general use of nutritionally rich media for in vitro cultivation of microorganisms can lead to a misunderstanding of the nutrient status of natural environments. In many natural environments, oligotrophic conditions are the rule rather than the exception. A continuing state of starvation or near-starvation is the natural state of many microorganisms. At limiting nutrient levels, there are a number of survival strategies. Expression of starvation genes results in a reduction in size and the formation of ultra small "microbacteria", usually by the process of reductive division. A rod-to-coccus transformation can take place. Protective starvation proteins are synthesized and substrate capture enhanced. In at least some cases, such cells are still culturable providing sufficient care is taken. All bacteria however, have a point at which exogenous nutrients can no longer be obtained and are thus effectively exhausted. At this point, cells may enter a dormant state that permits them to survive for long periods without division. During this state the cells are non-culturable.

Physiopathology

The bacterium multiplies in the small intestine, where it adheres to the mucosal brush border. The bacterium is not invasive, in other words it does not penetrate the intestinal wall or pass into the blood. It excretes a very powerful toxin, which causes active fluid secretion towards the lumen. This fluid is isotonic, ion-rich and protein free. There are no intestinal ulcerations and the faeces do not contain blood. There is little if any fever. There is no tenesmus. The faeces contain significant amounts of sodium, potassium and bicarbonate. Because of this the intestinal content is slightly alkaline ( V. cholerae thrives best in a slightly alkaline environment and the bacteria are therefore producing the conditions which are optimal for their own survival). The loss of large amounts of alkaline faeces results in metabolic acidosis. People with blood group O have an equal risk of infection but are at a significantly higher risk of clinically severe cholera if they become infected. The reason is unknown. Hypervirulent and hyperinfective strains play an important role in epidemics. Passage of Vibrio cholerae through the gastrointestinal tract results in a short-lived, hyperinfectious state of the organism that decays in a matter of hours into a state of lower infectiousness. Such strains have a much lower ID 50 than strains occuring in natural water reservoirs. The classic strain is associated with more severe illness. Faecal excretion of V. cholerae for up to two weeks has been documented and occasional asymptomatic carriers ocur. Asymptomatic patients typically shed bacteria in their stools at about 1000 V. cholerae bacteria per gram of stool, which is a low level of shedding, compared with the 100 million bacteria per gram in case of ricewater stools. Stool from cholera patients can harbor spirochete-like bacteria, such as Brachyspira pilosicoli and B. aalborgi , sometimes in densities equal to those of V. cholerae. The significance of such associations is unknown at present. The fastidious nature of these spirochetes limits culture as a research tool. The bacteria can sometimes be recognised as making up a pseudo-brushborder on colonic cells (the colon does not have a brush border, as opposed to the small intestine), see chapter on intestinal spirochaetosis.

Toxins

Vibrio cholerae produces several toxins: cholera toxin ( Ctx ), the zona occludens toxin ( Zot ) and the accessory cholera enterotoxin ( Ace ). The role of the two latter toxins is not entirely clear. The Ctx enterotoxin of V. cholerae consists of 2 parts: A and B, where A stands for a ctive and B for b inding. Part A is a monomer, while part B consist of 5 identical subunits (a pentamer). The polypeptides of part B bind to a receptor (G m1 ganglioside, a glycolipid) on the epithelium of the small intestine, after which part A can penetrate the cell. Part A binds covalently to an intracellular protein (G s -protein; s for stimulatory) which irreversibly activates it, leading to the persistent stimulation of another intracellular enzyme, adenylate cyclase. This latter enzyme increases intracellular cyclic-AMP, which inhibits salt absorption by the microvilli and promotes active chloride excretion by the crypt cells. Water and potassium bicarbonate passively follow the chloride. In the end there is an overall water loss to the intestinal lumen. Fluid loss originates in the duodenum and upper jejunum, the ileum is less affected. The colon is insensitive to the toxin and cannot absorb the large amount of fluid quickly enough. Catastrophic diarrhoea follows. The toxic A-subunit also has other effects such as disturbing the expression of some genes, increasing inflammatory cytokines and inhibiting antigen presentation by macrophages. On the other hand, the B-subunits of cholera toxin have anti-inflammatory properties. These are under intense study at present for possible therapeutic use in immune abnormalities. While cholera toxin adheres to the intestinal villus cells and disables the cellular saltwater pumps, the Zot toxin loosens the junctions that binds intestinal epithelial cells together. This contributes to the loss of water to the intestinal lumen.

The in-vivo detailed mechanism is probably more complicated. Cholera toxin also stimulates the nervous system in the intestinal wall, the myenteric plexus. This results in the release of 5-hydroxytryptamine (serotonin) from the enterochromaffin cells, leading to in additional fluid loss to the lumen. Granisetron, a 5-HT3 receptor blocker, partially reverses this effect. More research is needed to determine the role of this mechanism in the physiopathology.

Clinical aspects

The incubation period is brief: sometimes only hours, more commonly 1 to 5 days (average 2 days). It is one of the few infectious diseases where -in case of a very severe infection- you can be OK in the morning and dead by sunset the same day. Asymptomatic infections are common (about 93%), but chronic carriers are very rare. Sometimes there is an initial transient fever (more seen in children). Massive watery diarrhoea starts suddenly. The faeces very rapidly look like water in which rice has been boiled: watery with flakes of mucus. The faeces have a fish-like smell. The volume of faeces may rise to 500 ml per hour. Vomiting is common, but abdominal cramps are unusual. The onset of thirst, oliguria or anuria and weakness is rapid. In a short time the patient develops severe dehydration and can die within 24 hours. In other cases the diarrhoea is less severe, especially with infections with El Tor. As the patient's condition deteriorates, hoarseness of the voice and temporary deafness are often observed. Children with severe cholera may present with drowsiness or coma.

The signs of dehydration are thirst, dry mouth and lips (if the patient did dot vomit recently), hollow eyes and sunken fontanel in children. The skin turgor diminishes. The skin becomes wrinkled (washerwoman's hands). Often, the voice becomes weak and hoarse, the pulse quickens and is difficult to feel. The radial pulse might be impossible to detect. Blood pressure falls. There is little or no urine production (prerenal failure). Respiration becomes faster due to metabolic acidosis secondary to loss of bicarbonate in the faeces (bicarbonate is alkaline). This acidosis causes vomiting and muscle cramps. There is also significant potassium loss in the faeces. If rehydration is carried out using fluid without potassium, severe hypokalaemia may result. Nevertheless, quite often normokalaemia is found, together with an increased anion gap. The increase in anions (= negative ions) is multifactorial due to the hyperproteinaemia (hemoconcentration), hyperphosphataemia (internal shifts and renal failure) and lactate acidosis (shock). Ketones play little if any role. An elevated hematocrit (hemoconcentration) can be found in in nonanemic patients, as can neutrophil leukocytosis in severe cases. The mortality from classic cholera may reach 50 %, but can be brought down to < 1 % with correct therapy. Mortality is chiefly due to dehydration with kidney failure, hypokalaemia, hypoglycaemia and aspiration pneumonia during vomiting.

Note : anion gap

The anion gap is the difference in the measured cations and the measured anions in serum or plasma. The anion gap is a measure for negative ion such as phosphate, ketone bodies, sulfates, hippurate, uric acid and lactate, which are not measured routinely. The anion gap = (Na + + K + ) - (Cl - + HCO 3 - ) = normally 8 to 12-16 mmol/L). Calcium and magnesium are always left out, sometimes potassium is left out of the calculation. A high anion gap metabolic acidosis indicates lots of unmeasured anions. Common causes are can be summerized in the old acronym "KUSMALE". This stands for K : ketoacidosis(diabetic, ethanol or starvation) U : uremia (decreased acid excretion and reduced HCO 3 - reabsorption) S : salicylate poisoning M : methanol poisoning A : pAraldehyde administration L : lactate acidosis, which can be with normal amounts of oxygen or hypoxic E : ethylene glycol, a product which is used as antifreeze and in wine adultaration An alternative mnemontechnical expression is CUTE DIMPLES. Can you make one with MUDPILES? C : cyanide poisoning U : uremia T : toluene poisoning ("glue sniffing", multisystem toxicity, including kidneys) E : ethanol : ethanolic ketoacidosis D : diabetic ketoacidosis I : isoniazide M : methanol (check if sudddenly several people become blind after drinking homebrewed alcohol) P : propylene glycol, often used in radiator fluid, very low toxicity L : Lactate acidosis: hypoxic or normoxic E : ethylene glycol; forbidden sweeterer (wine!). Check urine for oxalic acid crystals S : salicycate overdose   Normal anion gap acidosis : the drop in HCO 3 - is compensated by increase in Cl - and is known as hyperchloremic acidosis. This is the classic situation in cholera where diarrhea predominates. If severe vomiting predominates, hypochloremic acidosis would be expected. Gastrointestinal loss of HCO 3 - (i.e. diarrhea) (note: vomiting causes hypochloraemic alkalosis) Renal loss of HCO 3 - i.e. proximal renal tubular acidosiRenal dysfunction i.e. distal renal tubular acidosis Ingestions NH 4 Cl = Ammonium chloride; Acetazolamide (a carboanhydrase inhibiting diuretic); Hyperalimentation fluids (i.e. total parenteral nutrition) Mineralocorticoid deficiency (Addison's disease), if associated renal damage  

Diagnosis

Cholera should be suspected in acute massive rice-water diarrhoea, certainly if there have been several cases in a short time (epidemic). The clinical picture of severe cholera is so spectacular that differential diagnosis does not present many difficulties. Milder cholera may be similar to other forms of gastro-enteritis (but not to dysentery). A child above the age of five years who develops acute dehydration, or dies as the result of acute diarrhoea, is always suggestive for cholera.

The vibrios are very small and can best be seen in a fresh faecal specimen with the help of dark field microscopy. There is characteristic motility ("star shooting") which stops immediately after adding anti-O1 antiserum. This does not give any information on possible toxin production. Confirmation is best made via a bacteriological culture. Culturing should preferably be on a special medium in a bacteriology lab, e.g. TCBS-agar [=Thiosulphate-Citrate-Bile salts-Sucrose], polymyxin mannose tellurite agar (PMT) or an other selective medium. TCBS agar is green before inoculation; sucrose-fermenting organisms such as V. cholerae turn it yellow. TCBS agar is important for rapid isolation and identification, but V. cholerae also grows on routine agar media. For routine media, large numbers of bacteria per gram stool should be present to allow detection. Patients or carriers with low burden of bacteria will be missed with routine culture media. Overgrowth by normal fecal flora limits recovery of colonies. In order to identify the serogroup and the serotype one subsequently finds out to which antibodies (antiserum) the colonies obtained exhibit an agglutination reaction. It is also possible to find out whether the vibrios are toxicogenic (produce toxin), e.g. by a PCR varianr called a loop-mediated isothermal amplification (LAMP) assay. Definitive identification is made in a reference laboratory.

Specimens may be transported in a transport medium, e.g. Cary-Blair. This is a kind of mild alkaline buffered gelatine in seawater with low redox potential in which the bacteria will survive for 4 weeks. If it is not available, a filter paper can be soaked with faeces and transported in an airtight bag to a well-equipped laboratory. A sample treated in this way remains usable for 1 week, but the recommendation is "the faster the analysis, the more reliable". Blotting paper, soaked with liquid faeces and if possible placed in a 1% saline solution, can be kept for several weeks at 37‹C (not in the freezer). This is useful if there are initial transport problems. Nevertheless it is better to have a fresh faecal specimen. For specimens from the environment or from food, in which the number of bacteria is much lower than in faeces, enrichment is necessary. The specimen can be incubated for 8 hours in alkaline peptone water, after which a TCBS agar is used.

About 10 days after infection with V. cholerae O1 the patient produces vibrocidal antibodies. They start diminishing after only one month and disappear within the year. Antibodies against cholera toxin are produced more slowly and remain for years. However, these cross-react with enterotoxin produced by ETEC bacteria [enterotoxic Escherichia coli ]. The immune response to V. cholerae O139 is not well understood. The detection of antibodies is not important for the urgent care of the individual patient, but does permit retrospective diagnosis.

Note: Other Vibrios

Sometimes other Vibrio species are responsible for diarrhea, e.g. Vibrio cholerae non-O1, V. parahaemolyticus , V. hollisae , V. minicus and V. fluvialis . Our knowledge of these latter bacteria is clearly insufficient. Vibrio vulnificus is an aggressive species present in seawater and filter-feeding organisms such as oysters. This bacterium may cause septicaemia and wound infections, certainly in patients with liver cirrhosis.

Note: Brownian motion

Not all movement which one can detect in a fresh stool sample is caused by flagellated bacteria. Do not confuse star shooting of Vibrio cholerae with Brownian motion.

During the formation of minerals, a drop of water can get trapped in a piece of igneous rock as the rock cools from its melt. In the early 19th century the Scottish botanist Robert Brown discovered such a drop of water in a piece of quartz. Brown reasoned that the water had been inaccessible for many millions of years to spores or pollen carried by the wind and rain. When he focused his microscope on the drop of water, he saw continuous movement. Suspended in the water were scores of tiny particles, ceaselessly oscillating with a completely irregular motion. The smaller the size, the more rapid its motion. The motion was familiar to Brown: he had previously observed such oscillations during his studies of pollen grains in water. The new experiment however, ruled out the explanation he had put forward earlier, namely that some kind of vitality is retained by the molecules of the plant long after death. Brown concluded that the agitation of the particles trapped inside the quartz inclusion had to be a physical phenomenon rather than a biological one. The explanation for this so-called Brownian motion is that a material particle (dust, pollen, bacteria) is continuously bombarded by the molecules of the fluid in which it is suspended. A single molecule hardly ever has enough momentum for this effect on the suspended particle to become visible under a microscope. However, this bombardment is not symmetrical. There is random fluctuation in the velocities of the nearby molecules. When many molecules collide with the particle from the same direction at the same time, they can noticeably deflect the particle. Atoms and molecules reveal their existence through the motions of a particle suspended in a fluid! The path of the particle is a random one. What a person can see through a microscope are the effects of relatively large fluctuations in the local molecular environment. If the resolving power of the microscope were increased by factors of, say, 10, 100, 1,000, the effects of bombardment by progressively smaller groups of molecules could be detected. The path would have a fractal structure (shape is self-similar at each magnification). This can be better appreciated when one knows that at room temperature, a dust particle will sustain about 10 21 collisions per second. Since the turn of the 19-20 th century, the study of Brownian motion had far-reaching consequences for physics, chemistry and mathematics (e.g. kinetic theory of gases, statistical mechanics, thermodynamics, entropy, diffusion, Avogadro's number...).

Treatment

Cholera treatment with IV-rehydratation. Copyright Alexander von Humboldt Institute, Peru

Cholera treatment with IV-fluids. Copyright Alexander von Humboldt Institute, Peru

Cholera treatment with IV-rehydratation. Copyright Alexander von Humboldt Institute, Peru

Cholera bed used by AIDS patients with chronic diarrhoea. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Rehydration is essential and must be instituted as soon as possible. Two phases are distinguished. First it is important to replenish what has been lost in the previous hours or days. Then one must compensate the persistent fluid loss (e.g. the amount of fluid that is lost every hour). In mild cholera without vomiting oral rehydration may suffice. In severe forms IV fluids should be administered.

There are several possible compositions of rehydration fluids. Solutions containing salt, sugar, potassium and bicarbonate are recommended. Lactate is also good because it is converted in the body to bicarbonate. In cholera it is preferable to use Ringer's lactate ( = Hartmann's solution). Normal physiological saline is second choice because it does not correct the acidosis nor does it contain potassium. Severe hypokalaemia may occur, with cardiac arrhythmias, kidney damage, paralytic ileus and significant muscle weakness with reduced or absent tendon reflexes. Dextrose (= glucose) 5 % without electrolytes is not advised as a rehydration fluid. A reminder: 1 gr KCl = 13 mEq KCl.

So: Hartmann = Ringer's lactate >Ringer>  physiological saline>>> not glucose infusion if there is an alternative

In severe cholera (fluid loss > 10 % of weight) the missing fluids should be administered quickly, e.g. 6 litres over 4 hours for a patient weighing 60 kg. The first 3 litres may each be administered in 10 minutes (total therefore 30 minutes). After administration of the lost volume, the losses are compensated for further IV and/or PO fluids (faeces volume + urine volume + 500 ml). Vomiting may make oral administration of fluids difficult. Generally a total of 6 to 10 litres per patient is necessary. When patients start to drink and do not vomit anymore, it is best to leave their IV lines in place for a while until one is sure that rehydration will not pose any more problems.

Special cholera beds are useful: they have a central opening to allow the liquid faeces to pass through, and they can be collected in a bucket. This makes it possible to quickly determine the amount of fluid loss. During an epidemic people who can still hold themselves upright can simply sit on a bucket and try to drink as much ORS [oral rehydration solution] as possible. Children quickly develop convulsions and coma. It is important that hypoglycaemia should be considered. For an adult 50 ml of a 50% glucose solution is given IV, for a child 2-4 ml/kg 25% glucose or 10 ml/kg of a 10% glucose solution.

Antibiotics are useful because they reduce the duration and thus the total volume of the diarrhoea. They are not essential, however, and resistance often occurs. At present a single dose of azithomycin 1 gram is the agent of choice. Classic V. cholerae is usually sensitive to tetracyclines. The dose of tetracycline is 500 mg x 4 daily PO for 2 to 3 days. Parenteral antibiotics are of no value (the bacteria are not invasive). Alternatives are doxycycline 300 mg once or erythromycin, cotrimoxazole and chloramphenicol. Single-dose ciprofloxacin is also effective. V. cholerae O139 is often resistant to cotrimoxazole (sulphamethoxazole-trimethoprim) but sensitive to tetracyclines. This resistance to cotrimoxazole is determined by a certain genetic element (the SXT [sulphamethoxazole] element). Antiperistaltic drugs such as loperamide may cause accumulation of fluid in the intestinal lumen with unfavourable consequences and shoud be avoided.

Prevention

In the West nowadays a patient with cholera will remain a sporadic case. In developing countries one case can lead to several secondary cases. It is not necessary to wear special masks, aprons or gloves, but washing hands (hospital staff, family, visitors) should be obligatory. The contamination of clothing and bedding is unavoidable. Boiling in water for five minutes is sufficient for disinfection. Mattresses and blankets can be dried in the sun. It is better to do this before washing them, to prevent infection of the washing area.

After surviving cholera a patient is probably immune for homologous biotypes for more than 3 years. There is some controversy: infection with the classic biotype seems to protect against recurrent infection by either biotype, but El Tor does not. No cross-immunity between V. cholerae O1 and V. cholerae O139 is seen, although they produce the same toxin. Immunity relies on antibodies in the intestinal lumen (the bacteria are not invasive). Systemic vibridocidal as well as anti-toxin antibodies develop during illness. Babies which are being breast-fed receive protective antibodies in their mother's milk.

Vaccination Parenteral vaccination with dead V. cholerae bacteria (IM administration) does not lead to sufficient formation of protective antibodies in the intestinal lumen. Approximately 50-65% of people living in endemic areas will be protected for 3-6 months. The IM vaccine is associated with local reactions in 50% and systemic reactions (fever, malaize) in 10-30%. Advice to vaccinate with this type of vaccine was discontinued in 1972 by the WHO [World Health Organisation]. Parenteral vaccination, mass chemoprophylaxis and "cordon sanitaire" (= restrictions on travel and trade) are not effective in preventing or limiting outbreaks. A newly developed oral cholera vaccine is based on a killed whole cell cholera vaccine combined with the recombinant B subunit of cholera toxin ( Dukoral TM ). The vaccine contains 1 mg of recombinant B subunit, as well as 25 x 10 9 bacteria each of V. cholerae O1 classic Inaba, V. cholerae O1 classic Ogawa, V. cholerae O1 El Tor Inaba (heat-inactivated), V. cholerae El Tor Inaba (formaline inactivated). Dukoral does not contain the A subunit of cholera toxin and therefore, no pathogenic toxin is present. Two to three doses need to be given. Protection against cholera serotype O1 is 61E6% for 4E months. Lower levels of protection continue for 3 years. Protection wanes rapidly in young children. A herd immunity effect is expected in areas where vaccine coverage is more than 50%. Because the risk of cholera for most travellers is extremely low, vaccination should be considered only for those working in relief or refugee settings or for those who will be travelling in cholera-epidemic areas and who will be unable to obtain prompt medical care. It is possible that in the future oral cholera vaccins might be considered, although traditionally there was fear that use of a vaccine might divert money and distract from the use of other meaningful interventions. Because cholera toxin and the heat-labile toxin of enterotoxicogenic Escherichia coli are closely related, the vaccine was also studied in the prevention of  travellersEdiarrhoea.  Bacteria are responsible for 50-85% of cases of traveller's diarrhea , with 15% or more due to ETEC. The vaccine has no effect on immunity against the heat-stable toxin of ETEC.  Unfortunately, the vaccine is expected to prevent 7% or less of cases of travellersEdiarrhoea and cannot be recommended for this purpose. Another oral vaccine (Mutachol or Orochol) is a live attenuated strain of V. cholerae O1 classic Inaba with 94% of the gene encoding the A subunit of cholera toxin deleted. It is administered as a single oral dose. It is not commercially produced at present.

Mass chemoprophylaxis is not effective because (1) the infection spreads faster than the organisation of drug distribution, (2) the effect of a drug only lasts 2 days, after which re-infection may occur, (3) the whole population needs to be treated simultaneously and people should then be isolated and (4) it is difficult to convince asymptomatic people to take a drug. Selective chemoprophylaxis of nearby contact persons can be given, certainly if there appear to be multiple secondary cases (the role of monitoring is important here).

Correct eating and drinking habits, safe stool habits and personal hygiene are the most effective means for individuals to limit their risk of cholera. Improved sanitation is the pre-eminent method of eliminating cholera and many other faeco-orally transmitted infections. This is directly linked, however, to the degree of poverty in a region. Boiling drinking water is often difficult since fuel may be scarce and expensive. Since a significant proportion of Vibrio cholerae can adhere to plankton, the drinking water can be filtered through a fine cloth, which removes both plankton and a lot of bacteria in a single operation. This is of course less effective than obtaining water from a clean pipe or pump, but it is cheaper. Chlorination of drinking water may be important (piped water or via water trucks). This is difficult to accomplish in rural areas. Chlorination is much less effective if the water is turbid due to organic debris.

Eating raw fish, shellfish (e.g. oysters, mussels) and crustaceans (such as crabs, shrimps) should be avoided. Washing hands is important for transmission control within a household. Infected faeces should not be disposed of in a poorly functioning drain (hospital: e.g. in pit with unslaked lime = CaO). When large groups of people come together (funerals, festivals, etc.) there should be latrines with facilities for washing hands and plenty of soap.

An attempt must be made to trace the source of small, local outbreaks (see John Snow). Contaminated water is the chief suspect in a sudden, local epidemic, while in isolated cases the cause should be sought in contaminated food. This is of course not an absolute rule. Food cooked by street vendors and in restaurants poses specific problems. Flies probably play an underestimated part in transmission, but their numbers also reflect the sanitary conditions in a region.

The following points should be emphasised during information campaigns:

Drink only clean water (boiled or chlorinated) Cook food completely and eat it while it is hot Avoid uncooked food, unless it can be peeled Wash hands after a bowel movement Wash hands before preparing food Wash hands before eating Correct use of a good latrine (also for children) With correct treatment cholera is rarely fatal If cholera is suspected medical help should be sought immediately In diarrhoea, give plenty of fluids (e.g. ORS) At present, cholera-vaccination is not stressed, unless better vaccines are developed in the future. Even the new oral cholera vaccine (Dukoral) induces limited protection during a short period. It can be considered for high-risk personel, e.g. relief workers.  

In case of an epidemic , it is important to have a large stock of IV rehydration fluid available as well as the means of preparing large amounts of oral rehydration fluid. Normally such buffer stocks should be stored at various strategic points. The stocks for cholera treatment should not be segregated in storage, but should be rotated during normal use to avoid allowing large amounts to run out of date. As soon as an epidemic is suspected, use as much oral rehydration as possible so that stocks of the IV solutions last as long as possible. Cholera beds should be made ready. In a normal epidemic an attack rate of 0.2% can be taken as a rule of thumb (i.e. 200 cases can be expected in a population of 100,000). This is useful for estimating the size of stocks that will be needed. Sometimes the attack ratios are higher (e.g. the Rwanda-Zaire border in 1994).

During an epidemic one should consider

clean drinking water in sufficient amounts,

sanitary provisions (latrines + hand washing with soap ± hand desinfection with hypochlorite solution)

treatment of the patients

direct contact persons of the patient (e.g. mother of a child) may take chemoprophylaxis, but mass prophylaxis is not advisable.

The risk for a traveller who observes elementary hygiene is very small (1/500,000).

Exercises

Chad . A woman weighing 60 kg suddenly develops severe watery diarrhoea in the morning. In the evening she is brought to you. What signs of dehydration do you look for? What do you do? Peru . During a cholera epidemic you are brought a child who for 2 days has had very excessive watery diarrhoea. It has just had a convulsion. What do you think and what do you do? Colombia . You are brought a 19-year-old man. He has had diarrhoea for 3 days. He has fever of 39°, looks toxic, and has to go to the toilet every half hour. The faeces are slimy. What do you think and what do you do? Bangladesh . A woman aged 20 years, 50 kg, has cholera. There was some dextrose 5% infusion in stock and an attempt had been made to rehydrate the patient. After 8 hours her fluid balance is clearly better, but as she is vomiting, her fluids policy needs to be continued. After 48 hours, however, she is having difficulty moving her arms and legs. Tendon reflexes are absent. Her heart beat is irregular. The intestinal peristalsis can no longer be heard. What do you think? Ecuador . A very weak, undernourished child aged 6 is admitted with rice water diarrhoea and vomiting. It is treated promptly with Ringer's lactate IV. The following day it develops fever to 39.7° and its respiration is fast and difficult. It is suspected that the acidosis has been insufficiently corrected so that the amount of bicarbonate is increased. Two days later the child dies. What should have been done? Mocambique . During a cholera epidemic vaccinations should be begun immediately and as a priority. What do you think of this statement? Cholera is an example of isotonic contraction of the blood volume. Quasi-isotonic fluid is lost (diarrhoea with the same electrolyte composition as plasma). There is thus little exit of water from the intracellular compartment, although the haematocrit increases. Compare with (1) hypertonic contraction of blood volume which occurs in a febrile sweating stuporous patient and (2) the hypotonic contraction which occurs during kidney failure (more sodium than water is lost) or a marathon runner in hot weather, with only pure water to slake thirst.

General

Tropical sprue is an acquired disease of unknown origin. An infectious origin appears probable and the term "post-infectious malabsorption" is also used. Possibly there is an initial insult at the level of the jejunal-ileal enterocytes, followed by bacterial overgrowth with enterotoxic strains. The disease is characterised by abnormalities of the mucosa in the small intestine with chronic malabsorption, multiple nutritional deficiencies and anaemia  The malabsorption is generalised and affects absorption of proteins, fat, carbohydrates, minerals and vitamins (typical is iron and folate deficiency). Good response to treatment with doxycycline and iron-folate supplements.

Tropical sprue occurs chiefly in the Caribbean, India, Nepal and Southeast Asia, in both the indigenous populations and immigrants. Cases have been reported from Mauritius, Fiji, southern Italy, Guyana and Central America. In Africa the disease is apparently very rare, although cases have been reported from Zimbabwe.

Map of areas with post-infective malabsorption, also known as tropical sprue. Copyright Wellcome History

Clinical aspects

Tropical sprue can have an insidious onset or can start acutely. The symptoms are those of chronic malabsorption. Generally it presents as a clinical triad of painful tongue, loss of weight and persistent abdominal discomfort with diarrhoea. Patients are noticeably tired, both physically and mentally. Amenorrhoea is very common. There is loss of weight with muscle atrophy. Hypoalbuminaemia leads to oedema. Due to malabsorption of carbohydrates there is increased gas production in the intestines, with borborygma, a bloated feeling in the abdomen and intestinal cramps. The D-xylose absorption test is abnormal in more than 90% of cases. Fat malabsorption leads to steatorrhoea with more than 10 g of fat in the faeces. This occurs in 95% of patients. The stools are pale, very odorous and quite voluminous, up to 5 times the normal amount. Dehydration, hyponatraemia and hypokalaemia are very common. Calcium deficiency may lead to tetany with positive Trousseau's sign [in latent tetany, the occurrence of carpopedal spasms accompanied by paresthesia elicided when the upper arm is compressed (diminished blood supply), such as by a tourniquet or a blood pressure cuff. It occurs when the concentration of ionised free calcium is below a certain treshold; this concentration is pH-dependent]. Hypokalaemia leads to reduced tendon reflexes and abnormalities on the electrocardiogram. There is usually a deficiency of vitamin B12, folic acid and sometimes also iron. Anaemia occurs and is typically macrocytic with megaloblastic bone marrow. In long-term cobalamine deficiency there may be peripheral neuritis and involvement of the spinal cord, chiefly of the dorsal columns (proprioception). The tongue is red and painful. As well as glossitis there may be stomatitis with superficial erosions. Deficiencies in fat-soluble vitamins (A, D, E, K) lead to prolongation of the coagulation time and osteomalacia. Vitamin A deficiency is characterised by a dry, rough skin with follicular hyperplasia and Bitot's spots on the conjunctivae. In severe deficiency night-blindness and xerophthalmia may occur.

Diagnosis

Tropical sprue should be suspected in anyone with megaloblastic anaemia and malabsorption who has lived in an endemic region. Biopsy of the jejunum shows typical abnormalities. Intestinal villi become shorter and broader. In the intestinal wall there is an inflammatory infiltrate, chiefly consisting of lymphocytes, plasma cells and a few eosinophils. The enterocytes exhibit large vacuoles. Radiography of the small intestine shows non-specific changes. There is flocculation of the contrast material and segmentation of the barium column, distension of the lumen and thickening of the mucosa. The mucosal folds in the small intestine are irregular and thickened, which gives the impression of a stack of coins. In advanced cases, no mucosal folds at all can be seen. There is no deconjugation of bile salts, unlike, for example, in the blind-loop syndrome. A flat mucosa is very unusual and should lead to suspicion of a different disease (e.g. gluten enteropathy). 

Differential diagnosis

The differential diagnosis is that of chronic malabsorption. In includes persistent giardiasis, isosporiasis, strongyloidosis, intestinal capillariasis, gluten enteropathy (coeliac disease), chronic pancreatitis, intestinal tuberculosis, intestinal amyloidosis, Whipple's disease, the blind-loop syndrome, bacterial overgrowth, diverticula and jejunocolic fistulae. Crohn's disease is rare in developing regions.

Treatment

Treatment is based on tetracyclines 250 mg QDS or doxycyclin 100 mg daily for three weeks to several months. Folic acid supplements (5 to 10 mg daily) and multivitamins and if necessary iron should be added to the treatment. Response to treatment is generally swift with an initial improvement within three days. Further recovery takes place in the course of the following three months.

Summary

A common and major problem; a major cause of mortality in children Mortality due to dehydration and invasive bacteria Etiology: viruses, Shigella sp ., Vibrio cholerae, Giardia lamblia, Entamoeba histolytica, ... Clinical: degree of dehydration, ± blood in the faeces, ± fever, ± acute/chronic Rehydration (PO or IV); nutrition, sometimes aetiological treatment necessary

General

Diarrhea is very common in the tropics. It is often self-limiting, but its general significance cannot be overestimated. It is a major cause of malnutrition and is one of the main causes of death, particularly in children. What precisely is meant by diarrhea varies between patients. An increased number of defecaetions per day (e.g. more than 3) , a decreased consistency of the faeces or an increased volume of stools (e.g. > 200 g/24h) all are used to define the problem. The WHO definition of diarrhoea is at least 3 evacuations every 24 hours of unformed faeces. Unformed means here that they take the shape of any container into which they are evacuated. WHO emphasises the importance of change in stool consistency rather than frequency, and the usefulness of parental insight in deciding whether children have diarrhea or not.

Diarrhea causes fluid loss resulting in dehydration. The patient also looses electrolytes, which can lead to ion imbalances, such as hypokalaemia. Acidosis develops due to the loss of bicarbonate in the stools, to reduced renal function (less acids are excreted) and to ketosis (breakdown of body fat due to reduced food intake). Often the patient has no appetite and the nutritional status which is sometimes already poor deteriorates further. Sometimes the mother thinks she is doing good by "letting the intestines rest" and temporarily not giving food. Moderate undernourishment can then develop into severe malnutrition (marasmus and kwashiorkor). The latter is often seen if a patient has had a number of episodes of diarrhea in quick succession.

Dysentery is a severe form of diarrhoea. Fever is common in bacillary dysentery, but rare in amoebic dysentery. Dysentery has three characteristics:

Abdominal pain Tenesmus (pain due to cramps in the rectum) and false defecation need Frequent evacuation of small quantities of faeces that are mixed with blood, mucus and/or pus

Steatorrhoea or fatty diarrhoea is characterised by large quantities of faeces with an increased fat content (the stools float on water). This occurs in certain malabsorption syndromes. The cause usually lies in disorders of the pancreas or small intestine.

Etiology

Etiology, general

Diarrhoea is usually caused by infections. Of the nearly 11 million deaths that occur annually among children under five years of age, diarrheal disease is the second leading cause (after respiratory tract infections). The most common cause of severe gastroenteritis worldwide, rotavirus accounts for 29 to 45 percent of nearly 2 million deaths. Bacterial intestinal infections (especially dysenteria) also contribute to the high mortality.

The following list is not exhaustive:

Preformed bacterial toxins, with the bacterium itself being no longer active in the intestine. Examples include staphylococcal diarrhoea ( Staphylococcus aureus toxin), the ingestion of Clostridium toxins after eating contaminated meat (pigbel) and Bacillus cereus toxins (contaminated rice, among other things). Incubation time very short (hours). Bacteria which multiply in the intestines: Salmonella, Shigella, Yersinia enterocolitica , a whole zoo of related Escherichia coli strains, toxicogenic Vibrio cholerae , Campylobacter jejuni , toxicogenic Clostridium difficile Protozoa: Giardia, Entamoeba histolytica, Balantidium coli , microsporidia, various coccidia ( Isospora belli, Cryptosporidia, Cyclospora, Sarcocystis ). Sometimes malaria is accompanied by diarrhea! Worms: only in case of serious infections, e.g. Schistosoma mansoni, Capillaria philippinensis, Strongyloides stercoralis, Trichinella spiralis ; rarely by other worms. C. philippinensis and S. stercoralis can remain several decades in the body and are able to multiply inside the human host, something that most other worms cannot achieve. They can be lethal. Since worm infections are so common in the tropics, worm eggs are often found in the stools. However, there is not necessarily an etiological connection between the presence of helminth eggs and diarrhea. Viruses: Rotavirus, Astrovirus, HIV, Noroviruses (Family Caliciviridae; the name refers to the 32 chalice-like depressions in the viral surface. They are also known as Norwalk-like viruses, referring to the outbreak in Norwalk, Ohio, 1968). Noroviruses cause gastro-enteritis, with important vomiting accompaying the diarrhea. Non-infectious causes such as laxative abuse, animal or vegetable toxins, marine biotoxins, mycotoxins and inflammatory intestinal diseases (Crohn's disease, ulcerative colitis) are much less common.  Endocrine problems (hyperthyroidism) and related problems (vipoma, carcinoid, etc) exist, but are present in a minority of persons who present with chronic diarrhea.

It is not always important to discover the exact cause of an episode of diarrhoea: for example, it is important to distinguish between amoebic colitis and bacillary dysentery, but the difference between Rotavirus and Norwalk virus enteritis is at present not clinically relevant in the tropics. Intestinal infections caused by protozoa occur everywhere, but they are more prevalent in tropical climates. The climate helps protozoa to survive in the outside world and poor hygiene promotes their transmission. Diarrhoea is often found together with a parasitic infection, but the causal connection must always be assessed critically. It is important to distinguish between infection and disease. Of the many protozoa that are found in faeces, only a few types are potentially pathogenic. Occasionally, Plasmodium falciparum and Leishmania donovani can cause digestive symptoms. The diarrhoea then displays no particular characteristics.

Campylobacter jejuni cultured in vitro. This bacterium is common cause of travellers diarrhoea. Copyright ITM

Giardia lamblia trophozoite. Copyright ITM

Entamoeba histolytica cysts. Cysts never contain red blood cells, but trophozoites occasionally do. Copyright ITM

Etiology, acute bloody diarrhoea with fever

This is the picture of a bacillary dysentery. Pathogens are Shigella , Salmonella , Campylobacter and some Escherichia coli . Some bacteria are very aggressive, while others give rise to milder infections, for example Shigella dysenteriae , S. flexneri , S. boydii, S. sonnei . Complications can occur: toxic megacolon, rectal prolapse, septicaemia, haemolytic-uraemic syndrome (TTP-HUS, often triggered by Shiga toxin produced by Escherichia coli O157:H7 or other verotoxin producing bacteria (VTEC), reactive arthritis, Reiter's syndrome [urethritis, arthritis, conjunctivitis, uveitis, hyperkeratosis of the palms of the hand (keratoderma blennorrhagicum) and painless ulcers in the mouth and on the glans (balanitis circinata)]. If HUS occurs, antibiotics are contraindicated because otherwise still more toxins are released from the bacteria that have been killed, which aggravate the clinical status. After using antibiotics an overgrowth of Clostridium difficile can occur in the intestine. The toxins that are produced by this bacterium cause a severe inflammation of the colon (pseudomembranous colitis). Ribotype 027 is especially virulent.

A very serious complication after Campylobacter enteritis is the Guillain-Barré syndrome, which is characterised by ascending paralysis caused by a demyelinating process of the spinal roots. Similarly there is Fischer's syndrome in which the cranial nerves are affected. The protein content of the cerebrospinal fluid is very high, but the fluid contains few cells. There are usually prodromata of headache, nausea, back pain and pain in the limbs. It can start very quickly with a progressively reducing strength in the legs and later in the arms. If the paralysis ascends to C3 (level of the phrenic nerve), paralysis of the diaphragm ensues. The seventh cranial nerve can also be affected. There are also sensory symptoms, but these are not prominent. Initially, treatment in an intensive care unit is necessary. The vital respiratory capacity must be monitored. Plasmapheresis and IV immunoglobulins (400 mg/kg/day x 5 days) are required. Steroids are no longer recommended. Most people make a full recovery, but this can take several months. In 10-20% of cases there are permanent neurological sequelae.

Guillain-Barré syndrome is due to an immunological process of molecular mimicry that is not fully understood. There is a connection with anti-GM 1 IgG antibodies following Campylobacter jejuni enteritis. Infection by C. jejuni carrying GM 1 -like LPS induces a high production of anti-GM 1 IgG antibodies. The autoantibodies bind GM 1 that is present on the nodal axolemma of the motor nerve and block electrophysiological conduction. After this, macrophages, guided by the anti-GM 1 IgG, enter the periaxonal space and degeneration of the motor axon follows.

In case of bacillary dysentery, examination of the faeces under the microscope shows numerous white blood cells (pus) and red blood cells. Bacillary dysentery is associated with a marked disappearance of the normal bacterial intestinal flora. It is not possible to distinguish between the different bacteria by microscopy alone (culture is needed for this). As always, fluid and electrolytes form the basis for treatment. With bacillary dysentery, antibiotics are an important part of therapy. The resistance of the various bacteria varies. Multi-resistant bacteria are becoming more common. Depending on the local conditions, cotrimoxazole, ampicillin or a quinolone (such as ofloxacin [Tarivid®]) should be used. The use of diarrhoea-inhibitors (loperamide) is not recommended.

Note: Classification of diarrhoeagenic Escherichia coli.

Diffusely adherent (DAEC)    Small intestine, watery diarrhoea Enterohaemorrhagic (EHEC)  Colon, often bloody diarrhoea. In 10% complicated by HUS. Incubation 3-9 days Enteroaggregative (EAggEC)           Small intestine. Watery mucoid diarrhoea (70%), bloody in 30%. Short incubation (8-18h) Enteroinvasive (EIEC)                    Distal ileum and colon. Watery diarrhoea, sometimes bloody. Enteropathogenic (EPEC)               Proximal small intestine. Watery diarrhoea. Incubation 6-48h Enterotoxicogenic (ETEC)     Small intestine, common. Watery diarrhoea. Incubation 14-30h. Testing for different pathogenic Escherichia coli strains is difficult. E.g. testing for enterotoxicogenic E. coli requires recovery of individual bacterial clones from an agar plate inoculated with a stool sample. This should be followed by molecular evaluation for detection of specific genes. This approach is not available in most laboratories, including most labs in the West.

Note: Shiga toxin, von Willebrand factor, TTP and HUS

In 1985, Karmali discovered a link between in haemolytic-uremic syndrome (HUS) and enteric infections with Escherichia coli that produce Shiga toxin. Such infections are very common in the tropics. HUS is mainly a disease of children. A combination of thrombocytopenia with an increased number of megakaryocytes in the bone marrow, microangiopathic haemolytic anemia with schistocytes and elevated LDH, renal failure with hypertension and fever is suggestive of HUS. The clinical distinction between thrombotic thrombocytopenic purpura (TTP, Moschcowitz's disease) and HUS is not always clear-cut, but neurological abnormalities are more common in TTP.

Shiga toxin is a 70-kD protein exotoxin encoded by Shigella dysenteriae DNA. Shiga toxins 1 and 2 (syn. Shiga-like toxin 1 and 2; also known as verotoxins) are encoded by bacteriophage DNA, which can be present in several E. coli serotypes. Verotoxins were first described in 1977. Their name refers to the cytopathogenic effect on Vero cells (in vitro cultivated monkey kidney cells). All these toxins have similar structures. They are so-called binary AB toxins. This group includes cholera, diphtheria and pertussis toxins. As their name suggests, AB toxins consist of two sub-units, A and B. The sub-unit B is a pentamer with five-fold symmetry. Each of the five sub-unit B monomers has three binding sites, which explains why Shiga toxin is so efficient. With these 15 sites it binds like Velcro to receptors on endothelial cells.  Sub-unit B of Shiga toxin binds with high affinity to a certain glycolipid (globotriaosylceramide, Gb3) present on glomerular, colonic and microvascular endothelial cells. This action stimulates glomerular endothelial cells to secrete unusually large von Willebrand factor multimers, which promote local platelet aggregation. The predominance of renal injury in HUS may be caused by differential expression of Gb3 on glomerular capillaries versus other endothelial cells. After it has been taken up into the cell, sub-unit A migrates through the cytoplasm and binds to ribosomes. Cell death follows. Antibiotics used in cases of diarrhoea might increase the risk of the haemolytic-uremic syndrome by causing the release of Shiga toxin from injured bacteria in the intestine, making the toxin more available for absorption. Oral administered porous particles which are coated with synthetic oligosaccharide receptors for verotoxin (Syncorb-PK) can be given.

HUS often resembles TTP. Von Willebrand Factor (vWF) is a heterogeneous multimeric glycoprotein which is produced by endothelial cells and megakaryocytes. The molecular weight varies from 400 kD to 20,000 kD. In platelets, vWF is stored in the alpha-granules. In endothelial cells vWF is stored in so-called Weibel-Palade bodies. The normal function of vWF is to stabilize factor VIII, as well as to be a carrier protein of factor VIII. Large vWF multimers also stabilise platelet adhesion to the subendothelial matrix in case of tissue injury. Secreted uncleaved unusually large multimers induce adhesion and aggregation of platelets in the circulation itself and have therefore considerable prothrombotic properties, especially in situations of high shear stress (arterioles and capillaries). Normally, these multimers are cleaved within seconds to a few minutes by the circulating plasma metalloprotease ADAMTS 13 (acronym for "a disintegrin-like and metalloprotease with thrombospondin type I repeats"). The function of ADAMTS 13 is therefore to cleave secreted vWF to limit the size of the multimers and to prevent platelet aggregation in the circulation. This enzyme is produced by the liver. Endothelial cells have a receptor for this enzym. When the unusually large multimers are cleaved so quickly, no thrombosis will occur. Platelets do not adhere to the smaller von Willebrand factor forms. In most types of TTP, plasma ADAMTS 13 activity is less than 5% of normal. The unusually large multimers are not cleaved on the surface of endothelial cells during more than 10 minutes in patients with TTP, leading to thrombosis. Acquired idiopathic TTP can occur when neutralising IgG auto-antibodies inhibit ADAMTS 13 activity. Multiple loss-of-function mutations in ADAMTS 13 are associated with congenital TTP. Testing for the activity of this enzyme could thus allow differentiation of HUS and TTP. Patients with idiopathic thrombocytopenic purpura (ITP), liver cirrhosis, chronic uraemia, new born babies and pregnant women tend to have low ADAMTS 13 in their plasma, but in these people, the activity is not less than 5%.

Etiology, acute bloody diarrhoea but little or no fever

The main causes are amoebic dysentery and, to a lesser extent, mild bacillary dysentery. Examination under the microscope of fresh (still warm) faeces is important in order to identify motile trophozoites. The normal bacterial intestinal flora is maintained in amoebic dysentery. In case of severe amoebic colitis there may be some fever. Amoebic dysentery is treated with medication against the trophozoites (tinidazole = Fasigyn®, metronidazole =Flagyl®) followed by medication against any remaining intestinal cysts (diloxanide furoate= Furamide®). Other, less common causes of bloody diarrhoea without fever are acute schistosomiasis (eosinophilia, worm eggs), massive trichuriasis (microscopy), ulcerative colitis (rare in the topics) and Balantidium coli (microscopy).

Ileocaecal intussusception can present with acute bloody diarrhoea followed by intestinal obstruction. The intussuscipiens forms the outer layer, the intussusceptum is the inner part. Occasionally there is intermittent haematochezia. A mass can sometimes be felt in the abdomen. The condition usually affects children between 3 and 18 months old. Rapid surgical intervention is required. Unlike children, 70-90% of adults with intussusception have an underlying condition. Malign tumours such as an adenocarcinoma of the colon or the small intestine (metastatic melanoma, lymphoma, sarcoma) have been reported. Carcinoid tumours, polyps, lipomas, neurofibromas, hyperplastic lymph follicles or Meckel's diverticulum may also be responsible. A firmly lodged foreign object (food, faecalith) or endometriosis in the intestine can be responsible for mechanical traction.

Food poisoning with Clostridium perfringens  causes necrotising enteritis. After the Second World War this became known as "darmbrand". In the dialect of Papua New Guinea the disorder is known as "pigbel". The anaerobic Gram-positive bacterium is frequently present in the flora of the colon, so there must be other factors present to cause the onset of the disease. The bacterium, better known as the causative agent of gas gangrene, can produce various toxins. The bacterial strains which produce toxins can be classified into types A, B, C, D and E. All types produce alpha-toxin, which is a lecithinase (phospholipase C). Clostridium perfringens type C, responsible for pigbel, produces alpha- and beta-toxins. The alpha-toxin is coded by a chromosomal gene ( cpa ), the beta-toxin is coded by a gene on a plasmid. These can be detected by PCR. Beta-toxins can be vaccinated against, considerably reducing the risk of disease. The toxins in the intestine are usually destroyed by proteases. In case of undernourishment there is an important deficiency in proteases such as trypsin, and as a result the toxins can remain active. If there are trypsin inhibitors present as well, such as are found in sweet potatoes, the remaining small amount of trypsin is neutralised. Adult Ascaris worms produce trypsin inhibitors. If the intestine has reduced motility, the toxin remains in contact with the wall for a prolonged period of time and cause transmural necrosis. It is often necessary to carry out a partial resection of the small intestine. The lesions tend to be more prominent in the jejunum, although lesions of the ileum also occur. Pigbel has been recognised in Papua New Guinea since 1961. The disorder is seen mainly in undernourished and parasite-infested children after eating a rich meal with sweet potatoes and infected pigmeat (pig intestines are also eaten). Meals such as this are sometimes prepared on the occasion of a great feast at which the host expresses his social standing by slaughtering and serving a large number of pigs. The illness can therefore occur in epidemics. Besides supportive therapy, treatment is based on antibiotics (chloramphenicol, benzylpenicillin or other, broad-spectrum antibiotics), type C antiserum and mebendazole. Sometimes surgery has be be performed. Vaccination against type C toxin is useful.

Etiology, acute non-bloody diarrhoea with fever

In children any infection of any type can be associated with diarrhoea, e.g. otitis media, tonsillitis, pneumonia, urinary infection, etc. The main pathogens are viruses, some Escherichia coli  and mild forms of bacillary dysentery ( Salmonella , Shigella , Campylobacter and Yersinia ). The possibility of malaria and typhoid fever must be considered. Acute diarrhoea with fever but without bloody stools, generally requires no antibiotics. The emphasis is on administering fluids and electrolytes. In small children a bacterial infection of the intestine can rapidly give rise to septicaemia. Antibiotics are therefore indicated in these children (<1 year).

Etiology, acute non-bloody diarrhoea with little or no fever

If the diarrhoea is very watery, the possibility of cholera must be considered. In these cases rehydration is very important. Antibiotics (such as tetracyclines) also help, but to a lesser extent. Cholera must be notified in view of the possibility of epidemics.

Food poisoning by bacterial toxins (including staphylococci) results in explosive diarrhoea shortly after a meal. The bacteria reproduce in food. These bacteria produce a thermostable toxin. The bacteria are usually killed when food is cooked or left over food is reheated. The toxin is not destroyed by the heat and enters the intestine, where it causes massive diarrhoea, probably by neurotoxic action on the autonomous nervous system. Antibiotics are therefore of no value here. Symptomatic treatment is indicated. Toxins produced by Bacillus cereus (often present in contaminated rice) can produce a similar picture or the "emetic syndrome". Some milder infections, such as traveller's diarrhoea, produce hardly any fever. In these cases bowel motion inhibitors (loperamide) can be given. Racecadotril (acetorphan) is an enkephalinase inhibitor with an antisecretory and antidiarrhoeic activity. The product reduces intestinal hypersecretion but not intestinal motility. It prevents the breakdown of endogenous enkephalins in the gastro-intestinal tract. These enkephalins are neurotransmitters in the intestinal wall. They are active on the d-opiate receptors and reduce the intracellular c-AMP concentration. Loperamide apparently acts only on the µ-opiate receptors.

Note: diarrhoea and neurological symptoms

In cases of diarrhoea and neurological complaints, with or without other non-systemic symptoms, one has to consider:

Clostridium botulinum (botulism) Scombroid, ciguatera or paralytic shellfish poisoning Sodium glutamate poisoning Guillain-Barré syndrome Fungal poisoning or contamination of the food with mycotoxins Pesticides Listeriosis Brucellosis Severe Trichinella spiralis infection Complication of AIDS

Etiology, chronic non-bloody diarrhoea with fever

Chronic diarrhoea, emaciation and persistent fever are important criteria for the clinical diagnosis of AIDS. Other clinical signs should be searched for, such as oral candidiasis, Kaposi's sarcoma lesions, chronic pruritus, severe or repetitive shingles. Serology can confirm the diagnosis. Intestinal parasites must be searched for.

Tuberculosis of the intestine is predominantly sited at the ileocaecal transition. A mass can sometimes be felt there on palpation. There is sometimes ascites due to concomitant involvement of the peritoneum. Pulmonary lesions can be present, but these are certainly not a requirement for the diagnosis of intestinal TB. It is difficult to differentiate intestinal tuberculosis from Crohn's disease because of similar clinical, pathological, radiological, and endoscopic findings. Histological interpretation of biopsies is of limited diagnostic value in the differentiation of intestinal tuberculosis from Crohn's disease, except when caseating granulomata are found. Mycobacterial culture (isolation of Mycobacterium tuberculosis ) and PCR are helpful in making the distinction between intestinal tuberculosis and Crohn's disease.

Etiology, chronic non-bloody diarrhoea without fever

There are a number of causes. Giardiasis is common. Other intestinal parasites (such as Cyclospora ) can cause a similar picture. At present there is still no effective treatment for cryptosporidiosis. Pellagra (niacin deficiency) is characterised by the DDD triad: d iarrhoea, d ermatitis, d ementia. This triad is not seen in all patients. Treatment with niacin brings about a rapid improvement. Overactivity of the thyroid gland ( hyperthyroidism ) can be accompanied by persistent diarrhoea. There is usually also tachycardia and tremor. Congenital lactase deficiency is common in Africa and in Asia in people who traditionally do not breed cattle and where milk is not included in the staple diet. It causes abdominal pain and diarrhoea in people who consume products containing lactose, such as milk. Because lactose breath tests and intestinal biopsies for determining enzymes cannot be carried out in most developing countries, a challenge test with milk can be performed instead. If the patient has no discomfort after drinking a large glass of milk (500 ml), there is no clinically relevant lactase deficiency. Coeliac disease is difficult to diagnose without proper medical infrastructure. Children and adults with any of the following symptoms or signs should be tested in a correct way: prolonged fatigue, chronic or intermittent diarrhea, failure to thrive or faltering growth in children, unexplained weight loss, aphthous stomatitis, persistent and unexplained abdominal symptoms including nausea, vomiting, recurrent abdominal pain, cramping or distension, unexplained constipation, suspected irritable bowel syndrome, unexplained iron deficiency. The presence of another auto-immune disease such as thyroid disease, dermatitis herpetiformis, type I diabetes, Addison's disease, auto-immune hepatitis or auto-immune myocarditis should heighten the suspicion. Other trigger factors for testing include a plethora of conditions, such as amenorrhea or unexplained subfertility, dental enamel defects, low trauma fracture, metabolic bone disease (rickets, osteomalacia), polyneuropathy, sarcoidosis, Sjögren syndrome. The testing for coeliac disease is accurate only if the person is eating a diet than contains gluten at the time of testing (best gluten in meal x 6 weeks prior to testing). When testing, one should use IgA tissue transglutaminase antibody as first choice test. Use IgA anti-endomysial antibody if IgA transglutaminase antibody result is equivocal. If serology is negative, rule out IgA deficiency. If there is IgA deficiency, one can order IgG tissue transglutaminase antibody. Intestinal biopsy can confirm or rule out diagnosis.

Campylobacter   infections and some strains of Esch. coli occasionally cause persistent diarrhoea. After acute infectious diarrhoea a patient can still suffer from a less serious form of diarrhoea for several weeks or months. In these circumstances there may be a secondary lactase deficiency. Malnutrition and a poor general condition in children prolong the symptoms.

Etiology, chronic bloody diarrhoea without fever

One has to consider persistent amoebic dysentery, severe schistosomiasis ( S. mansoni, S. japonicum ), inflammatory intestinal diseases, intestinal tumour and repeated intestinal invagination. Be aware of diarrhoea due to other causes together with bleeding haemorrhoids.

Etiology, chronic fatty diarrhoea

Causes of steatorrhoea include abnormalities of the small intestine and insufficiency of the exocrine pancreas. Calcification of the pancreas in chronic pancreatitis can be seen in 50% of cases (X-ray of the abdomen). Concomitant diabetes mellitus should be searched for. Non-infectious causes of intestinal abnormalities such as coeliac disease (hypersensitivity to gluten) and intestinal lymphoma are rare. Coeliac disease is associated with antibodies against gliadin (a component of gluten) and autoantibodies against tissue transglutaminase (oand/or anti-endomysium antibodies). People with this disorder are often HLA-DQ2-positive. Tropical sprue is a disease of unknown origin, common in Asia but less so in Africa. The disease responds to treatment with tetracyclines and folic acid.

Some infections may result in malabsorption:

Giardia lamblia : microscopy of the faeces. These are often asymptomatic infections, so their importance should not be overestimated. Giardia can also give rise to secondary lactose-malabsorption: dairy products can no longer be tolerated. Capillaria philippinensis : occurs mainly in the Far East but is rare. Infection is caused by eating raw fresh water fish. Like Strongyloides , this worm also leads to endogenous reinfection. It can therefore reproduce in the body, unlike most other worms. The eggs and larvae can be found in the stools (repeated analyses are necessary). Treatment of intestinal capillariasis is with mebendazole (Vermox®). It is a potentially fatal infection. Strongyloides stercoralis : Serious infections cause diarrhoea, eosinophilia, pruritus and larva currens. The stools contain seldom eggs but larvae are present. Cryptosporidia can cause malabsorption. The possibility of AIDS must be excluded in chronic cases. The parasite can be demonstrated using Ziehl stain. Cyclospora can be compared with "large cryptosporidia" with variable acid-fastness on Ziehl stain. Treatment with cotrimoxazole is effective.

Assessment of a patient with diarrhea

Medical history

 How long has the patient been suffering from diarrhea? Is it acute (<14d) or chronic (>14d)?  Is there blood or pus in the faeces (microscopy if necessary), or is it watery diarrhea ?  Is there fever?  Is there tenesmus? Suggests that the rectum has been affected by inflammation or ulceration.  Is there abdominal pain? Not with cholera.  Is the patient vomiting? Makes dehydration worse and makes therapy more difficult.  Are there a number of people in the area with the same symptoms? An epidemic? Acute diarrhea is often caused by self-limiting infections (beware exceptions). Chronic diarrhea is more often than not caused by non-infectious causes (beware exceptions, especially in immunompromised patients). Two intestinal helminths which as a rule persist (probably for life) even in untreated immunocompenent persons are Strongyloides stercoralis and Capillaria phillipinesis . Chronic diarrhea can be further classified by volume, where small frequent stools are suggestive of a distal colonic disorder. Large volume watery stools are suggestive for conditions involving the small intestine (but beware of a secreting villous colonic adenoma). Steatorrhea or fat-malabsorption suggests problems located in pancreas, bile ducts and/or small bowel. The presence of fecal leukocytes has a sensitivity of 70% for inflammatory diarrhea. A test for fecal lactoferrin has a higher sensitivity, but is rarely available. Continuation of diarrhea during fasting is suggestive for a secretory process. Normal osmolality of stools is about 290 mOsm. The calculated stool osmotic gap can be calculated as 290 - 2(stool sodium level + stool potassium level). A finding of less than 50 mOsm is consistent with secretory diarrhea, while a gap > 100 mOsm points to osmotic diarrhea (e.g. lactoluse laxative, lactose-intolerance). Common causes of chronic diarrhea include irritable bowel syndrome (painful, more in women than men, worse shen stressed), inflammatory bowel disease (painful), malabsorption syndromes and chronic infections in immunocompromised patients. Long term laxative abusus with products containing anthraquinones leads to melanosis coli, which can be detected during colonoscopy. People who ingest phenolphtaleine for its laxative properties will have a typical color-changing urine some drops of acid/base are added. Features that suggest an organic cause as opposed to a functional cause, include a duration less than 3 months, nocturnal diarrhea, abrupt onset, weight loss (> 5kg for an adult), stool weight more than 400 g/24h. Apart from colon carcinoma, amoeboma and classic amoebic colitis, conditions such as microscopic colitis, collagenous collitis, celiac disease, chronic intestinal capillariasis (low egg load, difficult diagnosis), strongyloidosis (often very few larvae), Whipple, intestinal lymphoma and tuberculosis, hyperthyroidism, carcinoid syndrome, mastocytosis, medullary carcinoma of the thyroid (C cells produce calcitonine) or even neuro-endocrine tumors (gastrinoma, vipoma) might be considered at some point. Don't be too hopeful to catch rare pathology, e.g. vipoma has an incidence of 1/10,000,000/year.

Physical examination (degree of dehydration)

The assessment of dehydration is most important. Dehydration is due to an insufficient intake of liquids (drinking, IV fluid) and/or excessive loss of fluid (vomiting, diarrhoea, polyuria, sweating). If loss of gastro-intestinal fluid is the cause, the patient will urinate less (oliguria) in order to minimise the loss.

If a child has lost < 5% of its body weight, the general condition is still quite good. The child is alert and thirsty. The mucous membranes (eyes, tongue, mouth) are moist and the turgor of the skin (elasticity) is maintained. Breathing is normal. Urine production is normal and if the child cries there are tears. The fluid deficit is < 50 ml/kg of body weight.

If 5-10% of body weight is lost the eyes are sunken, the fontanelle is hollow, the skin is no longer elastic, the lips and mouth are dry and sometimes cracked. The child is miserable, restless and cries. There are no tears. Breathing becomes more rapid (acidosis). This must be distinguished from an accompanying pulmonary infection. Urine production decreases. The fluid deficit is 50-100 ml/kg.

With a fluid loss of >10% the child is still and cold. The pulse is rapid and difficult to feel (circulatory collapse), especially the radial pulse. Skin folds do not disappear, the mucous membranes are very dry, the abdomen is hollow, the eyes are deeply set and the fontanelle is deeply sunken. Usually there is no more urine. The fluid deficit is >100 ml/kg.   Evaluation dehydration for children up to 36 months   .A rapid clinical dehydration evaluation can make use of the following items: general appearance, skin, eyes, tongue and tears. A more detailed evaluation can determine the following items: General appearance : Normal    Thirsty-restless-irritable      Drowsy-limp Capillary refill                  <1.5"                1.5-3"                        >3" Skin turgor                 instant recoil       <2 seconds                 > 2 seconds Fontanelle                 Normal                 Slightly sunken           Very sunken Eyes                          Normal                 Slightly sunken           Very sunken Tongue                      Moist                    Sticky                         Dry Tears                         Present                Decreased                  Dry Breathing (<1y)         <40/'                     40-50/'                         > 50/' Breathing (1-3y)        <30/'                     30-40/'                         > 40/' Heart rate (<6m)        <175/'                  175-185/'                     > 185/' Heart rate (6-36m)     <150/'                  150-165/'                     > 165/' Urine specific gravity < 1.015                 1.016-1.030                > 1.031

Treatment

Treatment, general

Two things must always be considered:(1) the degree of dehydration, (2) is drug treatment necessary? The most important thing with acute diarrhoea is to deal with dehydration and in the second place to correct protein and calory deficiency. Etiological treatment will only be possible in a minority of cases, but should not be disregarded.

Children are very sensitive to dehydration. Fluid loss can occur very quickly with vomiting and diarrhoea: 500 ml of fluid in a child weighing 5 kg means a loss of 10% of body weight and implies a high risk of death.

IV rehydration is not always possible nor even desirable. An important development has been the discovery that many cases of dehydration of whatever origin can be counteracted by oral rehydration. This is possible because despite the diarrhoea, the mechanisms for absorbing water, sodium and glucose in the intestine are maintained. The minimum ingredients for this oral rehydration solution ( ORS ) are clean water, glucose and salt. While this can indeed bring about rehydration or prevent dehydration, a disadvantage is that the diarrhoea itself continues. The volume of stools is not reduced. Alternatives to glucose are ordinary sugar (sucrose; this is a glucose-fructose disaccharide) or rice powder. Rice powder is better because it reduces the volume of stools. In ideal circumstances potassium (against hypokalaemia) and bicarbonate or sodium citrate (against acidosis) can be added. Citrate is easier to store than bicarbonate. In the future there may perhaps be better formulae which also contain neutral amino-acids (glycine and alanine) and perhaps dipeptides.

There are several formulae for ORS. The WHO has developed a standard formula in which each litre of water should contain:

KCl      1.5 gram NaHCO 3 2.5 gram or 2.9 gram sodium citrate NaCl 3.5 gram Glucose 20 gram or 50 gram of rice powder

Under field conditions the following guidelines can be used :

1 teaspoon of salt + 6 teaspoons of sugar + 1 litre of boiled water.

Treatment, in practice

Always weigh the child and assess its general condition.

Assess whether the weight loss is <5%, 5-10% or >10%.

Is it dysentery or not? If yes, is it amoebic or bacillary?

With mild to moderate dehydration use ORS. The volume that should be given is 1-2 times the fluid deficit. ORS is best given by the mother and should be given over a 4 to 6 hour period. It is best if it is given with a small cup and spoon. With very small children a syringe can be used to drip the fluid into the mouth. If the child vomits a few times the treatment should be continued nevertheless. Administration using a nasogastric drip infusion is rarely necessary. The success of the treatment should be monitored by assessing the general condition of the child and its weight. With severe dehydration (>10%) or if the treatment with ORS is not successful, IV rehydration should be used. If it is not possible to inject into a vein and a venous cut-down is not feasible and the situation is desperate, the intraosseous route can be used: the fluid enters the bone marrow of the tibia and is taken up in this way. The infusion can be rapid at first (70 to 100 ml/kg over 3 hours). If the pulse can be felt clearly again and the child has generally improved, the treatment can then be switched to oral therapy. Newborn children with a low birth weight are very sensitive to hypernatremia. Rehydration is achieved best with 2/3 ORS and 1/3 extra salt-free water. Food must continue to be given while the patient has diarrhoea. It used to be thought that a period of fasting (24 to 48 hours) was good for the child, but this is counterproductive. Breastfeeding should not be stopped. A balanced diet, best low in residue and semi-solid is indicated. A good diet is also important after the diarrhoea. During episodes of diarrhoea, patients are catabolic (they break down their own muscle proteins for energy).

Treatment, medication

Antibiotics for bacillary dysentery. However, antibiotics increases the risk of severe haemolytic-uremic syndrome (release of Shiga-toxins from lysing bacteria). The danger is real but seems limited in day-to-day clinical practise, justifying the use of antibiotics in this condition. Ofloxacine is not metabolised via the CYP450 system and has a halflife of 6 hours, making once daily dosing possible (as opposed to ciproxin with a halflife of 4 hours). Another commonly used antibiotic is azithromycin, which is also not metabolised via the CYP450 system, and has a intracellular halflife of 2-4 days. Antiparasitic agents for amoebiasis, giardiasis, malaria, isosporiasis, Strongyloides, capillariasis, etc. Zinc and vitamin A supplements Antimotility products reduce intestinal cramps and the frequency of bowel movements. They do not reduce fluid loss. They can aggravate dysentery and can easily be given to children in too high a dose, resulting in paralytic ileus. They are only indicated for uncomplicated diarrhoea. Loperamide (Imodium®) or opiates: codeine, laudanum or paregoric (= opium tincture). Anticholinergic medications. Adsorbents such as kaolin, pectin and charcoal produce better formed stools, without reducing fluid loss. Sometimes the main complaints is nausea. Domperidone can be used. Although the name of this molecule sounds like it should be the active ingredient in Dom Perignon champagne, but it's actually an anti-emetic drug. Lactobacillus concentrates are probably of little benefit, but more research is needed. The use of bacteriophages e.g. against Escherichia coli 0157:H7 is experimental but gave promising results in sheep. Enzym supplements in cystic fibrosis and exocrine pancreatic insufficiency. Experimental : sigma receptor agonists (e.g. igmesine). Encephalinase inhibitors (e.g. racedodril).

Prevention

Most diarrhoea is transmitted by the faeco-oral route. The prevention of these infections will therefore depend on improved general hygiene, which is determined by the general level of poverty (standard of living). Rotavirus disease kills approximately half a million children annually in developing countries and accounts for one third of hospitalizations for diarrhea worldwide. In 1999, the first licensed rotavirus vaccine (RotaShield) was withdrawn from the U.S. market less than a year after its introduction because it was associated with an uncommon but potentially life-threatening adverse event, intussusception, at an estimated rate of 1 incident per 10,000 vaccine recipients. The manufacture of the first licensed rotavirus vaccine was halted. In 2005, results were published of large clinical trials of two new vaccines, Rotateq from Merck and Rotarix from GlaxoSmithKline, were published. These are both live oral vaccines intended to be given to infants at the same time as their immunizations for diphtheria, pertussis, and tetanus, but they differ in their approaches, strains, and formulations. Rotarix is given in doses with minimum 4 weeks interval. Rotateq is given in 3 doses with minimum 4 weeks interval. Rotarix is a monovalent vaccine derived from the most common human rotavirus strain, G1P[8], that has been attenuated by serial passage and is administered in two oral doses one to two months apart. The vaccine strain replicates well in the gut, is shed by more than 50 percent of patients receiving the vaccine after the first dose, and like natural rotavirus infections) provides cross-protection against most other serotypes. Rotateq is a pentavalent vaccine based on a bovine strain, WC3, that contains five human–bovine reassortant viruses. WC3 is naturally attenuated for humans but is not broadly cross-protective, so each reassortant virus contains a single gene encoding a major outer capsid protein from the most common human serotypes. The bovine virus grows less well in the human intestine, so the aggregate titer required to immunize a child is greater. In addition, the vaccine strains are infrequently shed in the stool, and three oral doses are required, with at least a month between doses. Both vaccines demonstrated an impressive efficacy profile and a reassuring safety profile, particularly with respect to intussusception.

A few general tips and precautionary measures for avoiding diarrhea are recommended: Food should be completely cooked/boiled. Drinking water should be protected. This can be achieved in a village context (sand filters, protection of water-wells, etc). Water can be boiled and filtered, but boiling requires a lot of fuel, which is usually expensive. In some settings, if tap water cannot be relied upon, electric water condenser units can be useful. These machines take in ambient air, cool it in order to condense the atmospheric water, filter it, irradiate it with an UV-lamp and finally store it for consumption. One normal unit looks like a small refrigerator and fits easily in a corner of a room. It supplies enough water for the daily needs of an average family. Cfr waterex, see example at http://www.waterexsystem.com/   Wash hands with soap. Sanitary provisions: toilet and drinking water should be kept separate. Inexpensive, simple, build-it-your self, ventilated, odour-free, fly-free latrines that do not require any water can be made (the Blair latrine for example).

Diarrhoea: prevention for travellers

Food : avoid raw vegetables, fruit you cannot peel yourself, unpasteurised dairy products, fish, shellfish and meat that is raw or not cooked through. (Cook it, peel it or leave it). Avoid food from street stalls. Food should be protected against flies.

Drink : drink tea, coffee or bottled water, preferably sparkling (less risk of having been tampered with). Beer can quench the thirst, but large quantities of alcoholic drinks are not recommended. Avoid bottles sealed with reused crown caps. Ice cubes are not to be trusted. Drinking water can be filtered. This can be done in a number of ways (large porcelain filters such as Berkefeld, active charcoal filters, portable Katadyne filters). Afterwards the water can be boiled or purified chemically with silver salts such as Micropur®, Drinkwell® (not active against viruses), Chloramine (250 mg per 10-50 litres), sodium hypochlorite (Javel, Drinkwell chlorine®, Hadex®). An unpleasant taste of chlorine can be removed by adding the non-toxic sodium thiosulphate (Drinkwell-antichlorine® drops) work in for an hour. Lugol or 2% tincture of iodine (eight drops per litre) can also be used and is more active against amoebic cysts. Long-term use (more than 3 months) is not recommended. Thyroid disorders and pregnancy are contra-indications.

Chemoprophylaxis : This is normally not advised routinely, but does provide partial protection (e.g. ofloxacin). Only to be considered for short journeys where absolutely nothing should go wrong.

Note: flies Several species of flies live in close proximity to people and often eat organic waste and faeces as well as food intended for people. They lay eggs -often hundreds- on their food. When an adult fly feeds on infected material (on faeces for example), pathogens are taken in through the mouth and into the intestine. Pathogens can stick to the pads of the feet and on hairs of the legs and body. Later the insect can land on unprotected food. When walking over the food, the micro-organisms are deposited. Like all Diptera, flies eat liquid food. The fly vomits a little fluid to make the food liquid. This vomit can contain microbes from a previous meal. At the same time the fly defaecates the leftovers from a previous (infected) meal. Many pathogens causing enteritis can be transmitted in this way. Flies can also transmit trachoma, an infectious disease of the eye ( Chlamydia trachomatis ), which can cause blindness.

Exercises

Sudan . A mother asks you how she should prepare a drink that she can give her 2-year-old daughter in the case of diarrhoea. What is your answer? Mauritania . The supply of ORS bags from the WHO has run out. Someone asks you how they can make these themselves in the hospital. What is your answer? India . A medical assistant in a dispensary asks you what she should do if someone comes with diarrhoea. How would you reply? Zambia . A child weighing 25 kg had watery diarrhoea for 5 days. There is no fever, the child is drowsy and feels cold. You cannot feel a pulse. The eyes are sunken and folds in the skin remain for 10 seconds. What do you do? Gambia . A 50-year-old man has since 3 days a temperature of 39°C, and mentions sudden abdominal cramps and diarrhoea (14 stools a day, 4 at night). He appears toxic (seriously ill). Urine = 150 ml/24 hours. Microscopy of the faeces shows WBC +++ and RBC +++, no trophozoites. What is the probable diagnosis and therapy? Borneo . An adult has had a temperature of 38.5°C for 10 days. The stools contain mucus with some blood. His general condition is moderately good. Bacterial culture of the stools: Vibrio cholerae. What do you think and what do you do? A European tourist in Kenya. After 10 days suddenly he has a fever of up to 40°C, watery diarrhoea, general malaise, headache and muscular pain. What do you think and what do you do? Northern Kenya. A local man has had fever for 2 months, emaciation, asthenia. Three weeks ago he had repeated epistaxis. For 4 days his general condition has deteriorated with frequent diarrhoea containing bloody mucus. Physical examination reveals: cachexia, enlarged spleen and liver. What do you think and what do you do? In Brazzaville, Congo, you see a 24-year-old woman with chronic pruritus, persistent fever and emaciation. For 2 months she has had diarrhoea and difficulty swallowing. The stools do not contain any blood. Physical examination reveals cachexia and an area of depigmentation on the left thorax. What do you think? Guinea-Bissau . A child has had persistent diarrhoea for 2 months. There is no fever or other symptoms. What do you think? West Irian (New Guinea). At a village feast large quantities of beer, chicken, sweet potatoes, sago cakes and pigmeat were prepared. Two days later 100 people became ill, mainly children. They complain of sudden bloody diarrhoea/melena, fever, abdominal cramps, bloated abdomen and appear toxic. What could it be? Zambia . The local village idiot has chronic diarrhoea. He has dark coarse skin in the face and the arms with rash in the neck, arms and legs. He is very emaciated and there are no signs of AIDS (chronic candidosis, repeated shingles, Kaposi's sarcoma lesions, prolonged fever). Draw (to scale) a Giardia lamblia trophozoite, an Entamoeba histolytica trophozoite and cyst, a neutrophil. Ivory Coast . A man in stupor is sweating a lot and has severe diarrhoea, t° = 39°C. The central venous pressure is 0 cm, BP = 7/-. There are no abdominal masses. For 2 days urine production has been just 50 ml per day. His older neighbour in the bed to the left of him was admitted with subacute abdominal pain and also has urine production of just 75 ml per day. On palpation of this man's abdomen a mass can be felt suprapubically. His neighbour to the right is a boy of 8 years who was admitted with aching joints and fever. At present his urine production is just 80 ml per day. The urine contains protein ++++, red blood cells and many casts. Which of the patients is most likely to have pre-renal, renal and post-renal renal insufficiency? Can you think of a specific cause?

Summary

AIDS: Pandemic of progressive immunodeficiency AIDS is caused by virus infection (HIV-1 and HIV-2) Transmission principally via blood, contaminated needles, heterosexual and homosexual sex and mother-to-child Often co-infection with tuberculosis Some treatable opportunistic infections (cost price): toxoplasmosis, PCP, candidiasis Significance of prevention: no curative treatment Importance of perinatal medication to reduce mother-to-child transmission with AZT, nevirapine or HAART Importance of explanation, use of condoms, blood bank screening, STD control, male circumcision HAART principle: combination therapy of different antiretrovirals Generic cheaper antiretrovirals are now available Aim of HAART: total viral suppression, avoidance of mutant selection, increase CD4 count Rapid changing information: frequent updating essential

For extra info, see HIV manual (written 2006). Click on the book icon at the left side. It can also be consulted via www.HIVMedicine.com

Introduction

Introduction, general

In 1981 a new disease was described in the USA: AIDS ( A cquired I mmuno D eficiency S yndrome). Previously healthy male homosexuals became critically ill. They got diarrhoea, became emaciated and languished. Their lungs became infected with fungi that healthy people quickly overcome. Some of them also got Kaposi's sarcoma. In 1983 the causative retrovirus, HIV-1 (human immunodeficiency virus 1), was discovered by two French virologists, Francoise Barre-Sinoussi and Luc Montagnier (shared Nobel prize 2008). This was subsequently confirmed by the American investigator Robert Gallo. A blood test for detecting the virus was put on the market in 1985. A second, related virus, HIV-2, was discovered somewhat later. This latter virus also causes AIDS and occurs more in West Africa. Infections with HIV-2 develop into AIDS more slowly than infections with HIV-1

Introduction, origin and subtypes

HIV-1 cladogram. Copyright ITM

A whole series of HIV subtypes has been identified. HIV-1 is at present subdivided into 3 genetically different groups, each of which can be further divided into various subtypes that more or less have their own geographical distribution. The principal group is the M- (“major”) group with subtypes a, b, c, d, e, f, g and h. The second group, the O- (“outlier”) group, occurs principally in Cameroon. A variant (YBF 30), which represents a new group (N-group) that is more closely related to SIV (simian immunodeficiency virus) was discovered in Cameroon in 1998. This last HIV-variant was still not being detected even in 1999 by the standard AIDS tests used in hospitals. Data from old samples are important for a better understanding of the evolution of the virus and possibly for discovering the ancestral virus. In 1998 fragments of the HIV-1 genome were discovered in an old (1959) plasma sample taken from a patient from what was then Leopoldville (now Kinshasa).  Amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo was achieved. This information was used to conduct comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1. The diversification of all M types appears to have taken place in less than 50 years. A closely related virus, SIV, occurs in chimpanzees. HIV-1 probably originates from SIV that occurs in a certain subspecies of chimpanzees ( Pan troglodytes troglodytes ). Interspecies transfer of these viruses probably occurred historically on at least 3 different occasions. Apes and monkeys are hunted and eaten in many countries and there is a strong possibility of humans coming into contact with simian blood. HIV-2 is closely related to a virus that is found in another simian species ( Cercocebus torquatus atys or "sooty mangabey"). Another retrovirus, HTLV-1, possibly has its origin in mandrills ( Mandrillus sp.). HIV-2 is closely related to the sooty mangabey SIV (SIVsm) and its genetic material has 40-60% homology with HIV-1. In all known instances of infection of the natural primate host of SIV, neither a disease resembling AIDS nor a profound depletion of CD4 T-cells develops, despite the presence of very high viral loads in these animals. In contrast, transmission of SIV to unnatural hosts, such as rhesus monkeys ( Macacca mulatta ), causes progressive loss of CD4 T-cells and a high degree of susceptibility to opportunistic infections.

Introduction, impact

The very high case-fatality rate, the impact on health and society and the absence of any curative treatment or vaccine, make the HIV epidemic one of the biggest health problems at the end of the 20th Century and the beginning of the 21st. The chronic nature of the epidemic and the multiple accompanying life-threatening diseases result in enormous financial costs for patients and society. The patients occupy a large number of hospital beds for a long time and often require expensive palliative care, making heavy demands on already small health budgets. This money could otherwise have been used for the prevention or treatment of curable diseases. The emphasis will come to lie increasingly on home care insofar as possible. AIDS is a very complex problem of enormous dimensions. In addition to the purely medical aspect of the disease itself, there are the equally important social aspects of discrimination, ostracism, risk of transmission, orphaned children and financial problems, as well as burdensome psychological problems such as anxiety, feelings of guilt, anger and revenge, depression and attempted suicide.

Introduction, evolution of the epidemic

Projected population pyramid in 2020 with and without the HIV-AIDS epidemic, Botswana. This illustrates the massive impact of the epidemic. However, it is expected that the data will change if HAART becomes widely available in Africa. Copyright ITM

As is the case with other infectious organisms, transmission of an organism need not automatically result in an epidemic. An occasional infection with HIV somewhere in an African village may well have been followed by infection of the sexual partner(s), resulting in their death, but without further transmission. However, ideal conditions for an epidemic were created when the demographic and social conditions were altered (mass migration from rural to urban areas, migration for work, break-up of the traditional family, increased sexual promiscuity and prostitution, including the homosexual revolution in the West and contamination of blood reserves). An explosive increase in the number of cases in homosexual men, in intravenous drug users and in haemophiliac patients was initially seen in the USA and Europe. Heterosexual transmission later became increasingly significant in the West. The virus has spread into the general population. Heterosexual transmission was at first the principal route of transmission in Africa. It is an epidemic fundamentally determined by human behaviour. In Europe, before treatment with antiviral cocktails became available, the average time elapsing between infection and the appearance of AIDS in a patient was 10 years. After 10 years 50% of the infected individuals had developed AIDS. In Europe the case-fatality rate of AIDS was approximately 100% within 4 to 5 years following the diagnosis of AIDS (data from before the era of antiviral agents). In developing countries the progression of the disease is comparable with that in Europe, though patients in these countries will die more quickly after the appearance of symptoms than those in Europe due to the lack of treatment. In addition to the enormous human toll the epidemic also takes an economic toll in terms of the direct cost of medical care, the loss of productive working years from the patient and the costs of prevention.

Due to the success of the combined therapies the number of AIDS-related deaths in the USA fell by 44% between 1996 and 1997. At the same time the problem continued to spread unabatedly in Third World countries. If nothing fundamentally changes, the life expectancy in a number of countries will decrease further. Hence by 2010 the average lifespan in Zambia is expected to decrease from 66 to 33 years, in Zimbabwe from 70 to 40 years, in Kenya from 68 to 40 years and in Uganda from 59 to 31 years (extrapolation if HAART would not become available in the tropics). In mid-1999 AIDS was already the fourth most important cause of death in the world, after ischaemic heart disease, cerebrovascular diseases and lower airway infections. In Africa it is the principal cause of death. In this respect the disease has overtaken malaria and tuberculosis. The epidemic results in more or less selective infection of the 15 to 45-year-old population group (reproductive years) and young children. The problem of abandoned orphans has assumed dramatic proportions in certain areas. The total number of AIDS orphans in the world at the end of 1999 was estimated to be 13,200,000. The 30% decrease in mortality expected from the institution of the EPI Programme (a World Health Organization vaccination programme) will be annulled by the increase in neonatal AIDS. In some countries such as Zambia, Zimbabwe and Botswana the classic form of the population pyramid has been altered drastically.

Spread of AIDS

In the year 2000 AIDS was reported in practically every country. Approximately two thirds of all cases occurred in sub-Saharan Africa. In 2001 the cumulative mortality resulting from AIDS was estimated to be 21,800,000. Revision of the numbers is an ongoing process. The revisions are mainly due to improved methodology, surveilance and changes in key epidemiological assumptions. These figures may be understood better by noting that approximately 6800 new infections occur each day, of which 96% are in low and middle income countries. In Africa the ratio of infected men / women = 1. Over the whole world somewhat more men than women are infected. Approximately 10% of the total number of seropositive persons are children under 15 years of age. For update on the epidemiology, see http://www.unaids.org/en/HIV_data/  

Global summary of the HIV/AIDS epidemic, December 2008

Number of people living with HIV in 2008   Total 33.4 million    (31.1 - 35.8)   Adults 31.3 million    (29.2 - 33.7)   Women 15.7 million    (14.2 - 17.2)   Children under 15 years   2.1 million    (1.2 -   2.9) People newly infected with HIV in 2008   Total   2.7 million    (2.4 -   3.0)   Adults   2.3 million    (2.0 -   2.5)  Children under 15 years   0.43 million  (0.24 - 0.61) AIDS-related deaths in 2008   Total   2.0 million    (1.7  -  2.4)   Adults   1.7 million    (1.4  -  2.1)   Children under 15 years   0.28 million  (0.15 - 0.41)

Transmission

Transmission, general

HIV can be isolated from blood, semen, vaginal secretions, bone marrow, saliva, cerebrospinal fluid, tears, urine, amniotic fluid and breast milk. Transmission has been shown to take place only for blood, sexual secretions and breast milk. The low virus concentrations and the low isolation frequency from other body fluids may explain the absence of other transmission routes. In the year 2000, 90% of HIV transmission took place via heterosexual contact. There are no indications of the existence of other routes of transmission (such as food, water, blood-sucking insects, normal skin contact). The rate of HIV transmission through homosexual contact between men, intravenous drug use and haemophilia is much less significant in Africa than in Europe or the USA.

Transmission, sexual transmission

Transmission of the virus takes place through sexual (homosexual and heterosexual) intercourse. The risk of transfer per contact is approximately 0.3%, but is influenced by many factors. The presence of sexually transmitted diseases (STD) accompanied by ulcerations (syphilis, chancroid, herpes) increases the risk 5-fold. Hence the importance of treatment of other STDs in AIDS programmes. Transmission via oral sex has also been reported. Certain small groups ("core groups") such as prostitutes, truck drivers and military personnel, who are often away from home for long periods, have a disproportionate importance. These account for a substantial percentage of virus transfer. Having multiple sexual partners is a risk factor. This applies to several partners in the same period, as well as a number of consecutive partners spread over years. Seropositivity in prostitutes is very high in many countries. All too frequently people who know that they are HIV positive do not want to inform their partner.

Transmission, contamination of medical material

Preparation for endoscopy in Kinshasa. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Injections and medical procedures with non-sterile instruments (needles [including acupuncture needles], syringes, scalpels, endoscopes, dentistry material, etc.) are a second risk factor. The advice "do not recap needles" should be followed. Tattooing with contaminated needles as well as skin piercing can likewise be responsible for viral transmission. There is a ± 0.3% risk of becoming infected by needle stick injury with a contaminated needle, although the risk is higher with increasing blood volume which is injected. The risk of infection can be reduced about 80% by promptly taking antiretrovirals after such an accident (see post-exposure, prophylaxis, PEP). These data need to be reviewed when new information becomes available.

Transmission, intravenous drug abuse

Sharing needles and other materials used by intravenous drug users constitute a third risk factor. The importance of this transmission pathway varies greatly from country to country.

Transmission, blood transfusions

Blood transfusions form a fourth risk factor. In many countries blood that is used for transfusion has still not been sufficiently tested for HIV. Not only is there insufficient screening, but there is also still the problem of the "window phase". This is the time interval in which a person has been infected and is infectious, but does not yet have any detectable antibodies. Such persons are thus seronegative and yet infectious. Antigen can be detected in their blood. The more frequently AIDS occurs in the population, the greater the number of persons in the window phase. The injudicious use of blood should be discouraged as much as possible (also because of transmission of other infectious organisms). Patients with aplastic anaemia or with sickle cell anaemia who have to have repeated transfusions form a high-risk group. Blood should be regarded as a medicine with potentially very dangerous side-effects.

Transmission, mother-to-child transmission

Physical examination of a child. Copyright ITM

A fifth transmission pathway is from mother-to-child. A child born from a seropositive mother runs an approximately 25% to 33% risk of becoming infected at around birth (if no antivirals are taken). HIV has been isolated from breast milk. The chance of an HIV-infected mother passing on the virus to the child via breast-feeding is estimated to be 17%. HIV is not embryotoxic. It can be isolated from amniotic fluid, from the foetus after the 16 th week of pregnancy and from umbilical cord blood. HIV can obviously sometimes penetrate through the placenta and infect the foetus, though the percentage of children who are infected in the uterus is probably very low. There are strong indications that transmission often takes place at the moment of birth. Mothers in a very advanced stage of the disease, with low CD4 values and/or with a high viral load have a greater risk of transmitting the virus to their children. Mothers with vitamin A deficiency may have a higher risk. The virus usually cannot be detected in the baby's blood immediately after birth, but only 4 to 8 weeks later. This corresponds with the primary viraemia and confirms that most infections take place during childbirth. Elective Caesarean section reduces the risk of transmission to the child. An important reduction of risk is to be expected if HAART is given. Caesarean section is of less advantage when the amniochorionic membranes have broken and/or the mother is already in labour.

Pathogenesis

HIV-cell docking

The virus, approximately 0.1 µm in length, consists of 2 identical single-stranded RNA chains surrounded by capsule proteins (p24 and p18). Because of these two copies, the virus is known as pseudodiploid. The centre also contains reverse transcriptase. The capsule is further enclosed in an envelope that likewise contains certain virally coded proteins, namely gp120 and gp41. By means of gp120 (glycoproteins with a molecular weight of 120 kilodaltons) the virus attaches itself to certain cells (particularly CD4-lymphocytes, but also glia cells, monocytes and macrophages). Attachment of the virus takes place via coupling of the viral gp120 with well defined cellular receptors, namely CD4 plus a co-receptors such as CXCR4 (fusin) or CCR5 (chemokine receptors). Persons who are homozygotic for certain mutations in a co-receptor (for example a specific deletion of 32 base-pairs in CCR5) are resistant to infection with HIV. The mutant receptor is non-functional but does not appear to cause the person any immediate physiological disadvantage, although they are more susceptible to West Nile virus infection. The pharmaceutical industry is therefore looking for co-receptor-blocking molecules. HIV-variants can be classified according to their interaction with chemokine receptors. In vitro syncytium inducing [SI] HIV-variants use the alpha-chemokine receptor CXCR4 and are lymphocytotropic [T-tropic]. HIV-variants which are non-syncytium inducing [NSI] in vitro use the beta-chemokine receptor CCR5 and are monocytotropic [M-tropic]. These latter variants predominate early in infection and account for about 90% of the sexual transmission of the virus, whereas the lymphocytotropic variants tend to dominate in the later stages. It is thought that only one or a few HIV variants are responsable for sexual transmission. If these variants can be identified, it would be good news for vaccine researchers, since it avoids having to deal with antigenic instability.

Transfection experiments

There are only few kinds of human cells which contain the CD4 receptor. Transfection of mammalian cell lines with the gene for human CD4, did not make them susceptible to HIV infection. This implied that CD4 was not the sole receptor for HIV. Another piece of the puzzle was missing. Continuation of this work led to the discovery of fusin (CXCR4) and CCR5, the co-receptors for HIV.

Other molecules which play a role in binding HIV include DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 3 [ICAM-3]-grabbing nonintegrin). This impressive sounding molecule is found exclusively on dendritic cells. The DC-SIGN on the surface of the dendritic cells captures HIV, thereby allowing efficient cross-infection of CD4+ T-cells in the vicinity.

Macrophages express low levels of CD4 but abundant levels of heparan sulfate proteoglycans, especially syndecan. Syndecan binds to HIV efficiently. Another receptor expressed by macrophages, CD91, binds to heat-shock proteins including those on the HIV virion membrane. Human CD317 (tetherin) is a restriction factor that blocks release of HIV-1 from the cell surface. It is a part of the innate immune response directed against budding virusses. The viral protein Vpu counteracts tetherin, enabling the release of virions from the plasma membrane. 

Note: chemokine nomenclature

The idiosyncratic names of the various chemokines are very confusing (a menagerie of names such as IL-8, PF-4, MIP-1alpha, ENA-78, GCP-2, CKbeta11, RANTES, lymphotactin, etc.).

For a better terminology the chemokines can be divided into groups on the basis of the number of cysteines and the relative positions of the first two cysteines:  C, CC, CXC and CXXXC. The C indicates a cysteine residue and the X indicates an intermediate amino acid. In the alpha-chemokines (CXC) the first two cysteines are seperated from each other by an arbitrary amino acid, the CXXXC type chemokines have 3 intermediate amino-acids, the beta-chemokines (CC) have cysteines that are adjacent to each other, in the gamma-chemokines (C) the N-terminal part of the protein contains only 1 instead of 2 cysteines. Chemokine receptors are similarly divided into families based on the chemokine that they bind. The R indicates Receptor and the number is a serial number, for example CXCR4. There are, however, other receptors that are capable of binding chemokines, but there again one would at present be drowned in a morass of nomenclature.

Viral entry, general

When a virus attaches to a cell surface, it engages a specific viral receptor. Examples are the CD155 molecule, which functions as the receptor for poliovirus and the angiotensin-converting enzyme 2 as the receptor for the coronavirus that causes SARS. Such receptor molecules play a critical role in mediating the entry of the virus into the cell. The distribution of receptors across specific cell types helps to determine viral tropism.

The binding of the virus to the receptor induces the viral-enveloppe protein to undergo conformational changes that mediate fusion between the viral and cellular membranes in one of two ways. For some viruses, receptor-binding leads to endocytosis of the viral particle and delivery to an acidic compartment. There, the low-pH environment triggers conformational changes that lead to membrane fusion. Influenzavirus, West Nile virus and rabies virus are examples of viruses that use this pathway. For other viruses, the very process of binding to one or more receptors leads to the needed conformational changes. These pH-independent viruses can fuse at the cell surface. HIV is an example. Such data were essential for the development of enfuvirtide, a peptide that prevents the conformational change needed for fusion.

After entry

HIV particles surround themselves with a coat of fatty material known as the viral envelope (or membrane). Projecting from this are around 72 little spikes, which are formed from the proteins gp120 and gp41. Just below the viral envelope is a layer called the matrix, which is made from the protein p17. The viral core (or capsid) is usually bullet-shaped and is made from the protein p24. After fusion with the host-cell membrane, the viral RNA is released into the cell. The RNA of the viral genome is copied to DNA into two stages. A hybrid RNA:DNA duplex is first synthesised by means of a reverse transcriptase (RT), a protein that is present in the virus. The RNA of this hybrid molecule is very rapidly broken down by the virally coded enzyme ribonuclease-H (RNase H where H stands for hybrid). This enzyme activity is associated with the reverse transcriptase protein. A double-stranded DNA is then formed from the remaining single-stranded DNA. This DNA is then integrated into one chromosome of the cell (for example a CD4-lymphocyte) by means of a second enzyme, integrase . The cellular protein LEDGF (lens epithelium-derived growth factor) helps integrase to splice HIV DNA into the cell's genome. Integrase is also a constituent of the pro-RT protein, but is cleaved from the pro-RT protein by a viral protease so that it can function independently. This protease is an important target for treatment. During replication of HIV, several long strands of protein are formed which are then cleaved into functional fragments by the protease enzyme. The site of integration into a cellular chromosome - and even which chromosome is used - is rather arbitrary, although preferred sites appear to exist. The integrated DNA copy of the virus is called a provirus. The integrated viral DNA is transcribed and many RNA copies are made. Viral proteins are then translated from this RNA. These proteins are subsequently further modified by cleavage, glycosylation, etc. Many new viruses are thus formed with these proteins and the RNA. The lipid envelope is formed from the plasma membrane of the host cell. Proteins coded by the virus insert themselves into this membrane. The carbohydrate component of these glycoproteins is formed in the endoplasmatic reticulum and in the Golgi apparatus of the infected cell. Due to the presence of an envelope the virus is sensitive to detergents. HIV has just nine genes. Three of the HIV genes, called Gag, Pol and Env, contain information needed to make structural proteins for new virus particles. The other six genes, known as Tat, Rev, Nef, Vif, Vpr and Vpu, code for proteins that control the ability of HIV to infect a cell, produce new copies of virus, or cause disease. HIV-infected cells tether newly made virus to the surface with a protein called tetherin, but the viral protein Vpu (viral protein U) sets it free. A Vpu inhibitor might keep the virus from leaving the cell. Macrophages and lymphocytes contain the antiviral protein A3G (APOBEC-3G), a kind of editing enzym. A3G edits single strands of HIV DNA, introducing errors. Unfortunately, HIV has evolved a countermeasure to A3G. It makes a protein callef Vif (viral infectivity factor) which interferes with the protective function of A3G. Nef (negative factor) is a viral protein that interacts with host cell signal transduction proteins to provide for long term survival of infected T cells. Persons infected with HIV-1 strains that have deletions of the Nef gene develop AIDS symptoms much more slowly than those infected with standard HIV strains. The viral proteins Tat and Rev are positive regulators of HIV gene expression.

HLA class I molecules present antigenic epitopes to T-lymphocytes, so that a specific immune response follows. It was shown that alterations of one amino acid in HLA molecules can have a substantial effect on the progression to AIDS.

The function of the CD4-lymphocyte is impaired by HIV replication. The CD4-lymphocytes subsequently decrease in number. CD4-lymphocytes, however, have a central role in the immune system. Destruction of these cells results in the immune system being weakened. The patient becomes immunodeficient. As a result he/she is no longer capable of adequately reacting to certain infections. The cause of death of AIDS patients usually lies with these concomitant infections and is not due to infection with HIV itself. Infection with HIV causes a progressive weakening of the body's resistance to infection. AIDS is the most advanced form of this infection and is characterised by specific opportunistic infections and specific, well defined cancers. There are many more HIV-infected persons without symptoms than persons with AIDS. Th17 cells are a subset of CD4 cells that secrete interleukin 17. These cells reside in the gastrointestinal tract. They also secrete defensins that prevent bacteria from entering the bloodstream and lymph nodes. Destruction of these cells might make the gut "leaky", allowing gut microbes, or pieces of them, to enter the bloodstream. Th17 cells are severely depleted in the gastrointestinal tract of HIV-infected people and SIV-infected macaques (both species develop AIDS), but not in SIV-infected sooty mangabeys that suffer no harm from the virus.

An early hypothesis was that the virus can be present in a "dormant" form (without obvious active replication) for a long time in the body in asymptomatic persons. This hypothesis was abandoned as evidence emerged that rapid virus production is continually taking place, accompanied by a rapid turn-over of CD4-cells (every day very high numbers of CD4-cells are destroyed but also produced). Retroviruses, such as HIV, are capable of making frequent errors in their own reproduction in host cells in transcription of RNA (mutation frequency of reverse transcriptase is 1/1000 to 1/30,000 per base). This alters the virus very quickly (the virus is antigenically unstable), which makes treatment of the disease even more difficult. The population of virus within a single host is sometimes described as a "quasi-species". Recombination is another source of variability. During the infection various mutant viruses exhibit different tissue tropisms, so that some of them appear only in macrophages, lymph nodes, genital mucosa, peripheral blood cells etc. In the early phases of the infection the virus population is still more or less uniform (oligoclonal), which is an argument in favour of prompt aggressive combination therapy with several antiviral agents. This is, however, a hypothesis that has not yet been confirmed in practice. One can calculate that in a few years time, every possible single-base mutation on every position of the 10 Kbase genome of the virus will have occurred. Some double mutations or multiple mutations will, of course, also occur. If the virus can become resistant to a drug due to a single base substitution, monotherapy is doomed to fail. Combination therapy will reduce the risk of generating resistance, certainly if sufficient antiviral agents are combined and if the patient takes the drugs correctly.

Potent combinations of triple therapy with reverse transcriptase inhibitors and protease inhibitors can reduce the number of detectable copies of HIV to below the detection threshold in the peripheral blood in 80% of infected persons. There is likewise a spectacular reduction of virus in the lymph nodes. However, the virus is still present as provirus in various other cells. In this state the virus is invulnerable. However, these cells also have a certain half-life. Attempts are now being made to work out how long HIV can survive in these cells.

The largest reservoir of HIV is formed by the tissue in lymph nodes, tonsils, mesenteric lymphatic tissue and the spleen. It contains some 100 to 10,000 times as much virus as the blood and 50 to 100 times as many CD4 cells. The brain also forms a separate reservoir with a viral replication that appears to proceed relatively independently of that in the blood. The testes and the prostate probably form separate virus reservoirs, which could have important consequences for sexual transfer of the virus. When potent combination therapy is used the decrease in the quantity of HIV RNA in lymph tissue runs parallel to that in the blood. It may be possible to attack the virus reservoir in latently infected cells with immunostimulators. HIV replication is stimulated by activation of these cells and consequently kills the cells, whereupon potent anti-HIV therapy would inhibit further replication of the released virions (experimental).

Early clinical manifestations

Two to six weeks after infection with HIV an influenza-like syndrome comparable with mononucleosis (high temperature, rash, sore throat, swollen lymph nodes, sometimes meningitis) can appear. This phase sometimes proceeds asymptomatically. During this phase there is an explosive viral replication with a high viraemia. During this initial window phase, in which the person may be asymptomatic, there are as yet no antibodies. The person is, however, contagious. Antibodies become detectable some 4 to 18 weeks after the infection (the person is then seropositive) in the majority of infected people. It is important to realize that HIV can be isolated in the great majority of seropositive patients and that all seropositive persons should be considered contagious. After this first phase there is an asymptomatic period in which the patient's immunity very gradually diminishes. Lymph node swelling, emaciation, high temperature, nocturnal sweating, diarrhoea and pruritus (itching) gradually occur in adults after the asymptomatic period.

Diagram of the evolution of the viral load and CD4-count following untreated HIV infection. Copyright ITM

No HIV-related embryopathy is found, although disorders of the thymus in the foetus have been reported. For infected babies, the interval between birth and appearance of the first symptoms can amount to a few months to several years. Most signs and symptoms in children are non-specific, though frequently persistent and/or recurrent. AIDS is in children will often manifest itself as a pneumocystosis of the lungs, growth retardation, encephalopathy, chronically swollen lymph nodes, oral candidiasis or recurrent and/or persistent bacterial infections (e.g. otitis media).

Late clinical manifestations

Late clinical manifestations, opportunistic infections

An opportunistic infection is an infection due to an organism that does not cause long-term, chronic disease in healthy persons, but does cause disease in immunodeficient persons. These infections in AIDS patients are often serious and persistent. Relapse frequently occurs when treatment is stopped. Maintenance therapy is often necessary after initial therapy. As the cost of many of these treatments is very high and therapy is often not possible, the question arises whether it makes sense to try to detect infections if no therapy is available. Opportunistic infections frequently occur through reactivation of already present but "dormant" infective organisms (toxoplasmosis, pneumocystosis, herpes zoster, etc.). Fungal infections are frequent (candidiasis, aspergillosis, cryptococcosis, histoplasmosis, blastomycosis, coccidioidomycosis in endemic areas). Opportunistic infections and cancers are more likely when the immunosuppression becomes more severe. The absolute CD4-lymphocyte count reflects the immune status very well. If fewer than 200 CD4-lymphocytes are present per µl (microlitre) of blood, the risk increases quite quickly. However, determination of the CD4-cell count requires a well equipped laboratory with trained staff. The presence of concomitant infections is not always fatal. Simultaneous infection with the GBV-C virus (also known as hepatitis G virus) even slows down progression to full-blown AIDS. Persistent infection with this flavivirus, which is closely related to the hepatitis C virus, is quite frequent but in itself asymptomatic.

Late clinical manifestations, gastrointestinal system

Oral candidiasis during chronic HIV infection. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

CT-scan thorax, showing large abscess. Salmonella sp. were cultured from the pus. Copyright R. Colebunders - ITM

Necrotising gingivitis in AIDS patient. Photo Cochabamba, Bolivia

Hairy leukoplakia tongue in HIV patient. Photo Cochabamba, Bolivia

Oral candidiasis in AIDS. Photo Cochabamba, Bolivia

Persistent diarrhoea lasting longer than 1 month occurs in 40% of patients. This is frequently accompanied by nausea, vomiting and abdominal pain. Chronic diarrhoea is sometimes of bacterial ( Shigella, Salmonella, etc.) or parasitic origin (cryptosporidia, microsporidia, Isospora belli, Giardia lamblia ). Frequently, however, no clear cause for the diarrhoea is found. HIV itself is causes diarrhoea in a number of persons (HIV enteropathy). Dysphagia should prompt a suspicion of Candida infection of the mouth, throat and oesophagus. In cases of painful swallowing, oral and/or oesophageal ulcers caused by herpes simplex (most frequently) or cytomegalovirus (endoscopy and biopsies necessary for verification of diagnosis) should be suspected. It should be remembered that oral Candida infections can also occur in non-HIV patients, following the administration of antibiotics, in diabetes mellitus and when using steroids. White candidiasis plaques can easily be scraped off with a tongue spatula. A painless white discoloration of the edges of the tongue sometimes indicates "hairy leukoplakia", which cannot be removed with a tongue spatula. The lesions can also occur in the oesophagus (typically in the middle). Epstein-Barr virus and herpes simplex virus play a part in their occurrence. Hairy leukoplakia is strongly suggestive of HIV infection. It occurs very rarely in other forms of immunosuppression. Extensive aphtous lesions sometimes occur in the mouth. Their aetiology is unknown, though they sometimes respond well to short-term glucocorticosteroid therapy. Extensive necrotic gingivitis and Kaposi's sarcoma both occur frequently in the mouth.

Differential diagnosis of hairy leukoplakia in HIV patients:

Candida infection:  the white lesions are not adherent and tend to be more generalized. The condition may be painful. Frictional keratosis from rubbing upon poorly fitting dental work or jagged teeth may appear similar but is usually unilateral. Squamous cell carcinoma and papillomavirus-induced neoplasia. A biopsy will confirm the diagnosis. Geographic tongue. In general , there are more widespread lesions on the tongue. Lichen planus. Look for skin lesions and Wickham's striae. Tobacco-associated leukoplakia Syphilitic mucous patch. Serology is essential for diagnosis

Late clinical manifestations, liver - pancreas

The principal causes of liver failure in an AIDS patient are: infections (tuberculosis, atypical mycobacteria, CMV, hepatitis B and C), septicaemia, cryptosporidia cholangitis, extrapulmonary pneumocystosis), drugs (anti-TB medication, paracetamol overdose, antiviral medication), alcohol.  Pancreatitis can be induced by medication, such as Hivid® and Videx®.

Late clinical manifestations, fever

Fever can, among other things, be due to tuberculosis or opportunistic infections. Investigations in an AIDS patient with fever should be aimed at detecting treatable causes. Malaria is not an opportunistic infection, but can also occur in HIV patients. Malaria can adversely affect HIV infection by increasing the viral load. Recurrent Salmonella   septicaemia is frequent. The reason is that Salmonella bacteria are facultative intracellular pathogens. They are normally eradicated by T-cell-activated macrophages. This mechanism is deficient in AIDS patients. A "functional hypogammaglobulinaemia" exists despite the polyclonal B-cell stimulation and the accompanying hypergammaglobulinaemia. There is an increased risk of infections with encapsulated bacteria (e.g. pneumococci), but also with Branhamella , Haemophilus   and Staphylococcus . Infections with Mycoplasma and Legionella are not more frequent in seropositive persons. "Drug fever" occurs more frequently in seropositive than in seronegative persons.

Late clinical manifestations, respiratory

General

CXR of a patient with multidrug resistant pulmonary tuberculosis. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Patient with HIV infection and pneumococcal pneumonia of the left lung. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Pleural effusion due to Salmonella infection in an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

CXR: Pneumocystis jiroveci penumonia in AIDS patient. With special thanks to Dr Lut Lynen.

In Africa persistent cough lasting more than 1 month occurs in approximately one third of AIDS patients. Bacterial pneumonia (pneumococci, Haemophilus ) and tuberculosis are prominent in cases of respiratory problems. Atypical mycobacterioses and Pneumocystis jiroveci  pneumonia are rather infrequent in Africa. Deep fungal infections (histoplasmosis, cryptococcosis, blastomycosis) can likewise cause pulmonary lesions. Pneumocystosis usually develops subacutely, with dyspnoea (shortness of breath) and a dry cough. Recurrent bacterial pneumonia occurs frequently in AIDS patients and is a major cause of death. Vomiting blood (haemoptysis) and pleural effusions are principally caused by TB and Kaposi's sarcoma. Sinusitis is quite frequent in AIDS patients. Lymphoid interstitial pneumonitis occurs especially in children, but can also be found in HIV positive adults. It is characterised by diffuse interstitial infiltrates. The alveolar septa are infiltrated with lymphocytes, plasma cells and immunoblasts.

Pneumocystis jiroveci

CXR showing pneumocystosis in an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Pneumocystis jiroveci in BAL fluid (broncho alveolar lavage). Staining with monoclonal antibodies. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Pneumocystis carinii was originally described in 1909 by Carlos Chagas. He thought it was a cystic form of Trypanosoma cruzi . In 1910 Antonio Carini observed similar cysts in rats with experimental trypanosomiasis, but thought that it was a new, unknown organism. He sent material to his colleague Laveran for further investigation. In 1912 Laveran's student Delanoe described similar cysts in lungs of Trypanosoma -free sewer rats. He gave the organism the name Pneumocystis carinii. Later, similar organisms were found in immunosuppressed patients. These were considered Pneumocystis carinii , until later DNA analysis showed that the human pathogen is actually a different species, which was named Pneumocystis jiroveci. The taxonomic classification was long unclear, but on the basis of DNA analysis it is at present regarded as an unusual fungus.  The taxonomic positision of the genus Pneumocystis is near the base of the phylum Ascomycota, close to the Ascomycota/Basidiomycota divide. It frequently causes inflammation of the lungs in severe immunosuppressed individuals. Disseminated infections can occur, e.g. with retinal lesions and foci in spleen and kidneys (frequently calcified). It is not easy to confirm the diagnosis via microscopic investigation, as this has a limited sensitivity. The pathogenic organism is best shown via bronchoscopy and broncho-alveolar lavage, though it can also be found in sputum induced in a non-invasive manner. The latter however requires availability of a special aerosol appliance to create a very fine spray of 3% saline solution. This non-invasive technique is less sensitive. The organism can be shown with Giemsa staining and is recognizable as small, fine blue spots (the capsule is not stained with Giemsa). Gomori methenamine silver staining, which also stains the capsule, is better, but more complex.

 

Late clinical manifestations, neurological

Several neurological complications can occur in AIDS patients. These can be subdivided into diseases induced by HIV itself, by opportunistic diseases or by medication.

Acute aseptic HIV-meningitis, chronic HIV-meningitis, HIV encephalopathy (AIDS dementia), vacuolar myelopathy, predominantly sensory neuropathy, inflammatory demyelinising polyneuropathy, mononeuritis multiplex, myopathy.

Cerebral toxoplasmosis, Cryptococcus meningitis, tuberculous meningitis, CMV retinitis-encephalitis-radiculitis, herpes encephalitis, progressive multifocal leukoencephalopathy (JC virus), neurosyphilis, primary cerebral lymphoma, metastatic systemic lymphoma. Chagas disease reactivation should be considered as a differential diagnosis of meningoencephalitis in Chagas serological positive HIV patients with low CD4 T-cell counts.

AIDS dementia

HIV itself affects the brain. Glia cells are infected. HIV causes progressive behavioural, short-term memory and concentration disorders. These develop over the course of several months into presenile dementia with retention of consciousness. Aphasia, alexia and agraphia are usually absent (in contrast to Alzheimer's disease), though motor abnormalities frequently occur, with unstable gait and a feeling of weakness in the legs. The tendon reflexes are increased. The patient becomes forgetful, can concentrate only with difficulty, quickly loses the thread of a conversation or has to read a text repeatedly in order to understand it. He/she becomes clumsy, suffers from tremor, his/her handwriting becomes blurred (coordination problems) and the sense of balance can become somewhat disturbed, especially when the patient quickly turns his/her head. Awareness of the disease is retained for quite a long time and the patient can describe the symptoms well. Apathy occurs later and the patient becomes mentally retarded. The cerebrospinal fluid often contains a large number of cells, an increased protein level and a discretely reduced glucose level. Agitation, confusion, hallucinations and psychosis can occur. Pyramidal symptoms such as spasticity, hyperreflexia, clonus and the presence of Babinski's sign can occur late. Incontinence also occurs late.

One of the most feared consequences of HIV is AIDS-related dementia. The same genetic mutation (epsilon-4 mutation of apolipoprotein E) that increases the risk for Alzheimer's dementia in HIV negative elderly persons may place HIV positive persons at higher risk for AIDS-related dementia (AIDS dementia complex) and peripheral neuropathy. Larger trials are needed to confirm these results.

Vacuolar myelopathy

Not only the brain, but also the spinal cord can be affected by HIV (in approximately 20% of cases) resulting in vacuolar myelopathy, which leads to loss of strength in the legs, ataxia and incontinence.

Toxoplasmosis

MRI of the brain showing a focal lesion of cerebral toxoplasmosis. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

CT-scan brain, showing a focal lesion due to cerebral toxoplasmosis in an AIDS-patient.

MRI of the brain showing a focal lesions due to cerebral toxoplasmosis. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Patient with HIV infection, intravenous drug user, with cerebral aspergillosis abcesses. With special thanks to Prof Dr Colebunders. Copyright ITM

Cerebral toxoplasmosis in AIDS patient. MRI brain. Copyright Alexander von Humbolt, Peru

Toxoplasma gondii was first described by Nicolle in 1909 in a North African rodent, Ctenodactylus gondii . The pathogen derives its species name from this. It is a unicellular parasite that is very widespread in nature and can infect many animal species. It is localised and replicates in practically all nucleated cells. The parasite has 3 principal forms: (1) banana-shaped trophozoites (hence the generic name "toxon" = bow) which reproduce asexually in nucleated cells, (2) intracellular dormant cysts with bradyzoites. In the cells the parasite forms cysts that are held in check by the immune system. The parasite thus remains present in the body, especially in muscle cells and in the brain, throughout life. (3) oocysts that are the result of the parasite's sexual cycle in the intestine of the cat. These oocysts can survive in the outside world for several months. The infection can become active again due to diminished activity of the immune system.

People are infected primarily via cat faeces or by eating contaminated and insufficiently cooked meat. In adults with an intact immune system Toxoplasma usually causes few problems, apart from occasional lymph node swelling and/or hepatitis. Reactivated parasites can attack the brain and retina in immunodepressed persons. Headache, neurological problems, blindness and focal cerebral lesions can occur. Such focal lesions can lead to epileptic fits. A favourable clinical response to anti-toxoplasmosis therapy is suggestive of cerebral toxoplasmosis (a good clinical response usually occurs within 2 weeks). Cerebral biopsy and PCR (Polymerase chain reaction) can be performed if there is doubt about the diagnosis. Toxoplamosis is an infection that can be prevented by prophylactic therapy. Cerebral localisations of lymphomas as well as tuberculomas can resemble cerebral toxoplasmosis.

Differential diagnosis of focal CNS (central nervous system) lesions in AIDS patients

Toxoplasmosis:  by far the most frequent Primary CNS lymphoma Mycobacterium tuberculosis Mycobacterium avium  complex Progressive multifocal leukoencephalopathy (JC virus) Cryptococcus neoformans Candida sp. Listeria monocytogenes Nocardia asteroides Salmonella group B Aspergillus sp. Rhodococcus sp. Acanthamoeba sp. Trypanosoma cruzi Syphilitic gumma (syphilis, Treponema pallidum ) "Classical" bacterial cerebral abscess, e.g. otogenic, from sinus, secondarily to endocarditis.

Clinical manifestation of cerebral abscess according to localisation

Temporal lobe abscess: ipsilateral headache, aphasia, upper homonymic quadrant anopsia. Frontal lobe abscess:  headache, dizziness, confusion, diminished mental capacity, hemiparesis, motoric speech disorders. Parietal lobe abscess:  headache, visual disturbances. Cerebellar abscess:  nystagmus, ataxia, dysmetria, vomiting.

What can we learn from a MRI or CT scan?

Ring-enhanced single lesion:  abscess, with frequency toxoplasmosis > TB > cryptococcosis. The peripheral staining by the contrast medium shows the oedema zone around the abscess. On this basis a test therapy will often be started without having formal proof of toxoplasmosis. If there is no improvement within two weeks or if deterioration occurs, a stereotactic brain biopsy should be performed. In addition to these three etiologies, there can be other causes of an intracranial mass: herpes, Histoplasma, Nocardia, Candida , Kaposi, metastasis. In 10% of cases no specific cause is found. Non-ring enhanced lesion:  probably lymphoma > PML. In case of PML there is no mass effect. Bilateral diffuse lesions:  immune reconstitution syndrome (IRIS) if in the correct context: within 4 months of starting HAART. Multiple ring lesions:  toxoplasmosis >> tuberculosis. HIV atrophy: broadened sulci and enlarged ventricles (DD internal hydrocephalus:  flattened sulci and enlarged ventricles).

What can we analyse in cerebrospinal fluid?

Direct investigation: amoebae, trypanosomes, filaria Cell counts: type and number (nl < 3/mm3) Biochemistry:  protein content (nl 25-40 mg%), glycorrhachia (nl 40-90 mg%, nl 50-80% of the glycemia) Serology:  Syphilis, intrathecal production of various antibodies (compare with titre in serum sample) Antigen-detection:  Cryptococcus PCR:  Mycobacteria, JC virus, herpes, toxoplasmosis Stains:  Gram, Indian ink, Ziehl Culture:  virus, bacteria, mycobacteria, fungi Cytology:  only in case of suspected carcinomatous meningitis

Cryptococcal meningitis

Cryptococcosis is a cosmopolitan infectious disease caused by yeast of the Cryptococcus genus. There are at least four varieties: C. neoformans var. grubii (serotype A), C. neoformans var. neoformans (serotype D) and C. neoformans var. gattii (serotype B and C ) . These differ in their geographical distribution, ecology and biochemical characteristics. The antigenic specificity of the polysaccharide capsule determines four serotypes: A, B, C and D. Serotypes A and D are found worldwide in bird droppings (avian excreta), especially of pigeons. About 90% of the cryptococcal infections in the USA are due to C. n. grubii . Serotypes B and C are geographically associated with certain Eucalyptus trees . In Australia Eucalyptus camadulensis trees form the natural habitat for the var. gattii . C. n. neoformans occurs worldwide, whereas C. n. gattii is restricted to the tropics and subtropics.  The typical vegetative form of C. neoformans is a yeast with a diameter of 2.5 to 10 µm. The organism can also reproduce sexually. As it is a basidiomycete (Filobasidiella neoformans ) it forms sexual spores: " basidiospores ". [Basidiospore: Gr: "basidon": small base and "sporon":  seed. This indicates the morphology:  a club-shaped cell with the haploid spores at the far end]. Infection can probably occur as a result of inhalation of either dehydrated yeast form or basidiospores .

Cryptococcus neoformans , Indian ink stain. Notice the thick capsule and the refractile inclusions in the centre. Copyright ITM

Cryptococcus neoformans in biopsy. Notice the thick capsule around the yeast cells. Copyright ITM

Cryptococcus neoformans primarily causes a chronic meningitis. Systemic infections, inflammation of the lungs and cutaneous lesions also occur as a result of these yeasts. The demonstration of cryptococcal meningitis in a patient is considered proof of AIDS in HIV patients. Immunodepression due to other reasons (especially high dose of steroids such as in some transplant patients) also is a risk factor. The clinical picture can be very atypical, sometimes presenting as fever of unknown origin without a clear focus or with only  very mild neurological abnormalities, such as depression or diminished memory. Mild fever, headache, confusion and emaciation can be found in patients with cryptococcal meningitis. Neck stiffness is present in less than half of infected persons. Photophobia occurs in 25%. Confusion and coma occur later. Focal signs are seldom observed. Blindness can occur (possibly due to concomitant arachnoiditis), although this is less frequent in HIV patients than in immunocompetent individuals with cryptococcal meningitis. The diagnosis of a cryptococcal meningitis is made by centrifuging a few ml of cerebrospinal fluid and mixing the sediment with an equal quantity of East Indian ink. The yeasts are recognized quite easily as round organisms with a thick capsule. The saccharide capsule can be detected via an antigen-detection test (latex agglutination test for serum, cerebrospinal fluid and urine). The encapsulated organism can likewise be detected in tissue biopsies. The presence of low number of yeasts and the presence of poor-encapsulated strains will decrease the sensitivity of the antigen-detection test. Culture of the CSF might bring an answer here.

Tuberculous meningitis

Tuberculous meningitis usually presents with a non-specific clinical picture. The cranial nerves are frequently affected. Tuberculous meningitis induces an increase in the number of lymphocytes in the cerebrospinal fluid and a decrease in glycorrhachia. Acid-fast bacilli can sometimes be shown by direct examination, but in view of the low sensitivity of direct examination, a culture (Löwenstein-Jenssen) may be necessary. Nodular thickenings, i.e. tuberculomas of the meninges can be observed on CT scan or MRI.

Primary CNS lymphoma

Persons infected with HIV have a risk of primary CNS lymphoma that is 3600 times as high as the risk of persons in the general population. About 90% of these lymphoma are of B-cell origin. The majority of pathological processes in the brain are hypodens on CT scans and hyperintens on T 2 -weighted MRI as compared with normal gray matter. Primary CNS lymphoma is often isodense to hyperdense on CT scans and isointense to hypointense on T 2 -weighted MRI. This is due to the lower concentration of water (these tumors have a high cell density and scant cytoplasm). There is almost always enhancement with the administration of IV contrast material. Enhancement of the perivascular Virchow-Robin spaces is a highly specific feature. Treatment options include corticosteroids (relaps is frequent), surgery (little therapeutic gain), radiation therapy (often no permanent remission) and high dose methotrexate. Methotrexate 8 gram/m 2 is given initially over two weeks (induction therapy), followed by consolidation therapy and maintenance therapy. Calcium leukovorin will be started 24 hours before methotrexate. Combining radiation with chemotherapy often results in important neurotoxicity, especially in persons older than 60 years.

Progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinising disease caused by infection with a papova virus, the JC virus. The family of the Papovaviridae is divided into two genera:  Papilloma virus (e.g. wart virus) and Polyoma virus (including JC virus, BK virus and the SV40 virus). The name papova is derived from pa pilloma, po lyoma and va cuolating agent. They are small double-stranded DNA viruses that are potentially oncogenic. The infection preferentially destroys the oligodendrocytes, leading to demyelinisation since the myelin sheath around axons in the central nervous system is formed by concentric folds of the cytoplasma membrane of the oligodendrocytes (analogous to the Schwann cells in the peripheral nervous system). The disease was first described in 1958 by Aström. By injection into experimental animals the virus can induce a number of brain tumours (gliomas, meningomas, neuroblastomas, medulloblastomas). JC virus undergoes intranuclear replication in the astrocytes and oligodendrocytes, as well as in other cells (the virus can be found in epithelial cells in urine, liver, spleen, lymph nodes and lungs). Infections with this virus are frequent but nearly always remain without further consequences. However, in severe immunosuppresion (CLL, Hodgkin's disease, sarcoidosis, SLE, AIDS) a neurological syndrome can occur. Selective destruction of myelin takes place, but the axons are spared. There is little inflammation (in contrast to multiple sclerosis, where there is an inflammatory lymphocytic infiltrate). Multiple distinct foci of myelin destruction are observed in brain tissue obtained via stereotactic biopsy. The foci become confluent after a while. The lesions are asymmetrical without any preferred localisation, although lesions rarely occur in the spinal cord. The oligodendrocytes exhibit intranuclear viral inclusions. Giant astrocytes with pleomorphic, hyperchromatic nuclei strongly reminiscent of glioblastomas also occur. The clinical evolution is rapid, with an average course of disease of 2 to 4 months. Remission seldom occurs. Mono- or hemiparesis, speech disorders, mental deterioration with progression to dementia and death are the rule. Transverse myelopathy is rare. There is no fever. Headache and fits or convulsions are exceptional. The differential diagnosis includes other opportunistic infections (mycobacteria, fungi, Toxoplasma , cytomegalovirus), syphilis, cerebral lymphoma, endocarditis with embolism, HIV encephalopathy and multiple sclerosis. The EEG is usually diffusely disturbed and aspecifically slow. The cerebrospinal fluid is normal, though the virus can be detected in the fluid by PCR. CT brain scans and, even better, MRI scans show the subcortical lesions in the white matter. There is no mass effect and no staining of the lesions with contrast medium. As regards treatment, the results with various medications have been disappointing. The introduction of HAART was the single most significant development, reducing the rate of mortality from PML in HIV-positive individuals from 90% to 50%. Data from in vitro experiments show that mefloquine inhibits viral DNA replication after viral entry. If this data will have clinical consequences is unclear at present.

Note: JC virus and natalizumab

Infections with JC virus are very common (60-90%) in the general population, but nearly always asymptomatic. The virus is being held in check by the healthy immune system, although virus is often shedded in urine (data are variable, but up to 40% of asymptomatic people). Natalizumab is an antibody against alpha four integrins. It is being studied in inflammatory bowel disease such as Crohn's disease as well as multiple sclerosis.  It inhibits mechanisms necessary to control latent JC virus infection and the prevention of progressive leukoencephalopathy. Administration of natalizumab led to a marked increase of progressive leukoencephalopathy in the recipients. The administration of natalizumab as treatment for patients with multiple sclerosis can lead to flare up of latent PML in about 1/1000. Stopping the treatment with this monoclonal antibody (eventual removing any residual medication with dialysis) can lead to an immune reconstitution syndrome with initial worsening of symptoms, followed by improvement.

Various neurological problems

Mononeuritis (for example, facial paralysis) can occur at any stage of the HIV infection. Polyneuritis (often with severe pain and sensory disorders) in persons who are not taking any medication occurs almost exclusively in an advanced stage of the disease. Severe radiculitis with nerve pain can be caused by cytomegalovirus infection. Antiretroviral therapy, in particular Hivid®, Zerit® and Videx®, can cause neuritis. Neuritis can also be provoked by nicotibine (INH) and by alcohol, as well as vitamin B deficiency and diabetes. Neurosyphilis should be excluded in neurological problems. If peripheral nerve paralysis develops within 4 months of starting HAART, especially if there is also skin inflammation or ulcers, one has to consider the possibility of leprosy which becomes unmasked due to immune reconstitution syndrome (IRIS). IRIS can also lead to central nervous system lesions.

Kaposi's Sarcoma

Swollen cervical lymph nodes due to Kaposi's sarcoma in an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

AIDS patient with Kaposi's sarcoma. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Kaposi's sarcoma lesions on the index finger of an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Classic endemic Kaposi's sarcoma of the foot in a HIV-negative congolese patient. Copyright ITM

AIDS patient with Kaposi's sarcoma lesion in the conjunctiva. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Kaposi's sarcoma lesions on the palate of an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Classic Kaposi's sarcoma in a HIV negative patient from Congo. Lymphoedema resulted from this chronic tumor. Copyright ITM, photo Dr Van den Enden Erwin

Classic Kaposi's sarcoma in a HIV negative patient from Congo. Lymphoedema resulted from this chronic tumor. Copyright ITM, photo Dr Van den Enden Erwin

Classic Kaposi's sarcoma in a HIV negative patient from Congo. Lymphoedema resulted from this chronic tumor. Copyright ITM, photo Dr Van den Enden Erwin

Dark brown (on black skin) or purplish plaques and nodules (on white skin) on the skin or reddish-purple elevations in the mouth suggest Kaposi's sarcoma. This is a cancer that can affect any part of the body (lung, intestine, eye etc.) in AIDS patients, in contrast to the so called endemic Kaposi in Africa, which develops slowly and usually causes lesions only on the feet and legs. The disease was first described in 1872 by Moritz Kaposi, a Hungarian dermatologist. Kaposi's sarcoma occurs less frequently in Southeast Asia than in Africa. Since Kaposi's sarcoma occurs much more frequently in homosexual men than in patients infected with HIV via blood, an infectious cause was suspected. Kaposi's sarcoma also occurs more frequently in HIV-negative transplant patients. Immunosuppression appears to be needed for expression of the agent. A new herpes virus (HHV-8; human herpes virus 8) was found in the DNA of Kaposi's sarcoma cells and certain lymphomas. This virus apparently has a causal role in these cancers and also in a variant of Castleman's disease, a lymphoproliferative disease of B-cells. The virus is found in sperm, though much higher concentrations occur in saliva. This virus is also suspected of playing a role in pulmonary hypertension, but more research is needed.

Note: herpes viruses

KSHV is the first known human virus of the genus Rhadinovirus (?2-herpes virus). Other related viruses have been found in simians (monkeys and humanoid apes). In summary:  herpes viruses are divided into several groups: 

alpha-herpes virus, e.g. Herpes simplex virus 1 and 2, Varicella zoster virus (= HHV3)

beta-herpes virus, e.g. Cytomegalovirus (HHV5), Roseolovirus (= HHV6) and HHV7

Gamma 1-herpes virus, e.g. Epstein-Barr virus (= HHV4)

Gamma 2-herpes virus, e.g. Kaposi's Sarcoma Herpes Virus (HHV8)

Human herpesviruses Virus Common infections Site of persistence Mode of transmission Herpes simplex type 1 Herpes labialis, herpetic whitlow, herpetic keratitis, herpes encephalitis Neuronal cells, especially trigeminal ganglia Contact with secretions, especially oral Herpes simplex type 2 Herpes genitalis, herpes proctitis, neonatal herpes Neuronal cells, especially sacral ganglia Contact with secretions, especially genital Varicella-Zoster virus Chickenpox, herpes zoster (shingles) Neuronal cells, especially posterior root ganglia Contact with infected skin lesions, respiratory route for chickenpox Epstein-Barr virus Infectious mononucleosis, prolonged fever, multiorgan manifestations B lymphocytes Contact with oral secretions, blood or transplanted organs Cytomegalovirus Infectious mononucleosis, prolonged fever Monocytes, macrophages Contact with oral or genital secretions, urine, breast milk, blood or transplanted organs Human herpesvirus 6 Febrile illness, roseola (sixth disease) T lymphocytes Contact with oral secretions Human herpesvirus 7 Febrile illness, roseola, pityriasis rosea T lymphocytes Contact with oral secretions or breast milk Human herpesvirus 8 Kaposi's sarcoma Unknown Contact with bodily secretions      

Late clinical manifestations, cutaneous problems

Herpes simplex

Herpes genitalis is a sexually transmissible disease caused by herpes simplex virus type 2, sometimes by type 1. Primo-infection with HSV-2 is symptomatic in only 37% of cases. Transmission of the virus by asymptomatic carriers is possible, even after many years. A first acute episode is either a primo-infection in a person who has never been infected with HSV-1 of HSV-2 before, or a first clinical manifestation of herpes genitalis in a patient who has been carrying the virus for some time. This first episode is classically expressed as blisters followed by ulcerations accompanied by a burning sensation, discomfort and sometimes intense pain. Some patients also report headache and symptoms indicating a neurological disorder: neuralgia, sensory disorders in the calves and urine retention. Recurrences are generally less serious than the first episode. Recurrences are more frequent with HSV-2 infection than with HSV-1 infection. With time, however, the frequency of relapses decreases. Application of steroid-containing crèmes should be avoided, especially with eye lesions.

Merkel-cell carcinoma

Merkel-cell carcinoma is an uncommon skin cancer dervied from Merkel cells. These are mechanoreceptors in the basal-cell layer of the skin, in the root sheath of hair follicles, and in the mouth. In 2008, a polyomavirus has been described in association with these neoplasms. The disease can be locally aggresive, metastasize to regional lymphnodes or disseminate. Risk factors include long-term UV exposure, such as sun bathing, albinism, PUVA treatment for psoriasis and immunosuppression. In organ transplant recepients the incidence is 40 times higher than in age-matched controls, and in HIV infection, the risk is increased by a factor 13.

Herpes zoster

Herpes zoster in AIDS patient. Photo ITM, Dr Lut Lynen

Herpes zoster. Copyright Alexander von Humbolt, Peru

Herpes zoster in AIDS patient. Photo ITM, Dr Lut Lynen

Patient with HIV infection and numerous perianal and labial herpes simplex ulcers. With special thanks to Elly Katabira, Uganda.

Herpes zoster, zona or shingles results from reactivation of latent varicella zoster virus. The initial infection with this virus will result in chickenpox. Afterwards, the virus will stay dormant in the dorsal root ganglia and cranial nerve ganglia. The reactivation will result in a totally different clinical picture. There will inflammation of the posterior and anterior horns of the gray matter, the meninges, and the dorsal and ventral roots. This can proceed subclinical. Skin lesions will appear in a dermatome and sometimes in adjacent dermatomes. The initial symptom is often pain along the site of the future eruption. This pain precedes the rash by 2-3 days. Afterwards, characteristic crops of vesicles will appear. These have an erythematous base. Hyperaesthesia of the affected zone might develop. Dissemination to other parts of the skin and/or to visceral organs can occur, especially in immunodepressed people. Although fewer than 4% of non-immunedepressed people will experience a recurrence, new flare-ups are common in AIDS patients. Postherpetic neuralgia can persist for many months, even years. When herpes zoster affects the otic/geniculate ganglion, geniculate zoster, better known as the Ramsay Hunt syndrome will follow. Pain in the ear and facial paralysis will follow. There will be a vesicular eruption in the external auditory canal. Taste may be lost in the anterior two thirds of the tongue. If the gasserian ganglion in affected, ophthalmic herpes zoster will appear. A vesicular rash in the distribution area of this branch of the fifth cranial nerve is typical. Vesicles on the tip of the nose are a warning sign which indicate involvement of the nasociliary nerve. In this case, corneal lesions are to be expected (75% probability). If there is no lesion on the tip of the nose, the eyeball is involved in 30% of patients. Herpetic keratitis is vision-threatening.

Late clinical manifestations , Penicillium marneffei

Penicillium marneffei. Numerous yeast cells invade the bone marrow of this AIDS patient. Do not confuse with Leishmania amastigotes or histoplasmosis. Copyright ITM

Infection with Penicillium marneffei , resulting in typical skin lesions in this AIDS-patient (Southeast Asia).

Patient with HIV infection. Liver biopsy showing Penicillum marneffei yeasts. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

In Southeast Asia infection with Penicillium marneffei   should be included in the differential diagnosis of skin lesions. This fungus causes high fever (95%), hepatomegaly (40-90%), lymphadenopathy (50-90%), cough (50%), anaemia (40-80%), emaciation or weight loss (75%), splenomegaly (15-60%) and skin lesions (70%). The skin shows several papules with central umbilication. The chest X-ray can resemble tuberculosis. The organisms can be demonstrated in a smear from the skin, lymph nodes, sputum and/or from the bone marrow (a bone marrow analysis is the most sensitive). The white blood cell count varies greatly. The fungus is sometimes found in neutrophils in peripheral blood. Culture is also possible. The fungus is sensitive to amphotericin B and itraconazole. Do not confuse the yeast cells with other fungi or leishmania amastigotes.

The natural reservoir of Penicillium marneffei is still poorly known, though a connection with certain rodents (so-called bamboo rats such as Rhizomys sp. and Cannomys sp.) is thought to be likely. The organism was first isolated from the liver of a bamboo rat in 1956. It is named after Dr. Marneffe, a former director of the Pasteur Institute in Indochina. The first naturally infected human case was described in 1973. The mode of transmission has not yet been fully elucidated.

Late clinical manifestations, various cutaneous problems

Severe onychomycosis in an AIDS patient. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Tinea manuum due to infection with Microsporum langeroni . Copyright R. Colebunders - ITM

Sulfamide allergy in HIV. Copyright ITM

Molluscum contagiosum in AIDS patient. Photo copyright Cochabamba, Bolivia

Kaposi's sarcoma on the lower legs, after chemotherapy. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Patient with HIV infection. Fixed drug eruption triggered by cotrimoxazole. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

A papulopruriginous to nodular skin rash occurs in about 10 % of the seropositive population in the tropics. The cause of this nodular prurigo is not known. The itching is sometimes intolerable and difficult to treat. The differential diagnosis includes scabies (also frequent and sometimes very severe) and onchocerciasis. One gets the impression that HIV positive persons react more violently and with more itching (pruritus) to various insect bites. Cutaneous rash - not necessarily with itching - can occur as a result of side-effects of medication (sulphamethoxazole, thiosemicarbazone). Symptomatic treatment is often the only treatment for itch. Seborrheic dermatitis is very frequent (3% in the general population, but up to 50% in AIDS patients). This seems to be connected with a reaction to the skin yeast Pityrosporon ovale . Immunodeficient persons often develop a chronic, refractory, extensive but quite benign skin infection with a pox virus: molluscum contagiosum. This results in numerous small umbilicated papules. Psoriatic skin lesions are frequently activated and can be severe during late stage HIV infection. They can regress rather quickly with HAART. Lipoatrophy Lipoatrophy involves the loss of subcutaneous fat in the face, arms, legs, abdomen and/or buttocks. In contrast to the wasting syndrome of advanced AIDS, lipoatrophy is distinguished by the preferential loss of fat tissue without substantial loss of lean tissue mass. HAART, disease severity and host factors interact to produce this clinical entity. Thymidine analogues (with stavudine a greater risk factor than zidovudin), CD4-count of less than 200 and older age all contribute. Treatment with rosiglitazone and poly-L-lactic acid have been studied. Substitution of thymidine analogues to e.g. abacavir or tenofovir should be considered.

Late clinical manifestations, ocular abnormalities

Anterior segment

Keratitis can be caused by various organisms ranging from herpes viruses to microsporidia.

Posterior segment

Minor infarctions in the retinal nerve layer ("cotton-wool spots") are very frequent. They are transient and are of no further significance. In Europe before the era of antiviral combination therapy approximately 20% of AIDS patients developed ocular lesions due to infection with the cytomegalovirus. CMV retinitis is rare in developing countries as most patients do not survive a very severe immunodeficiency condition for a sufficiently long period of time (they die earlier of something else). CMV spreads from cell to cell in the retina and causes a peripheral or paracentral scotoma (retinal necrosis) that gradually becomes larger. Tunnel vision is one consequence of the destruction of the peripheral retina. Multiple minor haemorrhages and perivascular sheathing is characteristic of this infection. Retinal detachment can follow focal atrophy of the retina. If left untreated it can lead to blindness. Blindness is caused less frequently (3%) by toxoplasmosis retinitis. In cases of toxoplasmosis of the retina there is also a high risk (30%) of toxoplasmosis of the brain. Pneumocystis jiroveci choroiditis is very rare. Retinal necrosis can be caused by herpes simplex or herpes zoster virus.

Late clinical manifestations, slim disease and other problems

Weight loss is very frequent. It occurs in more than 90% of patients and can be very substantial ("slim disease"). It is probably multifactorial:  hypermetabolic patients with various infections, cytokine mediated effects, direct effects of HIV itself, malabsorption, anorexia, etc. Many patients die of extreme cachexia. In many cases tuberculosis is found in unusual sites upon autopsy. Many patients develop lymphadenopathy in the course of their illness. Tuberculosis should be excluded in cases of seriously enlarged (> 4 cm) or asymmetrically enlarged lymph nodes (lymph node aspirate with Ziehl staining). Syphilis, toxoplasmosis and non-Hodgkin's lymphoma can also cause lymph node swelling.

Surveillance (epidemiological monitoring)

In the West, various criteria are used for diagnosing AIDS. However, most of them are not practical in Third World countries as they are frequently based on complex laboratory procedures. Diagnosis of certain opportunistic infections is sometimes not possible without good laboratories. A clinical definition that can be used in Africa has therefore been proposed.

World Health Organization (WHO) clinical definition of AIDS in Africa:

AIDS in an adult is defined as the presence of at least 2 major criteria and at least 1 minor criterion (in the absence of any other cause of immunosuppression such as cancer, cytostatic therapy, prolonged steroid therapy or malnutrition). These criteria of course do not have 100% sensitivity and they are not 100% specific. They are only intended as guidelines. This definition should not be used by clinicians to declare the diagnosis without the result of a HIV-test. New definitions have been proposed since HIV-tests have become more available.

Major:

Weight loss > 10 % of earlier weight Chronic diarrhoea > 1 month Prolonged high temperature > 1 month (constant or intermittent)

Minor:

Generalised pruritic papulous cutaneous rash Generalised lymph node swelling Persistent cough > 1 month (when no TB) Chronic Candida infection in mouth/throat Recurrent Herpes zoster Chronic progressive and generalised Herpes simplex infection, resulting in painful genital ulcerations, not responding to antibiotics

Other:

The presence of disseminated Kaposi's sarcoma or cryptococcal meningitis is in itself sufficient for AIDS diagnosis.

CDC surveillance Case Definition for AIDS

In the diagnosis of the following diseases AIDS is assumed, even if the HIV status is not known. This concerns patients without other underlying immunodeficiencies. Candidiasis of oesophagus, trachea, bronchi or lungs Extrapulmonary cryptococcosis Chronic cryptosporidiosis (> 1 month) CMV infection of any organ, except liver, spleen or lymph nodes, when this has been present > 1 month. Herpes simplex infection with chronic (> 1 month) affection of mucocutaneous membranes, lungs or oesophagus. Kaposi's sarcoma in persons under 60 years old. Primary central nervous system (CNS) lymphoma in persons under 60 years old. Lymphoid interstitial pneumonitis (LIP) and/or pulmonary lymphoid hyperplasia in children < 13 years Disseminated Mycobacterium avium or M. kansasii Pneumocystis jiroveci pneumonia Progressive multifocal leukoencephalopathy (JC virus) Toxoplasmosis of the brain in persons over one month old.

Diseases diagnosed in known HIV positive persons

Multiple recurrent pyogenic infections in children under 13 years old. Recurrent Salmonella septicaemia Disseminated coccidioidomycosis or histoplasmosis Chronic isosporiasis Kaposi's sarcoma, irrespective of age Primary CNS lymphoma, irrespective of age Non-Hodgkin's lymphoma or immunoblastic sarcoma Extrapulmonary tuberculosis

Disease with suspected diagnosis in HIV positive persons

Oesophageal candidiasis CMV (Cytomegalovirus) retinitis Kaposi's sarcoma LIP in children under 13 years old Disseminated mycobacteriosis (without culture) Pneumocystis jiroveci pneumonia Toxoplasmosis of the CNS in a person for a period of over one month HIV encephalopathy HIV wasting syndrome

CDC classification of HIV infection

Group 1:  acute infection

Group 2:  asymptomatic infection

Group 3:  persistent generalised lymphadenopathy

Group 4:  other disease(s) present

A:  constitutional disease

B:  neurological disease

C:  secondary infectious disease (see list above)

D:  secondary tumours

E:  other diseases

WHO clinical staging system for HIV infection and disease

Clinical stage 1

Asymptomatic

Persistent generalised lymphadenopathy.

Performance scale 1:  aymptomatic and normal activity

Clinical stage 2

Weight loss > than 5% and < 10% body weight

Minor mucocutaneous symptoms (seborrheic dermatitis, prurigo, fungal nail infections, recurrent oral ulceration, angular stomatitis).

Herpes zoster within the previous 5 years

Recurrent upper respiratory tract infection (i.e. bacterial sinusitis)

And/or performance scale 2:  symptomatic, normal activity

Clinical stage 3

Weight loss > 10% body weight

Unexplained chronic diarrhoea > 1 month

Unexplained prolonged fever (intermittent or constant) > 1 month

Oral candidiasis

Oral hairy leukoplakia

Pulmonary tuberculosis within the previous year

Severe bacterial infections (i.e. pneumonia, pyomyositis)

And/or performance scale 3:  bed-ridden < 50% of the day during the last month

Clinical stage 4

HIV wasting syndrome (weight loss >10% body weight plus unexplained chronic diarrhoea or chronic weakness and unexplained prolonged fever (> 1 month).

Pneumocystis jiroveci pneumonia

Toxoplasmosis of the brain

Cryptosporidiosis with diarrhoea for > 1 month

Extrapulmonary cryptococcosis

CMV infection of an organ other than liver, spleen or lymph nodes

Herpes simplex virus infection, mucocutaneous > 1 month, or visceral (any duration)

Progressive multifocal leukoencephalopathy

Any disseminated endemic mycosis

Candidiasis of the oesophagus, trachea, bronchi or lungs

Disseminated atypical mycobacteriosis

Non-typhoidal Salmonella septicaemia

Extrapulmonary tuberculosis

Lymphoma

Kaposi's sarcoma

HIV encephalopathy

And/or performance scale 4:  bed-ridden >50% of the day during last month.

Diagnosis of HIV-infection

Inno-LIA test in the diagnosis (confirmation) of HIV infection. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

HIV quick test. With special thanks to Prof. Dr R. Colebunders. Copyright ITM

Two different blood tests are advised to confirm infection with HIV. In well equipped laboratories antibodies to HIV can be determined with an ELISA test. If the result is positive, a control is performed using a so called Western blot, though this is a very expensive test. The performance of a second (technically different) ELISA test or of a rapid test has been proposed for developing areas. A virus culture and PCR for HIV can be carried out if in doubt or for research purposes, though these are difficult techniques. Electricity is needed for ELISA tests, as well as the necessary apparatus and personnel who can use and maintain the equipment. If the tests are available and if priorities have to be set, the blood bank must be considered first (checking of blood donors). In the period before seroconversion, the serology is negative, even though there is infection. Viral proteins can be detected in the blood using antigen detection. The problem of this "window" in HIV detection (for example in blood donors) is especially significant in regions with high prevalence of HIV. These quite complicated tests may often not be available in rural areas. The "HIV check" test, which can be performed under quite primitive conditions, is easier to carry out than an ELISA. Current ELISA tests detect HIV-1 and HIV-2 simultaneously.

The diagnosis in children under 15 months is difficult with simple tests. All children born from seropositive mothers will be seropositive. Antibodies in the blood may originate from the mother or from the child itself. The maternal antibodies disappear spontaneously from the child's blood in the course of the following months. After 18 months (usually earlier) they are no longer detectable. Children who are infected with HIV produce their own anti-HIV antibodies and will thus remain seropositive, despite the disappearance of the maternal antibodies. There are other techniques besides serology to determine whether or not a child is infected. Detection of a viral antigen (p24 antigen) in the child's blood is a specific but rather insensitive test (only about 15% of infected children have a positive antigen test in their first year of life). Infection can be demonstrated by PCR (polymerase chain reaction) and virus culture, though these can also give false negative results in infected newborn babies.

Treatment

Treatment, general

In view of the rapidly changing availability of medications and the changing guidelines, it is advisable to acquire a recent copy of " The Sanford Guide to HIV/AIDS Therapy ". This is published annually and can be ordered via http://www.sanfordguide.com Updates on AIDS treatment : http://www.hivatis.org   HIV-patient websites : http://www.aidsmap.com http://www.medscape.com/hiv http://www.clinicaloptions.com/HIV British HIV association : http://www.bhiva.org  

AIDS is still an incurable disease, although patients' suffering can certainly be alleviated. Counselling, support and a multidisciplinary approach (social workers, nursing, physicians) are important and include:

discussing the diagnosis (pretest counseling, life expectancy) how transmission of the virus takes place how transmission can be prevented (safe sex, sterile needles, mother-to-child via avoiding breast-feeding and perinatal antiretrovirals) how one can best preserve health for as long as possible how to tackle the many AIDS problems that occur introduction to social workers and nursing staff

Above all, listen to the patient. Patients will benefit from a friendly empathic doctor listening to their story, being able to give a clear and logical explanation for their discomfort and reassuring them that they are not alone in this.

Treatment, lab tests for new diagnosed people

Besides two different serological tests performed on two different blood samples, a determination of the CD4-count and the viral load, some other tests are routinely performed in well-equipped settings.

Complete blood count: anemia may contraindicate use of zidovudine Electrolytes, blood urea nitrogen, creatinine, fasting blood sugar: abnormal renal function may contraindicate use of tenofovir or indicate need for adjustments of renally excreted nucleoside or nucleotide analogues. Diabetes may contraindicate use of protease inhibitors (which cause insulin resistance) Bilirubin, alkaline phosphatase, and transaminases: Indinavir and atazanavir can elevate indirect bilirubin levels. If liver function tests are disturbed, avoid hepatotoxic drugs. Creatine kinase: if not due to trauma, such as IM injections or heavy physical exercises, one should beware of HIV myopathy. A baseline value is useful to monitor zidovudine therapy. Amylase, lipase: beware of pancreatitis, e.g. didanosine Fasting lipid profile: certain protease inhibitors cause hyperlipidemia Serology syphilis, hepatitis A, B, C, toxoplasmosis, CMV Cervical Papanicolaou smear (± anal screening for HPV?): increased risk of cervical neoplasia in papiloma virus infections. The role of Cervarix (HPV vaccine) is this context is nor clear yet. Tuberculin skin test: if positive (induration > 5 mm) and active TB is ruled out, INH therapy for 9 months can be considered Chest X-ray to provide a baseline film Viral resistance profile is performed in high-risk settings

Treatment, overview antiviral agents

    Class HAART anno 2010   Entry inhibitors    Celsentri (maraviroc)   Fusion inhibitors    Fuzeon (enfuvirtide) Non-nucleoside reverse transcriptase inhibitors      Viramune (nevirapine)   Stocrin (efavirenz) Rilpivirine (Edurant)   Intelence (etravirine)   Nucleoside reverse transcriptase inhibitors      Retrovir (zidovudine)   Epivir (lamivudine)   Zerit (stavudine)   Ziagen (abacavir)  Videx (didanosine)   Emtriva (emtricitabine)     Nucleotide reverse transcriptase inhibitors    Viread (tenofovir)   Integrase inhibitors    Isentress (raltegravir) Protease inhibitors    Aptivus (tipranavir)   Reyataz (atazanavir)   Prezista (darunavir)  Telzir (fosamprenavir)   Crixivan (indinavir)   Kaletra (lopinavir + ritonavir)   Viracept (nelfinavir; removed from market)   Invirase (saquinavir)     Protease inhibitor booster    Norvir (ritonavir) Combined agents    Combivir (lamivudine + zidovudine)   Trizivir (lamivudine + zidovudine + abacavir)   Kivexa (lamivudine + abacavir)   Truvada (emtricitabine + tenofovir) Eviplera (rilpivirine, emtricitabine, tenofovir)   Atripla (emtricitabine + tenofovir + efavirenz)          

Entry inhibitors Two cellular receptors, CD4 and either CCR5 or CXCR4 are used by HIV to latch onto the CD4-positive host cell. CCR5 or CXCR4 are known as co-receptors. On the surface of the viral enveloppe, two sets of ligands attach to CD4 and CCR5/CXCR4. Entry inhibitors block the interaction between the ligands on the virus and their receptors on the cell surface. They act by binding to or altering the receptor sites. Since they act on the cellular level instead of the viral level, it is expected that there will be less resistance caused by viral mutations.  R5 viruses generally predominate early in infection and disease. Those using the chemokine CXCR4 receptor (X4 tropic strains) are more often present late in disease. The human co-receptor CCR5 can be blocked. This antiviral mechanism is different from that of other antivirals. Maraviroc (Celsentri) was the first in this class. Others are vicriviroc and aplaviroc. Fusion inhibitors Fusion inhibitors prevent HIV from entering a CD4 positive cell by blocking fusion of the outer viral membrane with the cell membrane. Fuzeon (enfuvirtide) is available as an injection, not under pill form. Non-nucleoside reverse transcriptase inhibitors NNRTIs bind to reverse transcriptase and inhibit this enzyme. This prevents the conversion of viral RNA to DNA, which is essential in the life cycle of HIV. Viramune (nevirapine), Stocrin (efavirenz) and Intelence (etravirine) are examples of this class. Nucleoside / Nucleotide analogues These molecules act as false substrates for reverse transcriptase, causing premature DNA chain termination. Retrovir (zidovudine), Epivir (lamivudine), Zerit (stavudine), Hivid (zalcitabine), Ziagen (abacavir), Emtriva (emtricitabine) and Viread (tenofovir) belong to this class. Combivir, Trizivir, Kivexa and Truvada are combinations of drugs in this class. Integrase inhibitors When viral RNA has been converted to DNA, the newly minted DNA chain needs to be integrated into the host's chromosomes. The enzyme which facilitates this integration is a target for anti-HIV drugs. Raltegravir (Isentress) was the first in this class to become available.  A tablet of raltegravir contains 400 mg and is given twice per day. Protease inhibitors Viral-coded protease is an enzyme needed for maturation of new HIV particles. When this enzyme is blocked, viral assembly is disturbed, and no new virus can be produced. Examples of this class are Norvir (ritonavir, often used for boosting), Crixivan (indinavir), Invirase (saquinavir), Kaletra (lopinavir boosted), Aptivus (tipranavir), Reyataz (atazanavir), Telzir (fosamprenavir), Viracept (nelfinavir), Prezista (darunavir).

Timeline of the development of different antiretroviral drugs (used in HIV-AIDS). Copyright ITM

AIDS medication, some examples. Combination therapy is essential. Photo Dr Van den Enden. Copyright ITM

The various antiviral drugs are becoming more and more available in developing countries, but the situation still needs to improve. 

Reverse transcriptase inhibitors

There are several types of reverse transcriptase inhibitor

nucleoside analogues non-nucleoside analogues nucleotide analogues

NUCLEOSIDE  ANALOGUES

A nucleoside consists of a sugar or saccharide (ribose or deoxyribose) and a base (pyrimidine or purine). Nucleoside compounds have to be metabolised to triphosphate compounds before they become active, in contrast to non-nucleoside compounds, which are directly active. As nucleoside analogues have to be phosphorylated in the cell before becoming active, antagonism between molecules that use the same phosphorylation pathways should be taken into account. Hence AZT and d4T are not combined with each other. Furthermore, combinations of drugs with a similar toxicity profile should be avoided.

Various drugs in this class can disturb the gamma-DNA polymerase in the mitochondria, resulting in mitochondrial dysfunction. This is characterised by myopathy, cardiomyopathy, neuropathy, liver steatosis and/or lactate acidosis. Note over terminology: Humans have 5 different DNA polymerases: alpha (replication lagging strand nuclear DNA), beta (repair nuclear DNA), gamma (replication mitochondrial DNA), delta (replication leading strand nuclear DNA), epsilon (repair nuclear DNA). Do not confuse these with the prokaryotic DNA polymerases I, II and III.

The intracellular half-life of the nucleoside-analogue triphosphates shows considerable variation. If the halflife is longer, it is "more forgiving" when a dose is forgotten or skipped.

  Drug     intracellular halflife (hours)  Zidovudine     3  Didanosine   25-40  Zalcitabine     3-4  Stavudine     3-4  Lamivudine   14-18  Abacavir   12-14  Emtricitabine     39  Tenofovir   50

Zidovudine  ( Retrovir ®). Nucleoside analogs in sponges eventually led to the design of zidovudine, which became available in 1987 (sponges do not make AZT itself). Azidothymidine or zidovudine (Retrovir®, AZT) is a substance resembling thymidine, one of the constituents of DNA.  After incorporation into a growing RNA-DNA heteroduplex, AZT prevents further synthesis of the chain. Hence virus replication in a newly infected cell is impaired and further infection of healthy cells is prevented since no progeny virus are produced. Its principal side-effect is bone marrow toxicity, though this is not very frequent (2% severe anaemia after 18 months' therapy). Macrocytosis is quite frequent. Reversible myopathy sometimes occurs. A blue discolouration of finger- and toenails and mucosae can occur. There are no major drug interactions, though other myelotoxic drugs such as pyrimethamine or ganciclovir are best avoided. Use of Retrovir® should be avoided if the patient suffers from severe anaemia, leukopaenia or persistent muscle pain. It must not be combined with D4T (Zerit®). The favourable effect of azidothymidine monotherapy is short-lived and resistant mutants quickly appear. Zidovudine is combined with some other antiviral substances in one tablet, e.g. Combivir® (AZT + 3TC) and Trizivir (AZT + 3TC + abacavir).

Lamivudine  ( Epivir ®). Lamivudine or 3TC is administered to an adult as two 150 mg doses per day or one 300 mg tablet per day. It can be taken with or between meals. Lamivudine undergoes renal excretion and the dose should be modified in cases of kidney failure (creatinine clearance <50 ml/minute). Due to its low molecular weight and low protein binding lamivudine can be eliminated by haemodialysis. It is usually tolerated well. Headache and nausea are undesirable side-effects. Occurrence of pancreatitis and paraesthesia is rare. The combination of lamivudine and zidovudine is available as Combivir®. Persons with chronic active hepatitis B can also be treated with lamivudine (Zeffix®) together with interferon alpha-2a, for example with Roferon®-pen. This has the significant advantage of allowing oral administration (interferon can only be injected). Resistance to Epivir® occurs quite easily, via a single point mutation. Lamivudine-resistant mutants can emerge within a few weeks if viral suppression and drug pressure are insufficient. Once again this shows the importance of combination therapy. The fixed combination of abacavir 600 mg with 3TC (300 mg lamivudine) is known as Kivexa® or Epzicom®. The combination of nevirapine, lamivudine and stavudine is known as Triomune®.

Emtricitabine ( Emtriva® , FTC) is the phosphorylated form of 3TC (lamivudine, Epivir®). It can be given once per day. The fixed combination of 200 mg emtricitabine with 300 mg Viread (tenofovir) is known as Truvada®. Truvada® is taken as a once daily tablet.

Stavudine  ( Zerit® ). This exists as capsules of 30 mg and as a solution of 1 mg/ml. The 40 mg tablets have been abandoned. The dose is 30 mg BD for adults. Combination with zidovudine is not advised. The principal side-effect is peripheral neuropathy, often occurring at a late stage and frequently irreversible.  Like all reverse transcriptase inhibitors, Zerit® also inhibits DNA polymerase, the enzyme that catalyses replication of mitochondrial DNA. The ratio of mitochondrial DNA to nuclear DNA decreases during treatment. A number of side-effects of the drug can be interpreted as "mitochondriopathy". Due to the impaired function of these energy-producing cell organelles there is an increased risk of hyperlactataemia, lipoatrophy and even lactate acidosis. Myopathy and neuropathy can also occur.

Didanosine   (Videx EC®). Didanosine (ddI or dideoxyinosine) became available in the West in 1992. The principal side-effects are pancreatitis as well as gastro-intestinal, liver and neurological abnormalities, and sometimes lactate acidosis. Pancreatitis is a contra-indication. The enteric coated formulation Videx EC permits the drug to be administered once per day on an empty stomach, i.e. 400 mg enteric coated tablet for an adult person weighing more than 60 kg. Beware of drug interaction with tenofovir.

Abacavir  (Ziagen®). The drug is chemically related to 2'-deoxyguanosine. Like all NRTIs (Nucleoside Reverse Transcriptase Inhibitors), abacavir is a prodrug that must be phosphorylated intracellularly in order to be activated. It is, however, not a substrate for enzymes that phosphorylate other NRTIs. An immunological mediated hypersensitivity reaction affects 5 to 8% of patients during the first 6 weeks of treatment. These side-effects include fever, skin rash, gastrointestinal tract symptoms and a fluelike illness with cough. The severity increases if the medication is continued. In case of hypersensitivity, immediate discontinuation is mandatory. Although symptoms of hypersensitivity can be difficult to distinguish from other conditions, oral rechallenge is contraindicated. An epicutaneous patch test can be used in patients who took abacavir in the past, but cannot be used as a screening tool in people who did not yet take abacavir. The HLA-B*5701 gene is strongly associated with a hypersensitivity reaction to abacavir (Ziagen, also in present in Kivexa and Trizivir). Patients who are considering treatment with abacavir should have a test to screen for the presence of this gene prior to starting treatment with abacavir. The drug is rapidly absorbed (both from tablets and from syrup). This is not affected by food. The product has a high bioavailability, 83%. Penetration into the cerebrospinal fluid is good (30-44%). Elimination from the body is not dependent on the P450 isoenzyme complex (reduced risk of drug interactions). The long intracellular halflife allows QD dosing and fixed-dose combination with lamivudine (cfr Kivexa, Trizivir).

Kivexa®. The fixed combination abacavir with epivir is known as Kivexa® or Epzicom (USA). The combination of efavirenz and Kivexa® makes QD therapy possible.

Chemical structure of abacavir, Ziagen. HIV - AIDS treatment. Copyright ITM

Trizivir ®. The combination zidovudine, lamivudine and abacavir (Trizivir®) has been available on the market since 2002. The patient should take one tablet twice per day. This scheme substantially improves patient adherence. It is probably less effective with high viral loads and when the CD4-cell count is less than 200. It is not used anymore as a first-line therapy, but sometimes as maintenance therapy when the viral load has become undetectable with other drugs. Atripla ®.  The combination of efavirenz 600 mg + emtricitabine 200 mg + tenofovir 300 mg in a single pill allows for one-pill-per-day treatment. It is equivalent to taking Stocrin + Emtriva + Viread. Side effects associated with Atripla include:  Dizzyness, unusual dreams, concentration problems and depression, more often in patients with previous mental illness Renal problems which can manifest al polyuria, thirst, myalgia and and muscle weakness Liver problems, more frequent in patients with viral hepatitis Osteopenia (due to the tenofovir component) Rash and nausea Eviplera ® The combination of rilpivirine, emtricitabine and tenofovir disoproxil fumarate can be administered once per day and has to be taken with a meal.

NON-NUCLEOSIDE  RT-INHIBITORS

These substances bind to reverse transcriptase and inhibit the enzyme.

Etravirine (Intelence) is usually given as 100 mg twice per day, following a meal. Etravirine was approved by the FDA in 2010 for treatment of HIV. Rilpivirine is another diarylpyrimidine which resembles pyrimidine nucleotides found in DNA. It is a potent inhibitor of HIV reverse transcriptase. Human immunodeficiency virus often mutates, thereby developing resistance to antiretroviral drugs. Rilpivirine tends to remain effective, seemingly because the drug shifts its shape enabling it to hold on to the binding pocket of the mutated target enzyme. At a dose of 25 mg, it is a part of Eviplera, a triple component drug.  

Nevirapine   (Viramune®). The recommended dose is one 200 mg tablet/day for 2 weeks, followed by one 200 mg tablet twice daily. This initial regimen is necessary as the compound induces its own breakdown. If initially 400 mg per day is given this would result in overdosing. Hypersensitivity reactions with skin rash occur. The blood level falls by 35% if St John's wort is taken at the same time (interaction via CYP3A4).  Nevirapine is not recommended in patients with a good immune status (CD4 > 250 in women and > 400 in men), because of enhanced risk of liver toxicity. When treatment is started, liver function tests should be monitored bimonthly during the first 3 months. In many countries, certain generic fixed drug-combinations, such as d4T, 3TC and nevirapine are available.

Chemical structure of nevirapine - Viramune. HIV - AIDS treatment. Copyright ITM

Efavirenz  ( Stocrin®, Sustiva®). The drug is administered in the evening as a single 600 mg dose.  Skin rash does occur in about 20%, so the patient needs to be warned on forehand. Alterations in the patients' psychological condition, such as dizzyness, restlessness, nightmares, etc.... are bothersome and can lead to discontinuation of the drug. These side-effects usually disappear within the first four weeks. Previous depression is a contraindication for efavirenz. It lowers the blood level of indinavir, so that the latter has to be given in higher doses. Efavirenz has a long half-life of 48 hours, and hence is more "forgiving" when a dose is missed. Efavirenz is not used in the early stages of pregnancy, as it is teratogenic in monkeys and apes in the first three months of gestation.  

Chemical structure of efavirenz - Stocrin. HIV- AIDS treatment. Copyright ITM

NUCLEOTIDE  ANALOGUES

Tenofovir   ( Viread ®). Tenofovir disoproxil fumarate (Viread®) is administered as a 300 mg tablet once a day, preferably with a light meal. It has less side-effects than most other drugs. Nucleotide analogues closely resemble nucleoside analogues such as Retrovir® or Epivir®. The only difference is that nucleotide analogues are chemically preactivated and thus have to undergo less biochemical processing in the body before becoming active.  The fixed combination tenofovir with emtricitabine is known as Truvada®.

FUSION INHIBITORS

Enfuvirtide  (T20, pentafuside, Fuzeon ®) prevents the fusion of viral and cell membranes, a critical step in the infection process. After locking onto a CD4 cell, the glycoprotein gp41 in the viral membrane must undergo a conformational change to expose a fusion peptide. Enfuvirtide, a 36-aminoacid peptide, binds to one of two heptad-repeats in gp41 and prevents this conformational change. Enfuvirtide needs to be injected SC twice per day (90 mg BD for an adult), which is a disadvantage. Patients can learn self-injection, which illustrates the importance of supporting nursing staff. Enfuvirtide is associated with increased headache, insomnia, peripheral neuropathy and depression. A typical side effect of enfuvirtide is an ennoying injection site reaction. Good patient education (rotation of injection sites) is important. It is rather expensive.

PROTEASE  INHIBITORS

In contrast to AZT and similar molecules, protease inhibitors inhibit maturation of HIV proteins by inhibiting a viral enzyme (protease) that cleaves viral protein precursors into their separate components. Protease inhibitors have the disadvantage that a large number of pills have to be taken. There are numerous side-effects. Multiple interactions with other drugs are possible. Lipodystrophy with redistribution of body fat, hypercholesterolaemia, hypertriglyceridaemie and diabetes mellitus can occur. Insulin resistance may occur due to the blockade of certain cellular saccharide transport molecules. There is evidence that protease inhibitors such as indinavir and saquinavir inhibit the development of Kaposi's sarcoma, not only because of their antiviral effect but also by direct interference with the angiogenesis in the tumour.

Saquinavir     (Invirase®).   An earlier formulation of soft gel capsules known as Fortovase has been withdrawn. The older hard gel capsules have been replaced by 500 mg tablets, which reduced the pill burden. Invirase® is poorly absorbed from the gastrointestinal tract (bioavailability about 4%). Boosting with ritonavir raises the blood level of saquinavir. Ingestion together with grapefruit juice considerably increases the absorption (see also cytochrome P450). Ritonavir-boosted saquinavir should not be taken with rifampicin (high risk of liver toxicity). The approved dosage of "Invirase 500 mg tablets" is 1000 mg taken with 100 mg ritonavir twice daily with a light meal. A once daily dosing (2000/100) is an alternative which can be used if patient adherence is problematic, but only if an undetectable viral load has been achieved previously.

Ritonavir   (Norvir®). This is very potent antiviral product, but is frequently poorly tolerated due to gastrointestinal side-effects. Nausea and perioral paraesthesias are frequent. Ritonavir needs to be given only twice per day, preferably with meals. The syrup contains 40% alcohol. The bitter taste can be masked by mixing it with chocolate milk. It is a potent cytochrome P450 (CYP3A4) inhibitor, both in the intestinal wall and liver. This is the most important enzyme for the metabolism of protease inhibitors. There is a substantial first-pass metabolism. There are many interactions with other medications, including, among others, saquinavir and indinavir, so that the dose of these latter substances can be reduced (inhibition of breakdown). It is at present mainly used for its "booster effect" and not so much for its direct antiviral effect. Kaletra® is an example of a fixed boosted combination.

Indinavir (Crixivan®). This substance is usually tolerated quite well. It should be boosted with ritonavir, otherwise the pill burden would be rather large. It is taken on an empty stomach. Indinavir penetrates into the cerebrospinal fluid and should also play a part in the prevention of AIDS dementia (HIV-encephalopathy). There are many interactions with other medications. If St John's wort is taken at the same time the blood level falls by 80%. Nephrolithiasis (indinavir crystals), renal insufficiency and haemolytic anaemia can occur. It is advisable to drink plenty of fluid when taking Crixivan®. Several studies demonstrated that the 400/100 bid formulation is as effective as the 800/100 in naive patients, but causes less side effects.

Nelfinavir   (Viracept®). This is not used as a first-line drug, except in pregnant women. The 250 mg tablets are initially taken in a dose of 750 mg three times a day, or even 1250 mg BD, depending on gastrointestinal tolerance. Undesirable side-effects of nelfinavir mesylate include gastrointestinal disorders such as diarrhoea. Monitoring liver function is advised. Nelfinavir and reyataz are unboosted protease inhibitors. Among the protease inhibitors, nelfinavir has been extensively studied in perinatal prevention. Nelfinavir also shows some promise as anticancer drug. It induces stress of the endoplasmatic reticulum which leads to apoptosis. The hyperlipidemia and hyperglycemia often seen in HIV-infected patients taking protease inhibitors, might be controllable / acceptable in oncologic conditions. Further study is needed.

Fosamprenavir   (Telzir®) is a prodrug of amprenavir, a now obsolete drug earlier known as Agenerase ® . It has the advantage of a convenient treatment regimen to encourage long-term patient adherence: one tablet of 700 mg, twice per day. It is best combined boosted with ritonavir.

Lopinavir/ritonavir  (Kaletra®). Lopinavir is a protease inhibitor. The compound has been marketed in a fixed combination with ritonavir (both drugs in one tablet). Ritonavir is not actually used here as an antiviral agent as such, but serves to raise the plasma concentration of lopinavir via inhibition of breakdown of the latter. Diarrhea is a frequent side-effect. Compared with the capsule formulation, Kaletra tablets require fewer doses to be taken each day (2 tablets of 200/50 mg BID), they do not need to be refrigerated, are better tolerated and do not need to be taken with food. Therefore, the tablet formulation is a clear improvement over the older capsule form.

Atazanavir   (Reyataz® ). Atazanavir is a protease inhibitor that needs to be given only once per day with a light meal. The medicament does not cause any hyperlipidaemia, in contrast to other drugs of the same class. Indirect hyperbilirubinaemia can occasionally occur without liver function disturbances. The genetic barrier against boosted atazanavir resistance is higher than for most other antiretroviral drugs. Often the combination atazanavir-ritonavir will be used ("boosted atzanavir)", especially in patients with increased cardiovascular risk.

Tipranavir (Aptivus®) .  Tipranavir shows no cross-resistance with the first-generation protease inhibitors. It is a potent and non-peptidic HIV-1 protease inhibitor, the first of its kind. It can be "boosted" by subtherapeutic levels of ritonavir (e.g. TPV/r 500/200 mg twice per day). The most common side effects are gastrointestinal disturbances (diarrhoea, nausea, vomiting). Initial studies showd tipranavir to be active against HIV isolates which where resistant to other protease inhibitors.

Darunavir (Prezista®) is a protease inhibitor. Prezista should be boosted with ritonavir. It is used at present in antiretroviral treatment-experienced adult patients, such as those with HIV-1 strains resistant to more than one protease inhibitor. Prezista has a high genetic barrier to resistance. The most common side effects are nausea, diarrhea, nasopharyngitis and insomnia.

INTEGRASE  INHIBITORS

Raltegravir : HIV-1 integrase is an essential viral enzymes. It represents a distinct therapeutic target, and blocking it prevents the insertion of HIV DNA into the genome of the host cell.  Without integration into the nucleus, there is no viral gene expression nor replication. As raltegravir targets a different viral enzyme, it would be expected to maintain activity against HIV-1 resistant to the other classes of ARV-drugs.  It was studied in both naïve and multiresistant patients, with extremely good results and a safety profile comparable with that of placebo. In treatment naïve patients under monotherapy a mean viral load drop of 2 log  after 10 days was observed. The dosing of 400 mg bid, food independent, will be taken further into studies. It’s primarily metabolised by glucuronidation and has no inductive or inhibitory potential in vitro. Its exact place in therapy will need to be defined.

CHEMOKINE  RECEPTOR  ANTAGONISTS

Maraviroc : Maraviroc (Celsentri TM ) is a chemokine receptor antagonist which blocks the CCR5 receptor. When the CCR5 receptor is unavailable, ‘R5-tropic’ HIV cannot attach to a CD4 T-cell. This variant of the virus is common in earlier HIV infection, while ‘X4-tropic’ viruses adapted to use the CXCR4 receptor gradually become dominant later in disease. Celsentri is indicated as part of rescue-therapy for people infected with CCR5-trophic HIV-1. The dose is 150 mg, 300 mg or 600 mg BD, depending on eventual interactions with other medication. It can be taken with or without food. Nausea, diarrhea, elevated liver enzyms, dizzyness, cough, rash, myagia, asthenia and headache are among the reported side-effects. Vicriviroc is another entry inhibitor, still in the experimental stage.

OTHER  ANTIRETROVIRAL  DRUGS

Etravirine (TMC-125) is under development. Rilpivirine (TMC-278) is under development. The skin glands of some frogs, like the Australian red-eyed tree frog Litoria chloris , secrete peptides with remarkable antiretroviral activity. More study is needed. Obsolete compounds include Hydroxyurea (Hydrea®) is not used anymore. It has an antiviral activity and is synergistic with ddI, but potentiates the toxicity of the latter (idem D4T). The mode of action is still not clear, though hydroxyurea probably inhibits the synthesis of deoxynucleotides by blocking the enzyme ribonucleotide reductase. Delavirdine  (Rescriptor®):  not available in Europe. It is associated with maculopapulous rash and itching, fever, conjunctivitis and joint pain. Loviride  is no longer used. Adefovir dipivoxil  (Preveon®). This is a nucleotide-analogue and has been abandoned. This substance contains a phosphoryl group and does not need to be phosphorylated (does not require any intracellulair transformation to form an active molecule). Due to possible side-effects on the mitochondria, it was best taken together with L-carnitine. The latter compound has an important role in the transport of long-chained lipids through the membranes of mitochondria. These latter have a role in energy production (beta-oxidation) and in the production of cardiolipine, a phospholipid (diphosphatidylglycerol). Amprenavir has been replaced by fosamprenavir. Zalcitabine (Hivid) is not used anymore. This was given in a dose of 0.75 mg three times per day, and did not need to be given with meals. It was not a very potent drug. The principal side-effects were peripheral sensomotor neuropathy. It could not be combined with Epivir®. Note: hepatitis B Co-infection with HIV and hepatitis B or C is common. Drugs for hepatitis B include interferon alpha-2b (available since in 1992), lamivudine (1998), adefovir dipivoxil (2002), entecavir (2005), pegylated interferon alfa-2a (2005) and telbivudine (2006). Especially entecavir needs more study to place it in this context (slight antiretroviral activity). Note: Bonemarrow transplant of CCR-delta32 donor An HIV-infected patient with acute leukemia who underwent stem cell transplantation from a donor who was homozygous for the CCR5 delta32 mutation had no HIV viral rebound despite discontinuation of antiretroviral therapy

FIXED  DRUG  COMBINATIONS

Combivir   : lamivudine + zidovudine Trizivir     : lamivudine + zidovudine +  + abacavir Triomune  : lamivudine + stavudine + nevirapine Kivexa      : abacavir + epivir Truvada    : emtricitabine + tenofovir Atripla      : emtricitabine + tenofovir + efavirenz Kaletra     : lopinavir + ritonavir (= fixed boosted PI)

Penetration of medication into CSF

    Low        Intermediate             High  Tenofovir             Stavudine  Zidovudine        Didanosine   Lamivudine  Abacavir  Zalcitabine  Emtricitabine  Delavirdine  Nelfinavir  Efavirenz  Nevirapine  Ritonavir  Amprenavir  Saquinavir  Fosamprenavir   Enfuvirtide  Atazanavir  Indinavir

Treatment, therapeutic schemes

Combinations of drugs are at present being used in the West (compare with tuberculosis and leukaemia chemotherapy). Various cocktails, that often have to be individually adapted, are used. As first-line treatment the WHO advises a scheme with 2 nucleoside analogues in combination with an NNRTI ("highly active antiretroviral therapy" or "HAART"). Alternatively, the combination of 2 nucleoside analogues and a protease-inhibitor can be used. Zidovudine, stavudine, lamivudine, abacavir, nevirapine and efavirenz penetrate into the cerebrospinal fluid and can thereby have a preventive effect on the occurrence of AIDS dementia. Any combination treatment should, therefore, contain one of these molecules. Videx® does not penetrate very well into the cerebrospinal fluid.

The recommended starting combination therapy in a drug-naive patient consists of: 

First line: two nucleoside analogues + one NNRTI or First line: two nucleoside analogues + one boosted PI

Examples of initial treatment regimens with minimal patient inconvenience:

Efavirenz (Sustiva) 600 mg once daily + Truvada (tenofovir 300 mg + emtricitabine 200 mg as fixed-dose combination)

Combivir:  2 x 1 per day, in combination with Kaletra® 2 x 3 per day

Combivir: 2 x 1 per day, with Viramune 2 x 1 per day (for the first two weeks 1 per day) Triomune is in wide-spread use in resource-poor settings

Clinicians are aware that the development of resistance is a significant problem in achieving long-term success with highly active antiretroviral therapy (HAART). However, resistance is not an all or none phenomena, but instead comes in gradations. These gradations are defined by the increase in the inhibitory concentration of a particular drug to a level higher