AFRICAN SLEEPING SICKNESS

INTRODUCTION: The Disease and Importance

Human African trypanosomiasis (HAT), also called sleeping sickness, is an illness endemic to sub-Saharan Africa. It is caused by the flagellate protozoan Trypanosoma brucei, which exists in 2 morphologically identical subspecies: Trypanosoma brucei rhodesiense (East African or Rhodesian African trypanosomiasis) and Trypanosoma brucei gambiense (West African or Gambian African trypanosomiasis). Both of these parasites are transmitted to human hosts by bites of infected tsetse flies (Glossina palpalis transmits T brucei gambiense and Glossina morsitans transmits T brucei rhodesiense), which are found only in Africa.

The reservoirs of infection for these vectors are exclusively human in West African trypanosomiasis. However, East African trypanosomiasis is a zoonotic infection with animal vectors. African trypanosomiasis is distinct from American trypanosomiasis, which is caused by Trypanosoma cruzi and has different vectors, clinical manifestations, and therapies.

The major epidemiology factor in African trypanosomiasis is contact between humans and tsetse flies. An increasing tsetse fly density, changing feeding habits, expanding human development into tsetse fly–infested areas, and an increasing number of immunologically naïve persons in previously endemic areas, influences this interaction. Major outbreaks from 1920-1950 led to extensive treatment and, apparently, immunity for 50 years. Now, infection is occurring again as the same populations lose their immunity.

Trypanosomes are parasites with a 2-host life cycle: mammalian and arthropod. The life cycle starts when the trypanosomes are ingested during a blood meal by the tsetse fly from a human reservoir in West African trypanosomiasis or an animal reservoir in the East African form. The trypanosomes multiply over a period of 2-3 weeks in the fly midgut; then, the trypanosomes migrate to the salivary gland, where they develop into epimastigotes. The metacyclic trypomastigotes infect humans.

Although human sleeping sickness may not seem as important on the world stage as diseases such as malaria and AIDS, it is nevertheless an important disease in Sub-Saharan Africa and is responsible for a considerable degree of suffering and mortality in countries where it is endemic. Some 55 million people in 37 countries are at risk, with an estimated 50,000 new cases reported annually. Left untreated, the outcome of the disease for the individual is death, but equally insidious is the effect on communities and quality of life resulting from the debilitating symptoms. In public health terms, the effects of the disease on the community life and, in particular, the contribution of individuals to food production and community support can be measured in terms of disability-adjusted life years lost (DALYS). Human sleeping sickness is responsible for 1.78 million DALYS, and the magnitude of this effect can be seen when compared with the related disease leishmaniasis, which has a value of 2.06 million DALYS despite there being 350 million people at risk in 88 countries.

Early History

The earliest recorded account of sleeping sickness comes from upper Niger during the 14th century in the historical writings of

Ibn Khaldoun, who wrote about the disease in his account of the history of North Africa. The next report came from Guinea in 1734. In 1803, the diseases that caused visible swollen lymph glands in West Africa came to be known as Winterbottom's sign, after the description of the disease by Cris Winterbottom. Slave traders who avoided trading and buying slaves who displayed those symptoms readily recognized such signs.

The earliest detection of trypanosomes in human blood was in 1902, when R.M. Forde discovered what was then thought to be filiaria in the blood of a steamboat captain who had traveled extensively along the River Gambia. J.H. Cook in East Africa made similar discoveries of filiaria-like organisms in the blood, but confusion arose as to how filiaria worms could cause such varying clinical symptoms. J.E. Dutton first correctly identified the parasite as a trypanosome and subsequently named it Trypanosoma gambiense. In 1902, A. Castellani observed the presence of trypanosomes in cerebrospinal fluid taken from a sleeping sickness patient, but it wasn't until 1903 that D. Bruce correctly recognized that trypanosomes were the causative agents of sleeping sickness transmitted to humans by tsetse flies, and that “trypanosome fever” and “sleeping sickness” - both thought to be different diseases at the time - were in fact the same.

Morphologically indistinguishable from the West African species as well as the animal infecting species Trypanosoma brucei brucei, Trypanosoma brucei rhodensiense was first discovered in Zambia by J.W.W. Stephens and H.B. Fantham in 1910. By 1926, T.b. rhodensiense could be found along the fly-belt between Tabora and Kigoma, Tanzania. The difficulties in identifying this virulent form of sleeping sickness lead to uncertainties today regarding the evolution and progression of T.b. rhodensiense through the continent, although it is generally agreed upon that it originated from the West African form.

The earliest recorded major epidemics of sleeping sickness took place in Uganda and Congo between 1896 and 1908, where roughly 500,000 people were estimated to have died in the Congo Basin and approximately 300,000 died in Busoga, Uganda. With the Rift Valley transecting the country, Uganda is in the precarious position of having foci of both forms of diseases which resulted in two other major epidemics of sleeping sickness - one in the late 1940's and another in 1980. Throughout West Africa, smaller epidemics of sleeping sickness rapidly spread from Senegal to Cameroon during the 1920's, and died down by the late 1940's.

ETIOLOGY

There are two clinical forms of African trypanosomiasis (also called sleeping sickness); each is named for the region of Africa in which they were found historically.

East African sleeping sickness is caused by the parasite Trypanosoma brucei rhodesiense, commonly found in Uganda, Kenya, Tanzania, Malawi, Ethiopia, Zaire, Zimbabwe and Botswana.

East African sleeping sickness is an acute or rapidly progressing disease that typically leads to death within weeks or months if not treated. The initial bite leaves a distinctive sore spot called a chancre. Symptoms, which appear one to four weeks after infection, may include swollen lymph nodes, irritability, fever, severe headache, fatigue, muscle and joint pain, and a skin rash. During the second stage of the disease, the parasite crosses the blood-brain

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barrier and attacks the central nervous system. Neurological complications include slurred speech, confusion, and difficulty with walking.

West African Sleeping Sickness also called Gambian sleeping sickness is caused by a parasite called Trypanosoma brucei gambiense commonly found in Democratic Republic of Congo, Angola, Sudan, Central African Republic, Republic of Congo, Chad and Northern Uganda.

Like East African sleeping sickness, West African (Gambian) sleeping sickness is a serious slow developing disease that is fatal if not treated. However, symptoms may not appear for months to years after the initial infection in a chronic disease. Initial symptoms include swollen lymph nodes, swelling of face and hands, skin rash, fever, severe headaches, muscle and joint pain, and fatigue. Weight loss is common as the disease progresses. Neurological impairment occurs during the second stage in which the victim can experience personality changes, slurred speech, changes in sleep patterns, progressive confusion, difficulty with walking, and seizures.

BACTERIOLOGY

The Trypanosome

Trypanosomes have been around for more than 300 million years. They are microscopic spindle-shaped unicellular protozoan of genus Trypanosoma, which are ubiquitous parasite of insects, plants, birds, bats, fish, amphibians and mammals. Because they have been around for so long, they and their natural hosts have evolved together to ensure their mutual survival. Trypanosomes can be found from Nairobi to New York, from Sydney to San Francisco, and from Birmingham to Buenos Aires. Fortunately, few species of trypanosomes are pathogenic. Trypanosomes, and other parasites, mainly cause disease when they spread to new hosts, like humans and their domestic animals, especially recent imports into endemic areas of species that diverged since continent separated.

African trypanosomes are extracellular organisms, both in the mammalian and insect host. T. b. gambiense and T. b. rhodesiense are morphologically indistinguishable, measuring 25-40 µ m in length. Infection in the human host begins when the infective stage, known as the metacyclic stage, is injected intradermally by the tsetse fly. The organisms rapidly transform into blood-stage trypomastigotes (long, slender forms), and divide by binary fission in the interstitial spaces at the site of the bite wound. The buildup of metabolic wastes and cell debris leads to the formation of a chancre.

Trypanosomes have a single specialized mitochondrion called a kinetoplast mitochondrion. One of its unusual features is that the entire DNA of the mitochondrion, which can be up to 25% of the total cell DNA, is localized in the kinetoplast, adjacent to the flagellar pocket. Kinetoplast DNA or kDNA exists in two forms: mini-circles and maxi-circles. Mini-circle DNA encodes guide RNAs that direct extensive editing of RNA transcripts post-transcriptionally. Maxi-circle DNA contains sequences that, when edited, direct translation of typically mitochondrially encoded proteins.

In the vertebrate host, trypanosomes depend entirely upon glucose for energy and are highly aerobic, despite the fact that the kinetoplast-mitochondrion completely lacks cytochromes. Instead, mitochondrial oxygen consumption is based on an alternative oxidase that does not produce ATP. When in the insect vector, the parasite develops a conventional cytochrome chain and TCA cycle.

The surface of the trypanosome has numerous membrane-associated transport proteins for obtaining nucleic acid bases, glucose, and other small molecular weight nutrients. None of these proteins reacts well with antibodies, because although they lie in exposed regions of membrane, they are shielded by allosteric interference provided by the variant surface glycoprotein (VSG) coat proteins. This flagellated stage enters the bloodstream through the lymphatics and divides further, producing a patent parasitemia. The number of parasites in the blood is generally so low that diagnosis by microscopic examination is often negative. At some point, trypanosomes enter the central nervous system, with serious pathological consequences for humans. Some parasites transform into the non-dividing short, stumpy form, which has biochemistry similar to those of the long, slender form and the form found in the insect vector.

The tsetse fly becomes infected by ingesting a blood meal from an infected host. These short, stumpy forms are pre-adapted to the vector, having a well-developed mitochondrion with a partial TCA cycle. In the insect vector, the trypanosomes develop into procyclic trypomastigotes in the midgut of the fly, and continue to divide for approximately 10 days. Here they gain a fully functional cytochrome system and TCA cycle. When the division cycles are completed, the organisms migrate to the salivary glands, and transform into epimastigotes. These forms, in turn, divide and transform further into metacyclic trypanosomes, the infective stage for humans and reservoir hosts. The cycle in the insect takes 25-50 days, depending upon the species of the fly, the strain of the trypanosome, and the ambient temperature. If tsetse flies ingest more than one strain of trypanosome, there is the possibility of genetic exchange between the two strains, generating an increase in genetic diversity in an organism that may not have a sexual cycle.

Flies can remain infected for life (2-3 months). Tsetse flies inject over 40,000 metacyclic trypanosomes when they take a blood meal. The minimum infective dose for most hosts is 300-500 organisms, although experimental animals have been infected with a single organism. Infection can also be acquired by eating raw meat from an infected animal. In East Africa, this mode of transmission may be important in maintaining the cycle in some reservoir hosts.

We generally associate trypanosomes with disease in Africa and South America. African trypanosomiasis is commonly known as Sleeping Sickness in humans and Nagana (meaning “loss of spirit” in Zulu language) in cattle.

Many trypanosomes do not appear to harm their hosts, but a number of species cause serious disease in humans or domestic animals. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense causes African sleeping sickness and is transmitted

Trypanosomes in the blood of vertebrates have been observed to have three body types; a short, broad form often without a flagellum, called the promastigote; a long and narrow form, the trypomastigote, and an intermediate between these two, the epimastigote. In arthropod hosts, all developmental stages have been reported to derive from an amastigote stage, which lacks a flagellum

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and has a circular shape. Trypomastigote form may be active and proliferative, at this stage they are referred to as metacyclic, which is presumed to be the infective stage for vertebrates.

Amastigote - Basal body anterior of nucleus, with a short, essentially non-functional, flagellum.

Promastigote - Basal body anterior of nucleus, with a long detached flagellum.

Epimastigote - Basal body anterior of nucleus, with a long flagellum attached along the cell body.

Trypomastigote - Basal body posterior of nucleus, with a long flagellum attached along the cell body.

These names are derived from the Greek “mastig”- meaning whip, referring to the trypanosome's whip-like flagellum.

T. brucei is found as a trypomastigote in the slender, stumpy, procyclic and metacyclic forms. The procylic form differentiates to the proliferitive epimastigote form in the salivary glands of the insect.

Subs-species of Trypanosome brucei

Trypanosoma brucei spp. is an extracellular gram-negative parasitic protist species that causes African trypanosomiasis (or sleeping sickness) in humans and nagana in animals in Africa. There

are 3 sub-species of T. brucei; T. b. brucei, T. b. gambiense and T. b. rhodesiense.

T. brucei gambiense - Causes slow onset chronic trypanosomiasis in humans. Most common in central and western Africa, where humans are thought to be the primary reservoir.

T. brucei rhodesiense - Causes fast onset acute trypanosomiasis in humans. Most common in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir.

T. brucei brucei - Causes animal African trypanosomiasis, along with several other species of trypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by human apolipoprotein L1. However, as it shares many features with T. b. gambiense and T. b. rhodesiense (such as antigenic variation) it is used as a model for human infections in laboratory and animal studies.

These obligate parasites have two hosts - an insect vector and mammalian host. Because of the large difference between these hosts, the trypanosome undergoes complex changes during its life cycle to facilitate its survival in the insect gut and the mammalian bloodstream. It also features a unique and notable variable surface glycoprotein (VSG) coat in order to avoid the host's immune system. There is an urgent need for the development of new drug therapies, as current treatments can prove fatal to the patient as well as the trypanosomes

MORPHOLOGY

A sound knowledge of the basic features of the various trypanosomes enables the identification of each species and so the exact cause of the disease. Once the basic features possessed by all trypanosomes are appreciated, the diagnostic differences can be recognized and the species identified.

Basic morphology of trypanosomes

Diagram of a trypanosome Fig. 1

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The parasite consists of a single cell varying in size from 8 to over 50 μm. All the activities associated with a living organism take place within this unicellular organism — nutrition, respiration, excretion, reproduction. The substance of which all living cells consist, the protoplasm, comprises three parts, an outer protective and retaining layer, the pellicle = cell envelope = cell membrane, within which the cytoplasm forms the bulk of the contents. Suspended in the cytoplasm are various structures, the most prominent being the nucleus, which may be regarded as the command centre of the cell and which also plays a major part in reproduction. It contains DNA (deoxyribonucleic acid), which is arranged in the form of genes and chromosomes; it represents the genetic information and is responsible for the manufacture of enzymes and other proteins of the cell.

Small granules (formerly called “volutin granules”) can sometimes be seen in the cytoplasm; they may have various origins, they may be food or nuclear reserves, or result from a reaction between the trypanosome and the host's immune system.

Trypanosomes are thoroughly adapted to living and moving in the blood plasma or tissue fluid of the host. They are elongated and streamlined, and tapered at both ends. The pellicle, the outer layer of the cytoplasm, is flexible enough to permit a degree of body movement, while retaining a definite shape. As shown in Figure 1 a flagellum arises near to the posterior end from a parabasal body, and runs the length of the trypanosome; it may be continued beyond the anterior end of the body as a whip-like free flagellum. Along the length of the body, the pellicle and cytoplasm are pinched up into a thin sheet of tissue called the undulating membrane, through the outer margin of which runs the flagellum, as shown in Figure 1.

Among other basic morphological features, a distinct well-defined body, the kinetoplast, is seen near to the posterior end of the trypanosome and differs in size and position according to the species. It is adjacent to the parabasal body (from which the flagellum arises), and so close to it that it cannot easily be seen separately with the light microscope. The kinetoplast has important functions in reproduction and metabolism and is probably essential for cyclical transmission by tsetse flies. (It is sometimes absent in a proportion of. Trypanosomes, especially of some strains of T. evansi, a species which has lost its ability of being cyclically transmitted.) The extent of the undulating membrane and the absence or presences of the free flagellum are also precious in specific identification of trypanosomes. Other morphological characters are the average length and the shape of the body.

Differential morphology

There are distinct differences in appearance, shape and size between the various species of trypanosomes, allowing specific identification. It must be remembered, however, that in any biological material there is some variability. In addition, trypanosomes are not rigid and continuously change their shape slightly; the individual parasite seen in the stained preparation presents the shape it had now of dying. It has also been subjected to the unnatural stresses of drying out and being fixed and stained. Many variations in appearance are therefore seen. It is thus necessary to observe carefully and systematically all the features in a sufficiently large number of individual trypanosomes: only after such an examination is it possible to arrive at a reasonably accurate diagnosis. There will be examples where trypanosomes are so few, or the staining so inadequate, that identification may not be possible or only after a prolonged search. It is also essential to examine several individual trypanosomes, because even if one specimen is perfect to establish its identity beyond any

doubt, further search may reveal another species and thus a mixed infection.

For specific identification, a number of trypanosomes should be examined systematically for the presence or absence, size and position of a number of features:

Presence or absence of trypanosomes of different appearance. If all individual trypanosomes are alike, the infection is called monomorphic (of one form); if there are distinctly different types it can be either a polymorphic (= pleiomorphic) species, or a mixed infection of different species.

Presence or absence of a free flagellum. In certain species, there may be some trypanosomes with, and some without, a free flagellum.

Size of the trypanosome (expressed in μm). The size and position of the kinetoplast. The position is

related to proximity to the posterior extremity (rear end) of the organism.

The degree of development of the undulating membrane. It may be conspicuous or inconspicuous.

The shape of the parasite, particularly the shape of its posterior part. The posterior extremity may vary from blunt to pointed.

Specific morphology

The sub genus Trypanozoon (the brucei group). This group comprises five members: T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. evansi and T. equiperdum. The three subspecies of T. brucei are normally transmitted by tsetse flies (in contrast to T. evansi and T. equiperdum) and are exactly similar in morphology, but only T. brucei gambiense and T. brucei rhodesiense are the cause of human sleeping sickness, the former mainly in West and Central Africa and the latter in eastern and southern Africa. T. brucei brucei is not infective to humans.

T. brucei (see Figure 2). T. brucei is polymorphic, with three main forms, all of which have a small kinetoplast and a conspicuous undulating membrane:

Long slender forms (23–30 μm in length) with a free flagellum, which may be up to one half of the length of the organism. The posterior end is pointed and the nucleus is central. The kinetoplast is placed up to 4 μm in front of the posterior extremity.

Short stumpy forms (17–22 μm in length) normally without a free flagellum, but in which there may occasionally be individuals with a short free flagellum. The kinetoplast is usually sub terminal. The position of the nucleus varies greatly and it is in some cases in the posterior part of the cell, sometimes so far posterior that the kinetoplast is anterior to it (so-called postero-nuclear forms). There is considerable variation in appearance between short stumpy forms, from broad, squat types (which include the postero-nuclear forms) to a form similar to T. congolense, although longer. In stained specimens, blue volutin granules are often present in the cytoplasm, often arranged in a line along the margin of the cell.

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Intermediate forms, varying in length between the two previously mentioned types. A free flagellum, of varying length, is always present. The nucleus is centrally placed. The posterior end is somewhat variable in shape, but usually bluntly pointed. The kinetoplast is close to the posterior extremity. Volutin granules are occasionally present but neither as common nor as plentiful as in the short, stumpy forms.

Trypanosoma brucei blood stream forms Figure 2

The cell structure

The structure of the cell is typical of eukaryotes, see eukaryotic cell. All major organelles are seen, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus etc. Unusual features include the single large mitochondria with a condensed mitochondrial DNA structure, and its association with the basal body of the flagellum, unusually the cytoskeleton organization mechanism of the cell. The cell surface of the bloodstream form features a dense coat of variable surface glycoproteins (VSGs) which is replaced by an equally dense coat of procyclins when the parasite differentiates into the procylic in the tsetse fly midgut.

The genome

The genome of T. brucei is made up of:

11 pairs of large chromosomes of 1 to 6 megabase pairs. 3-5 intermediate chromosomes of 200 to 500 kilobase

pairs.

Around 100 mini chromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome.

The large chromosomes contain most genes, while the small chromosomes tend to carry genes involved in antigenic variation, including the VSG genes. The genome has been sequenced and is available online.

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The mitochondrial genome is found condensed into the kinetoplast, an unusual feature unique to the kinetoplastea class. It and the basal body of the flagellum are strongly associated via a cytoskeleton structure.

The cytoskeleton

The cytoskeleton is predominantly made up of microtubules, forming a sub-pellicular corset. The microtubules lie parallel to each other along the long axis of the cell, with the number of microtubules at any point roughly proportional to the circumference of the cell at that point. As the cell grows (including for mitosis) additional microtubules grow between the existing tubules, leading to semi conservative inheritance of the cytoskeleton. The microtubules are orientated + at the posterior and - at the anterior. Microfilament and intermediate filaments also play an important role in the cytoskeleton, but these are generally overlooked.

Flagellar structure

The trypanosome flagellum has two main structures. It is made up of a typical flagellar axoneme, which lies parallel to the paraflagellar rod, a lattice structure of proteins unique to the kinetoplastida, euglenoids and dinoflagellates.

The microtubules of the flagellar axoneme lie in the normal 9+2 arrangement, orientated with the + at the anterior end and the - in the basal body. The cytoskeletal structure extends from the basal body to the kinetoplast. The flagellum is bound to the cytoskeleton of the main cell body by four specialised microtubules, which run parallel and in the same direction to the flagellar tubulin.

The flagellar function is twofold - locomotion via oscilations along the attached flagellum and cell body, and attachment to the fly gut during the procyclic phase.

Variant Surface Glycoprotein (VSGs)

Trypanosomes have a specialized mechanism to overcome the obstacles of the mammalian immune system.  Days after infection, host antibodies recognize surface glycoprotein that coat the protozoa—the antigenic determinants of the organism—and kill the organisms by labeling them destruction.  However a few protozoa escape destruction via a programmed system that changes their glycoprotein composition and thus enables them to evade immune recognition.  Again, a day later the immune system recognizes this change and mounts an immune response against the new glycoprotein.  This cycle is repeated with a pattern of high parasite load followed by a period of low parasite load.  For this reason, patients exhibit an irregular pattern of high-grade fevers followed by a febrile period throughout the course of a systemic infection.  For this reason, vaccine development has been thwarted.

The VSG coat

The surface of the trypanosome is covered by a dense coat of ~1x107 molecules of Variable Surface Glycoprotein (VSG). This

coat enables an infecting T. brucei population to persistently evade the host's immune system, allowing chronic infection. The two properties of the VSG coat that allow immune evasion are:

Shielding - the dense nature of the VSG coat prevents the immune system of the mammalian host from accessing the plasma membrane or any other invariant surface epitopes (such as ion channels, transporters, receptors etc.) of the parasite. The coat is uniform, made up of millions of copies of the same molecule; therefore, the only parts of the trypanosome the immune system can 'see' are the N-terminal loops of the VSG that make up the coat.

Periodic antigenic variation - the VSG coat undergoes frequent stochastic genetic modification - 'switching' - allowing variants expressing a new VSG coat to escape the specific immune response raised against the previous coat.

Antigenic variation

Sequencing of the T. brucei genome has revealed a huge VSG gene archive, made up of thousands of different VSG genes. All but one of these is 'silent' VSGs, as each trypanosome expresses only one VSG gene at a time. VSG is highly immunogenic, and an immune response raised against a specific VSG will rapidly kill trypanosomes expressing this VSG. This can also be observed in vitro by a complement-mediated lysis assay. However, with each cell division there is a possibility that one or both of the progeny will switch expression to a silent VSG from the archive. The frequency of such a switch has been measured to be approximately 1:100. This new VSG will likely not be recognized by the specific immune responses raised against previously expressed VSGs. It takes several days for an immune response against a specific to develop, giving trypanosomes, which have undergone VSG coat switching some time to reproduce (and undergo further VSG coat switching events) unhindered. Repetition of this process prevents extinction of the infecting trypanosome population, allowing chronic persistence of parasites in the host. The clinical effect of this cycle is successive 'waves' of parasitaemia (trypanosomes in the blood).

VSG structure

VSG genes are hugely variable at the sequence level. However, for them to fulfill their shielding function, different VSGs have strongly conserved structural features. VSGs are made up of a highly variable N terminal domain of around 300 to 350 amino acids, and a more conserved C terminal domain of around 100 amino acids. The C terminal domain forms a structural bundle of 4 alpha helices, while the N terminal domain forms a 'halo' around the helices. The tertiary structure of this halo is well conserved between different VSGs (in spite of wide variation in amino acid sequence) allowing different VSGs to form the physical barrier required to shield the trypanosome's surface. VSG is anchored to the cell membrane via a glycophosphatidylinositol (GPI) anchor - a covalent linkage from the C terminus, to approximately 4 sugars, to a phosphatidylinositol phospholipid acid, which lies in the cell membrane. VSGs form homodimers.

VSG archive structure

The VSG gene archive is the collection of silent VSGs in the T. brucei genome. Some of these are full-length, intact genes; others are pseudogenes) typically with omitted sections or premature stop codons. Expression of an antigenically novel VSG can occur by simply switching to a different full-length VSG gene. However, only

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5% of the archive is made up of such complete silent VSGs. To utilize the rest of the silent VSG archive, ‘mosaic’ VSGs can be formed by replacing part of the expressed VSG with a structurally homologous region from the archive. The combinatorial nature of mosaic formation in conjunction with the huge silent VSG archive gives the parasite a theoretically limitless VSG library, and is the major barrier to vaccine development.

VSG expression

One major focus in trypanosome research is how the majority of VSG genes are kept silent, and how these genes are switched. The expressed VSG is always located in an Expression Site - found at the telomeres of the large and intermediate chromosomes. Each is a polycistronic unit, containing a number of Expressions Site-Associated Genes (ESAGs) all expressed along with the active VSG. While there are at least 20 known expression sites, only a single one is ever active at one time. A number of mechanisms appear to be involved in this process, but the exact nature of the silencing is still unclear

The VSG can be switched either by changing the active expression (from the active to a previously silent site) or by changing the VSG gene in the active site. The genome contains many copies of possible VSG genes, both on minichromosomes and in repeated sections in the interior of the chromosomes. These are generally silent, typically with omitted sections or premature stop codons, but are important in the evolution of new VSG genes. It is estimated up to 10% of the T.brucei genome may be made up of VSG genes or pseudogenes. Any of these genes can be moved into the active site by recombination for expression. Again, the exact mechanisms that control this are still only partially known.

The Trypanosome cell cycle (procyclic form)

The mitotic division of T.brucei is unusual in terms of the cytoskeletal process. The basal body, unlike a centrosome of most eukaryotic cells, plays an important role in the organization of the spindle.

Stages of mitosis:

1. The basal body replicates, both remaining associated with the kinetoplast.2. The kinetoplast undergoes replication, and the daughter kinetoplasts are separated by the basal bodies.

3. The second flagellum grows while the nucleus undergoes replication.

4. The mitochondrion divides, and cytokinesis progresses from the anterior to posterior end.

5. The division resolves. The daughter cells may stay connected for a significant length of time after cytokinesis is complete

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The process of endocytosis in trypanosomes

Trypanosomes, like all organisms, need nutrients to live and grow. The parasites obtain these nutrients from their environment by taking up molecules and particles in a process known as endocytosis. Past studies using the electron microscope have shown that trypanosomes draw in molecules by way of a structure named the flagellar pocket. Molecules binding to the surface of the trypanosome, including antibodies, have been found also to be engulfed in this way. Therefore, in addition to changing its surface coat of proteins to avoid antibody binding, the parasite seems able to take up antibodies, and possibly in this way to neutralize their effect. It was demonstrated during the year that clearance of antibodies from the surface of the trypanosome in this way renders parasites in vitro resistant to the effects of “complement”, the collective name for a group of proteins in mammalian blood so named because they complement and amplify the action of antibodies to cause the lysis (rupture) of parasites.

One approach to learning more about the physical organization of the endocytic pathway is to determine the functional activity of defined fractions and subcomponents of trypanosomes. In 1990, in collaboration with scientists from the European Molecular Biology Laboratory (Heidelberg), a free-flow electrophoresis technique was used to separate microsomal trypanosome fractions. The fractions obtained were characterized to some extent based on demonstrations of enzymatic activity.

Progress was also made during the year in purifying and characterizing a receptor molecule of T. b. brucei that recognizes and binds to transferring circulating in the blood of the mammalian host. The receptor appears to be much smaller than its mammalian equivalent. The possibility that trypanosomes have, and make use of, receptors to mammalian growth factors will be examined in the coming year.

Parasite enzymes that break down proteins, called proteases, appear to be involved in the endocytic process. A

lysosomal thiol protease of T. congolense was purified in 1990. Antibodies to the enzyme have proved to be valuable markers of the lysosome. An important discovery made during the year was that this enzyme is better recognized by antibodies in the sera of trpyanotolerant N'Dama cattle recovering naturally from infections than in Zebu cattle that required treatment to recover from infection. Furthermore, the enzyme is detectable in the sera of cattle early in an infection and so is a possible cause of the immune and physiological dysfunctions that characterize trypanosomiasis in animals. The degree of protection afforded livestock by immunization with the purified molecule, and the possibility that it is involved in the pathogenesis of the disease, are subjects of current studies.

Locomotion

Trypanosomes move actively and progress by movement of the undulating membrane and the free flagellum (when present), which acts as a kind of propeller, thus drawing themselves through the blood plasma or tissue fluid. (The free flagellum, when present, arises from the anterior [front] end of the parasite.)

Reproduction

This is by a process of division to produce two daughter cells. However, as stated above, it has been shown that exchange and recombination of genetic material may take place in the tsetse fly between two trypanosomes, but it is unknown how frequently this occurs.

The division into two daughter cells (binary fission) follows the sequence of events illustrated in Figure 3. The kinetoplast divides first. A second parabasal body develops, from which a second flagellum develops. The nucleus divides next, followed by the rest of the trypanosome body duplicating all the structures present in the cytoplasm. The body then divides into two daughter cells, beginning at the anterior end. The process is rapid, and may result in a vast population in the host within a short period.

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Division of a trypanosome Fig. 3

Habitat

Blood Lymph channel throughout the body CSF Connective Tissue Intracellular spaces Brain

Life Cycle

The life cycle of the single-celled trypanosome parasite is complex. In both the tsetse fly vector and the mammalian host, trypanosomes undergo a series of transformations into different forms. The tsetse fly ingests trypanosomes when it feeds on an animal infected by the parasite. In the fly, the trypanosomes differentiate into several forms, culminating the metacyclic form,

which is able to infect mammalian hosts. When the infected fly next feeds, these metacyclic trypanosomes are injected into the skin along with tsetse saliva. In the animal, the parasites differentiate into a bloodstream form specially adapted to live in mammalian blood. The bloodstream parasites multiply by binary fission and enter the animal's lymphatic and blood circulation. As flies feed on animals infected with the parasite, they take up blood containing trypanosomes, which then completes the life cycle.

Insects are usually involved in the natural transmission of the African pathogenic trypanosomes with which we are concerned in this field guide. When this is the case, the life cycle has two phases, one in the insect vector and one in the mammalian host. Transmission by insects may be cyclical by tsetse flies, Glossina species, or mechanical by other biting flies (but apart from transmitting trypanosomes cyclically, tsetse flies can also act as mechanical vectors).

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The tsetse fly is large, brown and stealthy. While taking blood from a mammalian host, an infected tsetse fly (genus Glossina) injects metacyclic trypomastigotes into skin tissue. The parasites enter the lymphatic system and pass into the bloodstream

1. Inside the host, they transform into bloodstream trypomastigotes

2. are carried to other sites throughout the body, reach other blood fluids (e.g., lymph, spinal fluid), and continue the replication by binary fission

3. The entire life cycle of African Trypanosomes is represented by extracellular stages. A tsetse fly becomes infected with bloodstream trypomastigotes when taking a blood meal on an infected mammalian host

4. In the fly's midgut, the parasites transform into procyclic trypomastigotes,

5. multiply by binary fission,

6. leave the midgut, and

7. transform into epimastigotes

8. The epimastigotes reach the fly's salivary glands and continue multiplication by binary fission.

MODE OF TRANSMISSION

Cyclical transmission

When a tsetse fly hatches from its pupal case, it is free from trypanosomes. Until its first bloodmeal, it is called a teneral fly. It

acquires a trypanosomal infection when feeding on a parasitaemic (= having parasites in the circulating blood) mammalian host. The trypanosomes undergo a cycle of development and multiplication in the digestive tract of the fly until the infective metacyclic trypanosomes (metatrypanosomes) are produced. Different trypanosome species develop in different regions of the digestive tract of the fly, and the metatrypanosomes occur either in the biting mouthparts or in the salivary glands. The period from ingesting infected blood to the appearance of these infective forms varies from one to three weeks; once infective metatrypanosomes are present, the fly remains infective for the remainder of its life. During the act of feeding, the fly penetrates the skin with its proboscis. The rupture of small blood vessels forms a pool of blood in the tissues and the fly injects saliva to prevent coagulation. Infection of the host takes place at this stage, with infective metacyclic trypanosomes in the saliva.

Although no classical sexual processes in the life cycle of trypanosomes have been described, it has been shown that exchange of genomic material (DNA) between trypanosomes sometimes occurs in the tsetse fly, although it is not clear how significant this is.

Life cycle in the mammalian host. The infective metatrypanosomes undergo development and multiplication at the site of infection where a swelling or chancre may be detected in the skin, and finally the mature blood trypanosomes (or trypomastigotes) are released via lymph vessels and lymph nodes into the blood circulation.

Reproduction in the mammalian host occurs through a process of binary division.

Trypanosomes feed by absorbing nutrients, through their outer membrane, from the body fluids of the host. The proteins, carbohydrates and fats are digested by enzyme systems within their protoplasm. Oxygen dissolved in the tissue fluids or blood plasma of

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their host is absorbed in a similar manner, to generate the energy necessary for the vital processes.

Waste products are disposed of by a reverse process, through the outer membrane, into the body fluids of the host. They include carbon dioxide formed during respiration, as well as more complex metabolic products.

Life cycle in the tsetse fly. Blood stream forms (trypomastigotes) ingested by the fly undergo considerable changes, in morphology as well as in their metabolism. They change into long slender forms called epimastigotes, which multiply and finally give rise to the infective meta-trypanosomes.

Mechanical transmission

By biting insects. The process is purely mechanical. A biting insect passes the blood forms from an infected animal to another in the course of interrupted feeding. The time between the two feeds is crucial for effective transmission because the trypanosomes die when the blood dries. The importance of this mode of transmission is variable from place to place, depending on the numbers of hosts and biting insects present, and on the species of trypanosome. Large biting insects such as tabanids carry more blood and are more likely to act as mechanical vectors than for example mosquitoes. (Tsetse flies themselves can of course also act as mechanical vectors.) This mode of transmission has proved to be sufficiently effective to maintain Trypanosoma vivax and Trypanosoma evansi in South and Central America, and the latter species in North Africa and Asia as

well. No tsetse flies occur outside tropical Africa, apart from small tsetse pockets in the southwest of the Arabian peninsula.

By iatrogenic means. This can occur when using the same needle or surgical instrument on more than one animal, at sufficiently short intervals that the blood on the needle or instrument does not dry. It is not an uncommon occurrence when animals are vaccinated or treated by injection, or when blood is collected from several animals in a row, without changing or disinfecting needles or pins. It may also occur when several animals are subjected at short intervals to a surgical intervention (dehorning, castration, etc.) without properly disinfecting the instruments.

Transmission by other means

It is well known that carnivores may be infected with T. evansi and T. brucei by ingesting meat or organs from infected animals, as long as these are still sufficiently fresh to contain live trypanosomes. Infection occurs probably through the mucosa of the mouth (in which moreover bone splinters make wounds through which the parasites penetrate even more easily).

All trypanosome species are occasionally transmitted congenitally, or vertical transmission from the mother to the offspring, through the placenta either while the fetus is still in the uterus, or when bleeding occurs during birth. Congenital transmission of T. vivax, for example, has been observed in Latin America as well as in Africa, but its real importance is not well known.

High Risk Group

Hunters Rural villagers living in areas with a lack of infrastructure

Like running water, electricity Workers in game reserves or park Tourist who spend an extended time in rural areas or game reserves

Disease Course Infection with African trypanosomes can result in disease manifestations ranging from asymptomatic or mild to a severe fulminating disease. T. rhodesiense is more likely to cause a rapidly

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progressing and fulminating disease than T. gambiense. T. gambiense tends to cause either a slow progressing, which may be self-limiting, or the central nervous system (CNS).

The infection is initiated when metacyclic trypomastigotes are introduced from the saliva of the tsetse into the bite wound. Generally, there is an asymptomatic incubation period of 1-2 weeks in which the trypomastigotes are replicating within the tissue near the site of the bite. Occasionally, a local inflammatory nodule known as a ‘trypanosomal chancre’ is observed during this period. Chancres are usually tender and painful and ulceration may occur.

The trypomastigotes will invade the capillaries, enter the circulatory system during this incubation period, and continue to replicate within the blood of the human host. The establishment of this acute blood stage infection is characterized by irregular episodes of fever and headache. In the case of T. gambiense the number of parasites in the blood tends to be very low and often the infected person exhibits no symptoms, whereas most persons infected with T. rhodesiense will exhibit much higher parasitemias and a more pronounced fever sometimes associated with rigor.

Disease progression is often characterized by invasion of the lymphatics in T. gambiense infections. Symptoms during the lymphatic stage include enlarged lymph nodes (particularly post-cervical group), weight loss, weakness, rash, itching, and edema as well as the continued intermittent febrile attacks. Higher parasitemias are often associated with the symptomatic periods. The infection can spontaneously resolve during either the blood stage or the lymphatic stage. There is usually little evidence of lymphatic involvement in T. rhodesiense infections. In general, the symptoms during the earlier stages of the infection tend to be non-specific (fever, malaise, headache, weakness) and may infect multiple organs.

A hallmark feature of African trypanosomiasis is the invasion of the CNS and nervous system impairment. Trypanosomes crossing the blood-brain barrier result in a generalized meningocephalitis characterized by progressively worsening symptoms. Indications of nervous impairment include apathy, fatigue, confusion, somnolence, and motor changes (such as tics, slurred speech, and in coordination). The changes in sleep patterns are often characterized by extreme fatigue during the day and extreme agitation at night. Generally it is 6-12 months (or even years) after the infection before the neurological symptoms start to become apparent in the case the CNS stage of the disease will almost always progress to include convulsions or coma by death in both T. gambiense and T. rhodesiense infections.

Clinical Presentation

Incubation Period: The first clinical manifestation of African trypanosomiasis occurs a few days after infection as a chancre at the site of tsetse fly inoculation.  This is due to the localized proliferation of the pathogens within the subcutaneous tissue.  Incubation period for T.b. rhodesiense may be two to three weeks, while the incubation period for the Gambian species may last several weeks to months.

Dissemination: With the conclusion of the incubation period, the organisms have already disseminated into the bloodstream, leading to the emergence of a characteristic intermittent fever pattern that correlates directly with high versus low levels of parasitemia.  The reason for the oscillating levels of parasite load is linked to the ability of the organisms to change their variable surface glycoproteins (VSGs) and evade the host’s immune system.  Lymphadenopathy, the swelling of lymph nodes, especially in the posterior cervical nodes

(on the back of the neck) is characteristic sign of African sleeping sickness and is termed Winterbottom’s sign.

Invasion of the Central Nervous System:  Invasion of the central nervous system (CNS) occurs within several weeks in the Rhodesian species and months to even years in the Gambian species of trypanosomiasis.  Symptoms include headache, stiff neck, sleep disturbance, and depression, followed by progressive mental deterioration, focal seizures, tremors, and palsies.  This progresses to coma and the ultimate death of the patient often secondary to pneumonia or sepsis.  Without treatment, African trypanosomiasis is a universally fatal illness.

PATHOGENESIS

During the bite of an infected tsetse fly, the protozoan enters the bite wound in the fly saliva. The organism multiplies at the skin site and within a few weeks enters the lymphatics and blood circulation. The patient responds with fever and production of IgM antibody against the protozoa, and symptoms improve. Within about a week and at roughly weekly intervals thereafter, however, there are recurrent increases in the number of parasites in the blood. Each of these bursts of increased parasitemia, meaning parasite in the circulating blood, coincides with the appearance of a new glycoprotein on the surface of the trypanosomes. More than a thousand genes, each coding for a different surface glycoprotein, are present in the protozoan chromosome. Only one of these genes is activated at a time, and the patient’s immune system must respond to each gene product with production of a new antibody. The recurrent cycles of parasitemia and antibody production continue until the patient is treated or dies.

In Trypanosoma brucei rhodesiense infections, the disease tends to progress rapidly, with the heart and brain invaded within 6 weeks of infection. Irritability, personality changes and mental dullness results from involvement, but the patient usually dies from heart failure within 6 months. With Trypanosoma brucei gambiense, progression of infection is much slower, and years may pass before death occurs, often from secondary infection. Much of the damage to the host is due to immune complexes formed when antibody reacts with complement and high levels of protozoan antigen.

PATHOLOGY

T. brucei effectively evade the human immune system by a fascinating genetically programmed system of antigenic variation. When even a single parasite crosses the epithelial layer via the bite of an infected tsetse fly, local pain and inflammation result as the innate immune system responds to the invader. Nevertheless, when innate, non-specific mechanisms of destruction are not sufficient to clear the pathogen (as is often the case in humans), the parasite grows and multiples in the bloodstream, while a primary adaptive immune response is built. Indeed, the adaptive response is evident by the appearance of redness and swelling at the site of infection 1-2 weeks after the tsetse fly bite. VSGs on the surface of the parasite are recognized as foreign antigens by specific BCRs and TCRs. Generally, a highly specific B-cell mediated antibody response is generated against to certain VSG epitopes. The adaptive immune response eventually kills all of the clones of the original parasite via antibody-mediated adaptive immune response. However, some parasites spontaneously change the VSG coating by altering which VSG gen is expression. This process is known as gene conversion. In

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fact, 806 different genes encode for VSGs. Each VSG gene encodes for structurally similar, but extremely unique VSGs. Because different VSGs are distinct enough that they are each seen as representative of ‘new’ pathogens by the immune system, a separate, primary adaptive immune response must be generated to clear the parasites with new VSGs. The time required to mount a sufficient primary adaptive immune response allows the parasite to divide, multiply, and occasionally change its VSG surface again. Thus, although theoretically all T. brucei can eventually be cleared by the human immune system, spontaneous changes in which VSG is expressed allow the parasite to stay one step ahead of the adoptive immune system. Indeed, patients with African trypanosomiasis suffer from chronic cycles of infection. The disease progresses as these cycles lead to chronic inflammation, fever, and the build-up of immune complexes. Furthermore, the severe and often irreversible neurological damage occurs as parasites reaches deeper and into less protected areas of the body, such as brain. Fatigue is probably a result of the body constantly funneling of resources to fight a never-ending infection.

PATHOPHYSIOLOGY

Humans are infected with T brucei following a fly bite, which occasionally causes a skin chancre at the site. These injected trypomastigotes further mature and divide in the blood and lymphatic system, causing malaise, intermittent fever, rash, and wasting. Eventually, the parasitic invasion reaches the central nervous system (CNS), causing behavioral and neurologic changes such as encephalitis and coma. Death may occur.

The parasites escape the initial host defense mechanisms by extensive antigenic variation of parasite surface glycoproteins known as major variant surface glycoprotein (VSG). This evasion of the humoral immune responses contributes to parasite virulence. During the parasitemia, most pathologic changes occur in the hematologic, lymphatic, cardiac, and central nervous systems. This may be the result of immune-mediated reactions against antigens on red blood cells, cardiac tissue, and brain tissue, resulting in hemolysis, anemia, pancarditis, and meningoencephalitis.

A hypersensitivity reaction causes skin problems, including persistent urticaria, pruritus, and facial edema. Increased lymphocyte levels in the spleen and lymph nodes infested with the parasite leads to fibrosis but rarely hepatosplenomegaly. Monocytes, macrophages, and plasma cells infiltrate blood vessels, causing endarteritis and increased vascular permeability.

The gastrointestinal system is also affected. Kupffer cell hyperplasia occurs in the liver, along with portal infiltration and fatty degeneration. Hepatomegaly is rare. More commonly in East African trypanosomiasis, a pancarditis affecting all heart tissue layers develops secondary to extensive cellular infiltration and fibrosis. Arrhythmia or cardiac failure can cause death prior to the development of CNS manifestations. CNS problems include perivascular infiltration into the interstitium in the brain and spinal cord, leading to meningoencephalitis with edema, bleeding, and granulomatous lesions.

FREQUENCY

United States

All cases of African trypanosomiasis are imported from Africa by travelers to endemic areas. Infections among travelers are rare, with less than 1 case per year reported among US travelers. Most of these infections are caused by T brucei rhodesiense and are acquired in East African game parks.

International

African trypanosomiasis is confined to tropical Africa between latitudes 15°N and 20°S, or from north of South Africa to south of Algeria, Libya, and Egypt.

The prevalence of African trypanosomiasis varies by country and region. In 2005, major outbreaks were observed in Angola, the Democratic Republic of Congo, and Sudan.

In Central African Republic, Chad, Congo, Côte d'Ivoire, Guinea, Malawi, Uganda, and United Republic of Tanzania, sleeping sickness remains an important public health problem.

Fewer than 50 new cases per year are reported in countries such as Burkina Faso, Cameroon, Equatorial Guinea, Gabon, Kenya, Mozambique, Nigeria, Rwanda, Zambia, and Zimbabwe.

T. brucei transmission seems to have stopped and no new cases of African trypanosomiasis have been reported for several decades in countries such as Benin, Botswana, Burundi, Ethiopia, Gambia, Ghana, Guinea Bissau, Liberia, Mali, Namibia, Niger, Senegal, Sierra Leone, Swaziland, and Togo.

Sleeping sickness threatens millions of people in 36 countries of sub-Saharan Africa. The current situation is difficult to assess in numerous endemic countries because of a lack of surveillance and diagnostic expertise.

In 1986, a panel of experts convened by the World Health Organization (WHO) estimated that 70 million people lived in areas where transmission of African trypanosomiasis is possible. In 1998, almost 40,000 cases of the disease were reported, but this number did not reflect the true situation given the remoteness of affected regions and the focal nature of the disease. Between 300,000 and 500,000, more cases were estimated as remaining undiagnosed and therefore untreated.

During recent epidemic periods, the prevalence of sleeping sickness has reached 50% in several villages in the Democratic Republic of Congo, Angola, and Southern Sudan. Sleeping sickness was considered the first or second greatest cause of mortality in those communities, even ahead of HIV infection and AIDS. By 2005, surveillance had been reinforced and the number of new cases reported throughout the continent had substantially reduced; between 1998 and 2004, the figures for both forms of African trypanosomiasis together fell from 37,991 to 17,616.

The estimated number of cases is currently between 50,000 and 70,000. The current epidemic, which began in 1970, is thought to have been facilitated by factors such as the halting of screening programs, population migration, civil war, economic decline, and reduced health care financing.

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Mortality/Morbidity

The symptoms of East African trypanosomiasis develop more quickly (starting 1 month after bite) than the symptoms of West African trypanosomiasis, which can begin months to a year after the first bite.

Both types of African trypanosomiasis cause the same generalized symptoms, including intermittent fevers, rash, and lymphadenopathy. Notably, individuals with the East African form are more likely to experience cardiac complications and develop CNS disease more quickly, within weeks to a month. The CNS manifestations of behavioral changes, daytime somnolence, nighttime insomnia, stupor, and coma result in death if untreated.

In West African trypanosomiasis, the asymptomatic phase may precede onset of fevers, rash, and cervical lymphadenopathy. If unrecognized, the symptoms then progress to weight loss, asthenia, pruritus, and CNS disease with a more insidious onset. Meningismus is rare. Death at this point is usually due to aspiration or seizures caused by CNS damage.

Race

African trypanosomiasis has no racial predilection.

Sex

African trypanosomiasis has no sexual predilection.

Age

Exposure can occur at any time. Congenital African trypanosomiasis occurs in children, causing psychomotor retardation and seizure disorders.

CLINICAL SYMPTOMATOLOGY OF HUMAN AFRICAN TRYPANOSOMIASIS

The symptoms of two diseases are more pronounced in Caucasian than in the local African population.

After a painful tsetse bite, the chancre represents the initial lesion at the bite site, characterized by local erythema, edema, heat, tenderness and a lack of any suppuration. Trypanosomes are present in the inflammatory tissues. The chancre disappears within 2 or 3 weeks. The disease evolves in two distinct successive phases determining its two pathological stages. Within a few days after the tsetse bite, the patient enters the haemolymphatic stage I of the illness.

Stages 1, The Haemolymphatic Stage

Clinical signs appear very early. Intermittent fever develops because of the successive waves of invasion of the blood by the trypanosomes. Adenopathies, splenomegaly, or even hepatological signs mark the invasion of the reticulo-endothelial system. Skin eruptions or trypanides are commonly observed. Severe pruritus with scratching skin lesions becomes unsupportable for the patient.

Cardiovascular alterations are less prominent, especially in the Gambian form. Irregular febrile episodes are accompanied by headaches, malaise, exhaustion, anorexia, extreme thirst, muscle and joint pains, pruritus, anaemia, rash and often-deep hyperesthesia (the sign of the key of Kerandel). The lymph nodes are generally rubbery and mobile, painful at the beginning. Palpation of the subclavicular region (Winterbottom sign) is an important part of the diagnostic procedure in the Gambian form. Any adenopathy accompanied by fever should evoke the diagnosis of sleeping sickness in patients from endemic areas. Later, pruritus generalizes. Edema of the face and extremities appears early.

Few minor neurological and endocrine disorders may reveal the precocity of central nervous system (CNS) involvement, long before any detectable changes occur in the cerebrospinal fluid (CSF). Daytime somnolence or nighttime insomnia may already be reported and electroencephalographic (EEG) tracings may reveal abnormalities. Psychiatric signs, with the alternation of irritability, changes in personality or mood affecting the daily and professional life of the patients, constitute often the first manifestation of the disease. A permanent feeling of coldness marks the endocrine syndrome, lack of appetite or in contrast hyperphagia, polydipsia and impotence, amenorrhea or infertility, indicative of vegetative and sexual disturbances.

Stage 2, The Meningoencephalitic Stage

The meningoencephalitic stage appears slowly and insidiously over a period of months or years depending on the trypanosome. However, the clinical signs remain reversible for a long time with treatment attesting to the predominance of potentially reversible inflammatory lesions over irreversible demyelinating lesions. The general signs of the haemolymphatic stage do not completely disappear: spikes of fever (but sometimes hypothermia), adenopathies and splenomegaly, cardiovascular manifestations endocrine disturbances and typical pruritus.

The development of the neurological symptoms is progressive. As neurological signs occur already in stage I, biological criteria are the only means to confirm CNS invasion. The threshold criteria, which are commonly used, are based on CSF examination: more than 5 cells/µL and/or the presence of trypanosomes.

A wide variety of symptoms is encountered. The main symptoms from which sleeping sickness was named are daytime somnolence and nocturnal insomnia, the patients being "sleepy by day and restless by night". The sleep-wake cycle disturbances are accompanied with either or many of the following symptoms: headaches, sensory disturbances with diffuse superficial or deep sensations (muscle and bone hyperesthesia, either spontaneous or provoked; hyperpathia), presence of primitive reflexes (palm-mental reflex, sucking reflex), exaggerated deep tendon reflexes, psychiatric disorders (confusion, mood swings, agitation, aggressive behaviour, euphoria, absent gaze, mutism, indifference), and tremor (fine and diffuse without any myoclonic jerk at rest or during movement). Pyramidal alterations revealed by a Babinski sign can also be observed along with alterations in muscle tone, numbness or sensory deficit.

An abnormal number of monocytic cells is observed in the CSF. The early neurological symptoms correlate with the widespread meningeal inflammation, which occurs in both forms of HAT. The selective CNS locations explain in part the principal clinical neurological signs. Sleep-wake disturbances may result from invasion

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of the median eminence by a parasite, which also accounts for neuro-endocrine dysfunctions with the involvement of the suprachiasmatic nuclei. Disorders of the sleep-wake cycle are accompanied by a state of apathy in the patient, the loss of muscle tone especially in the neck muscles and a drooping of the eyelids. Extrapyramidal symptoms signal the involvement of the striatum. Deep sensory disturbances with hyperpathia may result from the involvement of the thalamus and the early invasion of posterior spinal roots.

Apart from the disruptions of the circadian rhythm of the sleep-wake cycle, other biological rhythms are disturbed, such as body temperature, cortisol and prolactin or growth hormone secretion. The invasion of the subthalamic and hypophyseal regions account for the persistence of ad-endocrine disturbances such as impotence, amenorrhea or infertility and the development of disturbed sensations of hunger and thirst, with often hyperphagia and polydipsia in contrast to the poor general state of malnutrition of the patients. At the terminal phase of the disease, CNS demyelination and atrophy are accompanied with disturbances in consciousness and the development of dementia with incoherence, incontinence and epileptic fits. The patient dies in a state of cachexia and physiological misery.

History

Stage 1 (early, or hemolymphatic, stage)o Painless skin chancre that appears about 5-15

days after the bite, resolving spontaneously after several weeks (seen less commonly in T brucei gambiense infection)

o Intermittent fever (refractory to antimalarials), general malaise, myalgia, arthralgias, and headache, usually 3 weeks after bite

o Generalized or regional lymphadenopathy (Posterior cervical lymphadenopathy [Winterbottom sign] is characteristic of T brucei gambiense African trypanosomiasis [sleeping sickness].)

o Facial edema (minority of patients)

o Transient urticarial, erythematous, or macular rashes 6-8 weeks after onset

o Trypanids (ill-defined, centrally pale, evanescent, annular or blotchy edematous erythematous macules on trunk)

Stage 2 (late, or CNS, stage)

o Persistent headaches (refractory to analgesics)

o Daytime somnolence followed by nighttime insomnia

o Behavioral changes, mood swings, and, in some patients, depression

o Loss of appetite, wasting syndrome, and weight loss

o Seizures in children (rarely in adults)

Physical

Stage 1 (early, or hemolymphatic, stage)o Indurated chancre at bite site

o Skin lesions (trypanids) in light-skinned patients

o Lymphadenopathy: Axillary and inguinal lymphadenopathy are more common in patients with East African trypanosomiasis. Cervical lymphadenopathy is more common in patients with West African trypanosomiasis. The classic Winterbottom sign is clearly visible (ie, enlarged, nontender, mobile posterior cervical lymph node).

o Fevers, tachycardia, irregular rash, edema, and weight loss

o Organomegaly, particularly splenomegaly (T brucei gambiense African trypanosomiasis)

Stage 2 (late, or CNS, stage)

o CNS symptoms: The CNS symptoms of West African trypanosomiasis have a slower onset of, ie, months to a year. Symptoms include irritability, tremors, increased muscle rigidity and tonicity, occasional ataxia, and hemiparesis, but rarely overt meningeal signs. East African trypanosomiasis usually has a faster onset, ie, weeks to a month, and does not exhibit a clear distinction between the two stages.

o Kerandel sign, including delayed pain on compression of patient's soft tissue

o Behavioral changes consistent with mania or psychosis, speech disorders, and seizures

o Stupor and coma (giving rise to the name sleeping sickness)

o Psychosis

o Sensory disorders, tremor, and ataxia

Disease Management

Disease management is performed in three steps: Screening for potential infection. This involves the use of

serological test and/or checking for clinical signs – generally swollen cervical glands.

Diagnosing shows whether the parasite is present. Staging to determine the state of progression of the

disease entails examination of cerebro-spinal fluid obtained by lumbar puncture and is used to determine the course of treatment.

Diagnosis must be made as early as possible and before the neurological stage in order to avoid complicated, difficult and risky treatment procedure.

DIAGNOSIS

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Laboratory Studies

Generalo In African trypanosomiasis (sleeping sickness),

the most significant laboratory abnormalities include anemia, hypergammaglobulinemia, low complement levels, elevated erythrocyte sedimentation rate (ESR), thrombocytopenia, and hypoalbuminemia, but not eosinophilia or abnormal liver function.

o In West African trypanosomiasis, the total immunoglobulin M (IgM) level is notably higher in blood and CSF (along with high CSF protein).

o A definitive diagnosis of infection requires actual detection of trypanosomes in blood, lymph nodes, CSF, skin chancre aspirates, or bone marrow. However, symptomatic improvement after empiric treatment is the usual confirmatory test in areas where diagnostic studies are not readily available.

Lymph node aspiration at a high dry magnification (X400) is commonly used as a rapid test for trypanosomes. It requires immediate search for parasites because they are mobile for only 15-20 minutes. This test has more utility in T brucei gambiense trypanosomiasis.

Blood smear

o A wet smear of unstained blood or Giemsa-stained thick smear (more sensitive) is used to evaluate for mobile trypanosomes, again for 15-20 minutes. Wright and Leishman stains are inadequate. This technique is most sensitive in early stages of disease, when the number of circulating parasites is highest (≥5000/mL), particularly in T brucei rhodesiense trypanosomiasis.

o Better assays are now available, including the hematocrit centrifugation technique for buffy coat examination and the miniature anion-exchange centrifugation technique (mAECT), which filters out the red cells but not the trypanosomes. This test can be used to detect parasitemia levels as low as 5 parasites/mL; the test can be repeated on subsequent days to increase the yield when results are negative.

Chancre aspirate can be used as a wet preparation, especially in East African trypanosomiasis, but a blood smear is more sensitive.

Bone marrow aspiration results may be positive in some patients.

CSF assay

o Lumbar puncture should be performed whenever trypanosomiasis is suspected. CSF examination helps to diagnose and stage the disease. However, a negative result does not necessarily rule out the diagnosis.

o The double centrifugation technique is the most sensitive method to detect the trypanosomes.

o Other CSF findings include elevated WBC count, elevated IgM levels, elevated total protein levels, and raised intracranial pressure. An uncommon characteristic finding is Mott cells, which are thought to be large eosinophilic plasma cells containing IgM that have failed to secrete their antibodies.

o Increased intrathecal synthesis of IgM has been found to be the most sensitive indicator of CNS involvement in African trypanosomiasis.

Imaging Studies

CT scanning and MRI of the head: Both head CT scanning and MRI reveal cerebral edema and white matter enhancement, respectively, in patients with late-stage African trypanosomiasis.

EEG in neurologic involvement usually shows slow wave oscillations (delta waves), a nonspecific finding.

Other Tests

General: Field serology-based diagnosis of African trypanosomiasis has been slow to progress over the past decades. Although many research tools are available for diagnosis, few are used clinically in endemic areas.

Serologic antibody detection

o The standard serologic assay to diagnose West African trypanosomiasis is the card agglutination test for trypanosomiasis (CATT).

o The CATT can be conducted in the field without electricity, and results are available in only 10 minutes. It is highly sensitive (96%) but less specific because of cross-reactivity with animal trypanosomes.

o Commercial antibody tests for Eastern African trypanosomiasis are not available.

Antigen detection tests based on enzyme-linked immunosorbent assay (ELISA) technology have been developed. They have shown inconsistent results and are not yet commercially available.

Culture of CSF, blood, bone marrow aspirate, or tissue specimens can be performed in liquid media.

Other tests developed but not frequently used clinically include antibody detection in the CSF and intrathecal space (low sensitivity), polymerase chain reaction (PCR), and serum proteomic tests.

Research tools such as isoenzyme analysis and restriction fragment length polymorphism (RFLP) are used for definitive subspecies identification.

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Procedures

Lumbar puncture: CSF fluid is used to detect trypanosomes and to measure WBC counts, protein, and IgM in patients with parasitemia or positive serologies or symptoms. Importantly, CNS disease can manifest early in East African trypanosomiasis.

Differential Diagnoses

Other Problems to Be Considered

Stage 1 (early) African trypanosomiasis (sleeping sickness) Symptoms

Differential diagnoses of recurrent fever include malaria, HIV infection, borreliosis, and brucellosis, typhoid fever, and other enteric fevers.

Differential diagnoses of lymphadenopathy include tuberculosis (TB) lymphadenitis, HIV infection, and cancer.

Stage 2 (late) African trypanosomiasis symptoms

Differential diagnoses of mental status changes include TB, meningitis, and HIV-related opportunistic infections, including cryptococcal meningitis.

TREATMENT

Medical Care

Prehospital care of African trypanosomiasis (sleeping sickness) centers on management of the acute symptoms of fever and malaise while closely monitoring the patient’s neurologic status.

In the emergency department, if CNS symptoms are severe, then airway management to prevent aspiration becomes important, along with an immediate blood smear, CBC count, and lumbar puncture for trypanosome detection.

Medication

The type of drug treatment used depends on the type and stage of African trypanosomiasis (sleeping sickness).The drugs used in the first stage of the disease are less toxic, easier to administer and more affective. Treatment success in the second stage defends on a drug that can cross the blood –brain barrier to reach the parasite. Such drugs are quite toxic and complicated to administer. Four drugs are registered for the treatment of sleeping sickness and provided free of charge to endemic countries through a WHO private partnership with anofi-aventis (pentamidine, melarsoprol, eflornithine) and Bayer AG (suramin).

Type of Trypanosomiasis

MedicationsStage 1

(Hemolymphatic Stages)

MedicationsStage 2

(Neurologic [CNS] Stages)

East African trypanosomiasis(caused by T brucei rhodesiense)

Suramin 100-200 mg IV test dose, then 1 g IV on days 1,3,7,14,21

Melarsoprol 2-3.6 mg/kg/d IV for 3d; after 1 week, 3.6 mg/kg/d for 3days; after

10-21 d, repeat the cycle

West African trypanosomiasis(caused by T brucei gambiense)

Pentamidine isethionate 4 mg/kg/d IM for 10 d or

Suramin 100-200 mg IV test dose, then 1 g IV on days 1,3,7,14,21

Melarsoprol 2-3.6 mg/kg/d IV for 3d; after 1 week, 3.6 mg/kg/d for 3 days;

after 10-21 d, repeat the cycle or

Eflornithine 400 mg/kg/d IV in 4 divided doses for 14 days

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Suramin (Metaret)

Antiparasitic agent used IV in early-stage African trypanosomiasis and onchocerciasis. Suramin is a polysulfonated naphthylamine derivative of urea. Suramin is trypanocidal and works by inhibiting parasitic enzymes and growth factors. Highly bound to serum proteins and, thus, crosses the blood-brain barrier poorly. Serum levels are approximately 100 mcg/mL. Suramin is more effective and less toxic than pentamidine. Excreted in the urine at a slow rate.

Suramin was introduced in 1920 to treat the first stage of the disease. By 1922, Suramin was generally combined with Tryparsamide (another pentavalent organo-arsenic drug) in the treatment of the second stage of the gambiense form. It was used during the grand epidemic in West and Central Africa in millions of people and was the mainstay of therapy until 1969.

Adult

100-200 mg test dose, then 1 g IV on days 1, 3, 7, 14, 21

Pediatric

1-2 mg test dose, then 20 mg/kg IV on days 1, 3, 7, 14, 21

Melarsoprol (Melarsen Oxide-BAL, Mel B, RP 3854)

Trivalent arsenical used in the late or CNS stage of African trypanosomiasis. Trypanocidal, inhibiting parasitic glycolysis. Water insoluble and has a half-life of 35 h. Serum levels range from 2-5 mcg/mL, but CSF levels are 50-fold lower. The kidneys primarily excrete the drug. Clinical improvement is usually observed within 4 d after starting the drug. Therapy is as high as 90-95% successful in clearing the parasitemia. However, it can be toxic and even fatal in 4-6% of cases. Studies have now demonstrated the effectiveness of 10-day melarsoprol treatments for late-stage African trypanosomiasis. In addition, melarsoprol resistance has become a concern in the Congo and Uganda; up to 30% of cases do not respond to the drug.

The organo-arsenical melarsoprol (Arsobal) was developed in the 1940s, and is effective for patients with second stage sleeping sickness. However, 3 - 10% of those injected have reactive encephalopathy (convulsions, progressive coma, or psychotic reactions), and 10 - 70% of such cases result in death; it can cause brain damage in those who survive the encephalopathy. However, due to its effectiveness, melarsoprol is still used today. Resistance to melarsoprol is increasing, and combination therapy with nifurtimox is currently under research.

Adult

2-3.6 mg/kg/d IV for 3 d; after 1 wk, 3.6 mg/kg/d IV for 3 d; after 10-21 d, repeat the cycle; always administer each dose slowly on an empty stomach

Pediatric

0.36 mg/kg IV initially; increase gradually to a maximum 3.6 mg/kg at intervals of 1-5 d for a total of 9-10 doses; average dosing is 18-25 mg/kg over 1 month

Eflornithine (Ornidyl)

Recommended for treatment of patients with West African trypanosomiasis, especially late (or CNS) disease. Selective and irreversible inhibitor of ornithine decarboxylase, which is a critical enzyme for DNA and RNA synthesis. Generally tolerated better and is less toxic than arsenic drugs. Available via World Health Organization. Initial response time is 1-2 wk, used for patients in whom melarsoprol fails.

Eflornithine (difluoromethylornithine or DFMO), the most modern treatment, was developed in the 1970s by Albert Sjoerdsmanot and underwent clinical trials in the 1980s. The drug was approved by the United States Food and Drug Administration in 1990, but Aventis, the company responsible for its manufacture, halted production in 1999. In 2001, however, Aventis, in association with Médecins Sans Frontières and the World Health Organization, signed a long-term agreement to manufacture and donate the drug.

Adult

100 mg/kg IV q6h for 14 d

Pediatric (Not established)

Pentamidine isethionate (Pentam 300, Pentacarinat, NebuPent)

Antiprotozoal agent usually used for early (or stage 1) African trypanosomiasis as well as Pneumocystis carinii pneumonia and leishmaniasis. Works by inhibiting dihydrofolate reductase enzyme, thereby interfering with parasite aerobic glycolysis. Because of poor GI absorption, the drug is administered IV/IM and is strongly bound to tissues, including spleen, liver, and kidney. Clinical improvement usually noted within 24 h of injection. Reported to have a >90% cure rate. Pentamidine does not penetrate the blood-brain barrier effectively and, therefore, does not treat CNS infection.

Pentamidine, a highly effective drug for the first stage of the disease, has been used since 1939. During the fifties, it was widely used as a prophylactic agent in Western Africa, leading to a sharp decline in infection rates. At the time, it was thought that eradication of the disease was at hand.

Adult

4 mg/kg/d IM/IV for 10 d

Pediatric

Administer as in adults

During the first half of the 20th century, there were very few efficacious medications. The only one, which was known and used at

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first, was an arsenical by-product named Atoxyl it had to be administered over a several week period of time through course of subcutaneous injection. As it was a swift killer of trypanosomes roaming the blood stream, its use during the first stage prevented the risk of transmission with new fly bites. However, as this medication does not pass through the meningeal barrier it is not efficacious against trypanosomes which are already present in the cerebro-spinal fluid so that the disease can continue to progress in patients who are experiencing the second stage of this condition.

In 1926, a new arsenical by-product named Tryparsamide became available. It is given intravenously once a week over a few weeks. This medication has a great advantage over Atoxyl as it penetrates into the cerebro-spinal fluid. It is therefore a medication for the second stage of the disease. Nevertheless about 20% of all patients are not going to recover and their outcome will be fatal one.

When mass therapy campaigns against Tyrpanosomiasis got started, only these two medications were available. They are arsenical by-products with deleterious side effects for the optic nerve, as a result, most of the time patients are cured but some complain of ocular side effects and may even wind up fully blind. The need of repeated injections is another shortcoming as it is difficult to have patients stick to the program unless they are inpatient over two or three month period. Then incomplete treatment course give rise to strains which are resistant to these medication. These first generation arsenical drugs are obsolete now and not used any longer with the availability of new molecules, which were developed through research and development.

An international research team working in the Democratic Republic of the Congo, New Sudan and Angola involving Immtech International and University of North Carolina Hill has completed a Phase IIb clinical trial and commenced a Phase III trial in 2005 testing the efficacy of the first oral treatment for Sleeping Sickness, known at this point as “DB289”.

Combination therapy

Combination therapy may be more effective than monotherapy for the treatment of late-stage T brucei gambiense trypanosomiasis. An open randomized trial involving 278 patients compared melarsoprol monotherapy (two different dosing regimens), nifurtimox monotherapy, and melarsoprol-nifurtimox combination therapy. The 48 relapses reported in the study were limited to patients receiving one of the 3-monotherapy regimens. The trial concluded that a consecutive 10-day low-dose melarsoprol-nifurtimox combination is more effective than the standard melarsoprol regimen.

A trial that compared the efficacy of eflornithine monotherapy to nifurtimox-eflornithine combination therapy in patients with late-stage T brucei gambiense infection found that cure rates were similar. However, adverse effects were more common in patients who received eflornithine monotherapy (25.5% vs 9.6%). Thus, the nifurtimox-eflornithine combination appears to be a promising regimen for use in late-stage T brucei gambiense trypanosomiasis.

Anthelmintics

Parasite biochemical pathways are sufficiently different from the human host to allow selective interference by chemotherapeutic agents in relatively small doses.

Follow-up

Further Inpatient Care

If late or CNS disease complications and coma occur, intensive care unit (ICU) staff are needed while treatment is administered (i.e., melarsoprol for East African trypanosomiasis or eflornithine for West African trypanosomiasis). Monitor potential adverse effects from such drugs, including hematologic, renal, and hepatic function.

Further Outpatient Care

In both early- and late-stage trypanosomiasis, treatment usually resolves symptoms and clears the parasitemia on repeat blood smears.

Patients who have recovered from late-stage East African trypanosomiasis should undergo lumbar punctures every 3 months for the first year. Patients who have recovered from West African trypanosomiasis should undergo lumbar punctures every 6 months for 2 years. A relapse is suggested if symptoms return, the CSF WBC count is above 20 cells/µL, CSF pleocytosis occurs, or if trypanosomes are still present in blood or CSF. A persistently elevated CSF WBC count can be observed in recovering patients, however, so the change (increase or decrease) in WBC count is more helpful diagnostically. If a relapse is noted, consider repeat treatment with melarsoprol or eflornithine.

Prognosis

In early, or stage 1, trypanosomiasis, most patients experience full recovery following treatment.

In late, or stage 2, trypanosomiasis, the CNS manifestations are ultimately fatal if untreated. The cure rate approaches 95% with drugs that work inside the CNS (eg, melarsoprol).

MEDICAL ECOLOGY OF SLEEPING SICKNESS: THE VECTOR

Distribution

Tsetses (pronounced se’tse’, tse’tse’), sometimes-spelled tzetze, are large biting flies inhabiting much of mid-continental Africa between the Sahara and Kalahari deserts. They live by feeding on the blood of vertebrate animals and are the primary biological vectors of trypanosomes, which causes human sleeping sickness and animal trypanosomiasis, called nagana.

Tsetse flies are 6-14 mm long, excluding the proboscis. The broad head bears three ocelli and two large compound eyes, which are widely spaced in both sexes. The pair of short antennae has three segment and each an arista with its characteristically branched hairs. Hygro-, thermo-, chemo- and mechanoreceptors are concentrated on

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the antennae. The mouthparts consist of three unpaired components, the piercing labium, the labrum enclosing the food channel, and the hypopahrynx with the salivary channel. All are sheathed by two modified maxillarypalps. On the back, the thorax ends in a caudal, triangular scutellum. The eight abdominal segments can distend greatly after feeding. Genital organs, males possessing a button-like hypopygium under the dorsal end of the body, which covers the clasper, can only distinguish males and females. The white larvae are elongated, without eyes and legs, at the posterior end possessing a pair of black polypneustic lobes.

There are 31 species and subspecies of tsetse flies under the genus Glossina, family Glossinidae, and order Diptera. Tsetse flies are largely classified into three subgenera based on morphological differences in the structure of the genitalia: Morsitans (Glossina), Palpalis (Nemorhina), and Fusca (Austenina) groups. Although the tsetse flies can be found over some 9 million squared kilometers of the African continent, presence of glossina populations throughout the continent are far from continuous. In general, the Sahara and Somali Deserts limit the populations in the north, extending across the entire continent from Senegal in the west to southern Somalia in the east. Tsetse populations are denser in West and Central Africa, and are found more sporadically to the East and down to the borders of the Kalahari and Namibian Deserts in Southern Africa. Although tsetse fly habitats may vary considerably, climate and altitude - through their direct effects on vegetation, rainfall, and temperature - are still the primary determinants for proliferation. Unlike other insects, there are no seasonal interruptions in the life cycles of tsetse flies. However, both adult longevity and puparial duration are related to temperature, and a significant seasonal decline in tsetse populations is normal, particularly in savannah habitats during the dry season.

Behavioral Ecology of the Vector: Mating

The mating behaviors of tsetse flies have received much attention because of the development of the Sterile Insect Technique (SIT) for tsetse fly control. The existences of tsetse flies at low densities in certain areas suggest highly specific mating mechanisms involving visual and olfactory responses. Female tsetse flies only need to mate once in their life time, but multiple mating have been known occur occasionally. Cross mating is possible in areas where habitats of different species overlap, however male hybrids are infertile. Mating of female is mostly confines to early life with mean duration of mating declining by age. Most female flies are successfully inseminated even at very low population densities; usually during their first blood meal right after emerge from the pupal stage.

Behavioral Ecology of Vector: Host Feeding

Tsetse flies use visual and olfactory characteristics to recognize potential hosts before initiating host-oriented responses. There are series of behavioral responses involved in the process of obtaining a blood meal. Host-seeking behaviors are influenced by endogenous and exogenous factors. Endogenous factors include circadian rhythm of activity level of starvation, age, sex, and pregnancy status of the fly. Exogenous factors include temperature, vapor pressure, visual and olfactory stimuli, and mechanical stimulation.

Four Stages of Host-locating Behavior

RangingFlying in search of a host in the absence of an

external cue. Activation

Change in behavior caused by perception of external stimulus

OrientationUpwind anemotaxis in response to complex chemical and visual directing the insect to the host

LandingGenerally, the tsetse fly will detect an odor plume upwind until it visually recognizes the host. After landing on the host, heat stimulation cause a probing and feeding response.

Life Cycle of the Vector

Unlike most insect species (r-strategists) that produce large quantities of eggs, fertilized female tsetse flies (k-strategies) “give birth” to one larva. A typical female tsetse fly will produce one full grown larva approximately every 9-10 days depending on the temperature and humidity. A single egg will hatch and develop to a third-stage larva in the uterus of the female fly, where it is nurtured and supplied with nutrients. This reproductive process is known as adenotrophic viviparity.

MEDICAL ECOLOGY OF SLEEPING SICKNESS: THE HUMAN

Although epidemics as large as the ones in Uganda at the turn of the century have not been repeated, there is much concern over the re-emergence and increase in the number of sleeping sickness cases being reported every year in Africa. In 1994, there were an estimated 150,000 cases in Congo, with prevalence as high as 70% in some villages. Despite the WHO projection of 60 million people at risk in Africa, only a fraction of the population at risk is currently under surveillance, and relatively few cases are accurately diagnosed annually. Although sleeping sickness was largely under control during the 1960s, recent epidemics have been strongly associated with political and civil unrest in West and Central Africa resulting in mass movement of populations into areas formerly uninhabited by humans.

In 2005, major outbreaks have been observed in Angola, the Democratic Republic of Congo and Sudan. In Central African Republic, Chad, Congo, Côte d’Ivoire, Guinea, Malawi, Uganda and United Republic of Tanzania sleeping sickness remains an important public health problem. Countries such as Burkina Faso, Cameroon, Equatorial Guinea, Gabon, Kenya, Mozambique, Nigeria, Rwanda, Zambia and Zimbabwe are reporting fewer than 50 new cases per year. In countries such as Benin, Botswana, Burundi, Ethiopia, Gambia, Ghana, Guinea Bissau, Liberia, Mali, Namibia, Niger, Senegal, Sierra Leone Swaziland and Togo transmission seems to have stopped and no new cases have been reported for several decades. Nonetheless, it is difficult to assess the current situation in a number of endemic countries because of a lack of surveillance and diagnostic expertise.

Epidemiology of West African Sleeping Sickness

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Both protozoa species are morphologically indistinguishable but have drastically different epidemiological features

West African sleeping sickness is typically a chronic disease, making it a difficult disease to diagnose in the field. Low levels of trypanosomes in circulating blood make it difficult to detect the presence of parasites in blood smears, requiring more sophisticated means of detecting trypanosomes such as with the use of miniature anion-exchange/centrifugation (mAEC) technique. In comparison to the East African form, T. b. gambiense has a longer evolutionary history with humans, having successfully adapted to establishing infections in human hosts without manifesting severe symptoms. Astonishingly, infection rates of T. b. gambiense in wild glossina populations are as low 0.1%, even in areas with an epidemic of sleeping sickness.

Vectors of West African sleeping sickness are species of the palpalis group, most of which are in close contact with humans. Several different reservoirs for T. b.gambiense have been identified, strongly suggesting that other animals, such as the African domestic pig, may maintain the persistence of sleeping sickness in human populations. However, T.b.gambiense has not been observed or proven experimentally to reach significantly infectious levels of parasetemia in other reservoir host. Although it is widely accepted that the human-to-fly contact is the main route of transmission, some suggest a minor cycle involving an animal reservoir may help explain the re-emerge and persistence of the disease in West Africa.

The epidemiology of T.b. gambiense sleeping sickness is far from being fully understood. Despite the low levels of parasitemia in humans, the disease has successfully established endemicity in many regions of West Africa. It has also long been observed that the incidence of disease is not related to the density of the glossina populations, and that epidemics often occur in areas where the density of the vector is low. In Nigeria, sleeping sickness occurred in the north where the distribution of G.p. palpalis and G.tachinoides were scare and restricted to vegetation close to watercourses during the dry season. In Southern Nigeria, the same species of tsetse flies are found in abundance due to favorable climatic conditions, yet cases of sleeping sickness have never been observed. It is thought that the nature of the human-fly contact is of particular importance in the transmission of T. b. gambiense and the distribution of the disease, and that human-fly contacts can be classified as “personal” or “impersonal” depending on the ecological circumstances of the interaction.”Personal” contact refer to situations where fly movements are restricted to areas where exposure to humans are frequent, such as a watering hole or a stream, and single tsetse fly can have multiple opportunities to feed on humans. “Impersonal” contact occurs when fly movements are less restricted, and where repeated contacts are not likely. In general, ecological isolation of tsetse flies in the vicinity human populations leads to increased “personal” contact. Climatic stress, lack of natural host where humans have destroyed wild animals close to villages, or clearing vegetation for cultivation are all examples of restrict movements of palpalis group vectors.

Epidemiology of East African Sleeping Sickness

East African sleeping sickness differs from West African sleeping sickness in both its epidemiology as well as its clinical manifestations in mammalian host. The clinical symptoms of East African sleeping sickness are more severe, and the onset of disease is rapid. In contrast to T. b. gambiense, T.b. rhodesiense occurs with higher levels of parasitemia in ungulates, and humans are the adventitious hosts. The vectors of T.b. rhodesiense and the

G.morsitans subspecies, G.pallidipes and G.swnnertoni species from the morsitans group, and on lesser occasions the peridomestic vectors from the palpalis group, G.fuscipes and G.tachinoides. Sporadic cases usually arise from among those in the population whose activities bring them into contact with the savannah woodland habitats of the morsitans group. Although the vectors normally feed on game animals, under extreme situations where “personal” contact is increased due to social and environmental factors, a human-fly-human transmission cycle may ensue resulting in an outbreak. Droughts and political turmoil are known to increase the number of cases when entire communities relocate to hitherto unoccupied areas in search of safety or fertile lands and water.

PREVENTION AND CONTROL

Prevention

There is no available vaccine and Chemoprophylaxis in unavailable. Trypanosomes are able to switch at random to over 1000, different antigens making it difficult for the body to create the appropriate antibodies

Preventive measures can help:1. Wear protective clothing, including long-sleeved shirts and

pants. The tsetse fly can bite through thin fabrics, so clothing should be made of medium-weight material.

2. Wear neutral-colored clothing. The tsetse fly is attracted to bright colors and very dark colors.

3. Inspect vehicles for tsetse flies before entering. The flies are attracted to moving vehicles.

4. Avoid bushes. The tsetse fly is less active during the hottest period of the day. It rests in bushes but will bite if disturbed.

5. Use insect repellant. Though insect repellants have not proven effective in preventing tsetse fly bites, they are effective in preventing other insects from biting and causing illness.

Control

In the absence of vaccine for trypanosomiasis and the looming threat of further trypanocidal drug resistance, the most theoretically desirable means of controlling the disease is through the vector population. Although complete eradication of the vector is impossible, the most successful attempts at controlling tsetse flies are likely to be at the extreme limits for survival of the fly, where both the density of the fly is low and “personal” contact with humans may be highest.

Chemical Control

There are several different control techniques available today, but the use of chemicals in controlling tsetse populations is still the most common method. In brief, whether aerial or from the ground, residual insecticides such as organochlorines (DDT, Dieldrin, Endosulfan), pyrethroids (deltamethrin, pyrethoids (deltamethrin, permethrin, and alphamethrin), and avermectins (ivermectin) are used as a target areas where human-to-fly contact are likely. Pyrethroids are preferred because they are rapidly degraded in soil and are environmentally safe, unlike organochlorines, carbamates and

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organophospates that a bioaccumulate in the food chain and are highly toxic to mammals and other vertebrates. Despite being effective, the use of organochlorines and organophosphates are now banned for widespread outdoor spraying. Susceptibility to insecticides varies from one species to another, and between the different classes of species.

Targets and Traps

Traps and target are mechanical devices used to kill or weaken tsetse flies through insecticides or various trapping methods. The use of traps and targets to control tsetse populations have been successful primarily because tsetse flies are k-strategies with a low rate of reproduction, and require very little sustained mortality pressure to bring about a reduction in population or even eradication from an area. Hargrove estimated that an additional mortality of 4% per day imposed on female flies was enough to cause extinction, in the absence of immigration. The traps and targets attract tsetse flies by taking advantage of their host-seeking behaviors, visual and olfactory stimulation. The developments of potent attractants in the last 20 yrs as well as the production of second-generation synthetic pyrethroid insecticides are making this form of control technique highly successful.

Bush Clearing

Exploiting the knowledge that tsetse flies concentrated in certain areas lead to numerous bush-clearing projects all over West and East Africa to drastically alter and maintain the area unsuitable for tsetse fly habitation. Discriminative bush clearing was used in Uganda to control G.m. centralis is by clearing taller Acacia trees in the Ankole district. In Tanzania, between 1923 and 1930, bush-clearing methods were also widely employed to stop the spread of sleeping sickness epidemic in Maswa district, where G. swynnertoni was prevalent. Similar tactics were used in Ghana to control sleeping sickness around villages were human-fly contacts were high. Despite the apparent success of these methods, it is widely accepted that bush-clearing is unsuitable as a long term control measure due to the expense and speed of reinvasion, as well as the environmental damage it causes through soil erosion, decreased soil fertility, and its adverse effects its adverse effects on water supplies

Sterile Insect Techniques

One of the modern methods of non-insecticidal control is the Sterile Insect Technique (SIT), which was first considered as a means to control tsetse by Simpson in 1958. This technique relies on the mating of wild females with sterile male flies. Physiologically, female tsetse flies only required to mate once to store sperm in its spermathecae in sufficient quantity such that fertilization can occur over its entire reproductive life. Mating with a sterile male would thus result in no offspring.

Sterilization of male Tsetse flies can be carried out by: Irradiation

Gamma rays, Beta rays Chemo sterilization

Bisazir, Metepa, Tepa, Apholates, Phytosterols Physiologic sterilization

Pyriproxyfen, Sulphaqunizaline, Chlordimeform

The Future of Sleeping Sickness Control

After the publication of works such as “Silent Spring”, public awareness of the dangers associated with insecticides are increasingly changing the way we treat our environment, and the way we institute environmental controls. Consequently, efforts to introduce more environmentally friendly methods of vector control, such as the use of traps without insecticides, challenges us to understand more about the vectors that transmit the disease, as well as the ecological balance that we - as humans - strike with them.

We live in a world where various technical means of control are available to address the spread of the disease. However, sleeping sickness is a disease of the developing world, where despite the multitude of control strategies, the issues have widely been neglected and abandoned. One of the key components required to bring about effective change is to consider the sustainability of the control strategy, and to encourage local communities to take ownership over the process, thereby empowering people to take an active role in an environmentally conscious solution. Increasing knowledge through culturally sensitive education, providing technical support, and a long-term commitment of basic resources to beneficiary communities is essential for large-scale tsetse control.

Alongside efforts to reduce the spread of disease through environmental controls, there is also an urgent need to improve current surveillance and diagnostic procedures. Mortality can be drastically reduced when cases can be diagnosed early enough to prevent the progression of late-stage sleeping sickness. Training and resources are desperately needed in endemic areas for proper diagnostics and sero-surveillance.

Perhaps the most mysterious aspect of this disease relates to the issue of treatment options, and the availability of drugs in Africa. Drug and vaccine development for diseases in developing countries have always been lagging, and unfortunately, trypanocidal drugs are no exception. An estimated 300,000 – 500,000 people are currently infected and suffering from the disease with no hope for treatment. In 2000, the USFDA approved the use of eflornithine by the Bristol-Myers Squibb Company and The Gillette Company in a product called Vaniqa TM, a topical eflornithine HCl cream to remove facial hair. Perhaps some of the profits generated from the sale of this form of the drug will be used to underwrite the free use of the drug in Africa; similar to what has already happened at Merck, who donates invermectin for the treatment of river blindness, and at Pfizer Inc for their azithormycin give away program for the treatment of trachoma.

SURVEILLANCE

Sleeping sickness is one of the few communicable diseases where systematic population screening is necessary, particularly for gambiense sleeping sickness which has a very long almost asympotomatic period. There are several reasons for this including the difficulty of diagnosis, which cannot normally be made in remote primary health care facilities (4), the difficulty and high risk of treatment for the late stage, for which special skills are required, and the near impossibility of vector control. Therefore, the control measure most often used for gambiense sleeping sickness is systematic screening of the population to detect all cases, including those in both the first and second stage of disease, and then curing them. Guidelines for sleeping sickness surveillance have been

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developed by WHO in collaboration with sleeping sickness endemic countries (5).

______________________________________________________

(4) Serological detection with the CATT test (Card Agglutination Trypanosomiasis Test) is commonly used in screening. However, this test has insufficient specificity (too many false positives) to be used as a definitive diagnosis. Parasitological examinations are sufficiently specific, but are not sensitive enough unless done over a period of several successive days, because the level of parasites in the blood oscillates rapidly. If the blood is taken during the part of the cycle when few parasites are circulating, then a parasitological examination is likely to be negative, even though the disease is present. Appropriate treatment depends on whether or not the parasite has passed the blood/brain barrier. A spinal tap is needed for determining this.

(5) Trypanosomiase Humaine Africaine: Surveillance epidemiologique et systeme d'information geographique (S.I.G), Geneva, World Health Organization, 1996.

Surveillance Strategies

The five alternative surveillance strategies. These were the classic mobile teams, fixed-post surveillance and the less widely used innovative techniques of filter paper sampling by trained community animal health workers, either visiting the community or based at rural health centers.

Fixed-post or passive surveillance, where patients presenting with symptoms that are difficult to diagnose or don't respond to treatments, say for malaria, are eventually referred to a treatment centre and tested for a variety of disease, including trypanosomiasis, and those with the disease are eventually diagnosed. The initial screening test is performed on wet blood.

Filter paper sampling at rural health centers, where community health workers based at rural health centers receive some training in collecting samples on filter paper and then routinely test any new patients presenting themselves at the health centre for whatever reason.

Filter paper samplings by community health workers, who have been trained in collecting filter paper samples and then spend 20% of their time collecting samples and following up seropositive individuals. This was based on experience in Uganda and Côte d’Ivoire.

Monovalent mobile teams, the classic surveillance teams; in the scenario the CATT is performed on whole blood and all the parasitological tests except for lumbar punctures are done by the team in the field. The monovalent teams work only on trypanosomiasis.

Polyvalent mobile teams, which operate in the same way as monovalent teams, except that only a third of their work consists of screening for trypanosomiasis.

In order to standardize the results, these were calculated for an area with a human population of 100,000 people, containing ten rural health centers, and where 20 community animal health workers were operating.

REFERENCES

www.who.int

www.cdc.gov

www.new-medical.net

www.thefreedictionary.com

www.microbiologybyte.com

www.wikipedia.org

www.pathmicro.med

www.iaea.org.at

www.nhm.ac.uk

www.medicalecology.org

www.medicalhelthcare.info

www.nature.com

www.virtualcentre.org

www.dictionary.com

www.wrongdiagnosis.com

www.healthsystem.virginia.edu

Journal

1 December 2007

Volume 45, Number 11 Clinical Infectious Diseases 2007; 45:1435–1442© 2007 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2007/4511-0006$15.00DOI: 10.1086/522982CSE THEME ARTICLE MAJOR ARTICLE

Nifurtimox‐Eflornithine Combination Therapy for Second‐Stage Trypanosoma brucei gambiense Sleeping Sickness: A Randomized Clinical Trial in Congo

Gerardo Priotto, 1 Serena Kasparian,1 Daniel Ngouama,2 Sara Ghorashian,3 Ute Arnold,3

Salah Ghabri, 1 and Unni Karunakara3

Alanine aminotransferase, bilirubin, and creatinine levels were measured by a colorimetric method (Randox) in a subgroup of patients. The reagents for alanine aminotransferase were different (Human) for the last 39 patients because of logistical constraints. Abnormal values were defined as follows: bilirubin, >17 μmol/L, with a >1.5‐fold increase; alanine aminotransferase, >12 IU/L (as

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determined for 43 patients using the Randox reagents) or >32 IU/L (for female subjects) and >42 IU/L (for male subjects, as determined for 39 patients using the Human reagents), with a >2.5‐fold increase; and creatinine, >80 μmol/L (for female subjects) and >97 1Epicentre, Paris, France; 2Programme National de Lutte contre la Trypanomose Humaine Africaine, Ministry of Health, Republic of Congo; and 3Médecins Sans Frontières, Amsterdam, Holland

Background. Human African trypanosomiasis caused by Trypanosoma brucei gambiense is a fatal disease. Current treatment options for patients with second‐stage disease are either highly toxic or impracticable in field conditions. We compared the efficacy and safety of the nifurtimox‐eflornithine drug combination with the standard eflornithine regimen for the treatment of second‐stage disease.

Methods. A randomized, open‐label, active‐control, phase III clinical trial comparing 2 arms was conducted at the Sleeping Sickness Treatment Center, which was run by Médecins Sans Frontières, in Nkayi, Bouenza Province, Republic of Congo. Patients were screened for inclusion and randomly assigned to receive eflornithine alone (400 mg/kg per day given intravenously every 6 h for 14 days) or eflornithine (400 mg/kg per day given intravenously every 12 h for 7 days) plus nifurtimox (15 mg/kg per day given orally every 8 h for 10 days). Patients were observed for 18 months. The study's outcomes were cure and adverse events attributable to treatment.

Results. A total of 103 patients with second‐stage disease were enrolled. Cure rates were 94.1% for the eflornithine group and 96.2% for the nifurtimox‐eflornithine group. Drug reactions were frequent in both arms, and severe reactions affected 25.5% of patients in the eflornithine group and 9.6% of those in the nifurtimox‐eflornithine group, resulting in 2 and 1 treatment suspensions, respectively. There was 1 death in the eflornithine arm and no deaths in the nifurtimox‐eflornithine arm.

Conclusions. The nifurtimox‐eflornithine combination appears to be a promising first‐line therapy for second‐stage sleeping sickness. If our findings are corroborated by ongoing findings from additional sites (a multicenter extension of this study), the new nifurtimox‐eflornithine combination therapy will mark a major and multifaceted advance over current therapies.

Received 30 March 2007; accepted 23 May 2007; electronically published 22 October 2007.

(See the editorial commentary by Chappuis on pages 1443–5)

Reprints or correspondence: Dr. Gerardo Priotto, Epicentre, 8 rue Saint‐Sabin, 75011 Paris, France (gpriotto@epicentre.msf.org).

Human African trypanosomiasis (HAT), or sleeping sickness, remains a public health challenge in sub-Saharan Africa, with an estimated 50,000–70,000 new cases per year, of which 20,000 are detected and reported [1]. The disease is caused by the protozoan parasite Trypanosoma brucei gambiense, which is transmitted by the tsetse fly (Glossina species), and it progresses from the hemolymphatic first stage to the meningoencephalitic second stage. It is invariably fatal without appropriate treatment. Since 1949, melarsoprol is the most commonly used treatment for second‐stage HAT. This arsenical derivative is associated with severe toxic effects

—in particular, reactive encephalopathy, which is fatal in 10%–70% of cases and affects 5%–10% of treated patients [2, 3]. Moreover, increasing melarsoprol failure rates (up to 30%) have been reported in several countries [4–6].

Eflornithine (diethylfluoromethylornitihine), the only new drug registered in 58 years for the treatment of second‐stage HAT, is a trypanostatic that inhibits ornithine decarboxylase, an enzyme essential for cell multiplication and differentiation [7–9]. It is better tolerated than melarsoprol, and its toxic effects—mainly seizures, gastrointestinal disorders, and myelosuppression—are reversible if well managed. Its efficacy is comparable to that of melarsoprol. However, a major disadvantage of eflornithine is the mode of administration, requiring 1 slow infusion every 6 h for 14 days (56 infusions in total), a regimen imposed by its short half‐life of 1.5–5 h [10, 11]. The difficulty in administering eflornithine in resource‐poor settings explains why melarsoprol continues to be the first‐line treatment.

Nifurtimox is an orally administered drug used in the treatment of Chagas disease (American trypanosomiasis) at dosages of 8–20 mg/kg per day for 90–120 days. Although it has not been approved for treatment of HAT, nifurtimox is used for compassionate treatment of relapsed disease. Its toxicity, which has been poorly documented, includes mainly neurological (headache, sleep disorders, agitation, and confusion) and gastrointestinal (anorexia, nausea/vomiting, and dyspepsia) dysfunctions [12, 13], some of which increase with the duration of intake [14, 15]. In the 1970s and 1980s, nifurtimox was tested empirically in several HAT case series, and the results were conflicting [14–17]. These studies, which applied different treatment regimens and evaluation criteria, are difficult to compare.

Drug combinations can potentially avert or delay the emergence of drug‐resistant organisms. Dosage reductions of each drug combined may reduce the overall toxicity while maintaining good efficacy. Combinations may also allow for a simpler administration of treatment, improving the feasibility of therapy in remote areas with logistic and staffing limitations.

A first attempt to assess various combinations for the treatment of second‐stage HAT in Uganda was interrupted because of excess fatality in the melarsoprol‐nifurtimox arm. Although the results were inconclusive, the trial showed promising safety and efficacy results with the nifurtimox‐eflornithine combination [18]. Assessment of this combination was therefore extended to a case study of 31 patients that yielded similar results [19].

In 2003, Médecins Sans Frontières and Epicentre, in collaboration with the Ministry of Health, initiated a randomized, open‐label clinical trial in Nkayi, Republic of Congo, to evaluate the efficacy and toxicity of the nifurtimox‐eflornithine combination with doses equal to those in the 2 previous studies, with additional simplification of the administration schedule. In 2005, the study was extended to the Democratic Republic of Congo and Uganda, in partnership with the Drugs for Neglected Diseases initiative, the World Health Organization Special Program for Training and Research in Tropical Diseases, the Swiss Tropical Institute, and national HAT programs.

Methods

To facilitate external comparability, the study methodology was similar to the methodologies in previous clinical trials involving second‐stage HAT [18–22].

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Participants. Study participants were identified among persons with cases diagnosed at the treatment center or during active screening. Inclusion criteria were as follows: confirmed second‐stage infection, with trypanosomes detected in specimens of blood, lymph, or CSF, with >20 leukocytes/μL in CSF specimens. Exclusion criteria were as follows: age, <15 years; pregnancy; history of second‐stage HAT treated during the preceding 36 months; severe comorbidities likely to lead to early death during the follow‐up period; hemoglobin concentration, <5 g/dL; or inability to complete 18 months of follow‐up for other reasons.

Three ethics committees approved the study protocol and protocol amendments: (1) the Médecins Sans Frontières Ethical Review Board, (2) Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale (Saint‐Germain‐en‐Laye, France), and (3) the World Health Organization Research Ethics Review Committee (Geneva, Switzerland). The Congolese Ministry of Health issued authorization. All participants provided written informed consent.

A data safety monitoring board consisting of 4 independent experts was formed before study initiation. The data safety monitoring board received regular reports and issued recommendations for the study continuation.

Interventions. Participants were randomized into 1 of 2 arms: the eflornithine arm or the nifurtimox‐eflornithine arm. A scientific committee established the dosages on the basis of published and unpublished evidence. The investigational arm received eflornithine (400 mg/kg per day given intravenously every 12 h for 7 days) plus nifurtimox (15 mg/kg per day given in oral tablets every 8 h for 10 days), and the active comparator arm received eflornithine (400 mg/kg per day given intravenously every 6 h for 14 days). Eflornithine was infused over a 2‐h period and was diluted in 250 mL of normal saline. Nifurtimox doses were repeated if vomiting occurred within 30 min. All doses were directly administered and observed by the medical staff. Before commencement of treatment, all patients with malaria (as determined by microscopic evaluation and rapid diagnostic testing) received arthemeter‐lumefantrine for 3 days; study treatment was started at least 1.5 days after the last dose of antimalarial therapy. The administration of drugs for all other concomitant conditions was postponed until the end of the hospitalization, unless the clinical need warranted immediate treatment. Patients and attendants received a food ration of at least 2100 kcal/day each.

All patients were examined daily and hospitalized for 7 days after the end of treatment (or longer, if deemed necessary). A lumbar puncture was performed on the day after the patient received his or her last dose, and CSF specimens were examined for trypanosomes. Laboratory studies, including lumbar puncture examinations and examinations of blood and lymph specimens, were performed at 3, 6, 12, and 18 months. At each follow‐up visit, the CSF specimen was examined for parasites after double‐centrifugation, a parallel CSF leukocyte count was determined, and IgM titers in CSF were determined using the Latex/IgM reagent (Institut de Médecine Tropicale) [23], which aimed at increasing the sensitivity of detection of relapses. Blood samples were examined by capillary tube centrifugation and quantitative buffy coat [24]. Lymph was aspirated from any palpable posterior cervical lymph node.

The study follow‐up period was set at 18 months, as recommended by the Informal Consultation on the Conduct of Clinical Trials in HAT (World Health Organization, 2004) on the basis of data that suggested that 70%–90% of relapses occur 18 months after the

completion of treatment. Although the study end point was determined at 18 months, the national protocol required a last follow‐up visit at 24 months for all patients.

The definition of relapse is not currently standardized. Relapse was diagnosed if trypanosomes were seen in body fluid specimens or if CSF leukocyte counts increased twice consecutively by at least 20 cells/μL any time after the completion of treatment. Patients who presented with a single increase were reexamined 1 month later. At the month 18 examination, relapse was diagnosed if the CSF leukocyte count was 20 cells/μL, regardless of previous counts. Distinction between relapse and reinfection was not made. The probability of reinfection was considered to be minimal, because disease transmission had been substantially reduced after 3 years of intensive disease‐control activities by Médecins Sans Frontières.

Safety was assessed with the international Common Toxicity Criteria [25], which grade adverse events by intensity from 1 to 4 (for mild, moderate, severe, and very severe, respectively), drug‐event relationship (unrelated, unlikely, possible, probable, and definite), and outcome (complete recovery, still present, sequelae, and death). All patients had a blood sample taken before and after treatment; the sample was examined for hemoglobin concentration, total and differential leukocyte counts, total bilirubin level, creatinine level, and alanine aminotransferase level.

Anemia was defined as a hemoglobin concentration <13 g/dL for male patients and <11 g/dL for female patients that had decreased by >20%. Leukopenia was defined as a leukocyte count <4000 cells/μL that had decreased by >30%. Neutropenia as a neutrophil count <2000 cells/μL that had decreased by >30%.

μmol/L (for male subjects), with a >1.5‐fold increase.

Pharmacokinetic analysis of plasma and CSF drug concentrations was performed in an ancillary study related to the clinical trial. The methodology and results will be reported elsewhere.

Outcomes. The primary outcome was cure. The following end points were regarded as therapeutic failures: (1) death in temporal relation to treatment (i.e., 30 days after the commencement of treatment), and (2) relapse of HAT or death compatible with HAT within the 18 months of follow‐up. Deaths due to disease without clearly established alternative causality were regarded as compatible with HAT. Secondary outcomes were the adverse events in temporal relation to treatment, particularly the major adverse events graded as severe (grade 3) and very severe (grade 4).

Sample size. The sample size to test non inferiority in cure rates for the complete multicenter study was set at 280 subjects. This report analyzes the data of the 103 patients enrolled in Nkayi.

The randomization list (in blocks of 10) was electronically generated. The list and the block size were concealed from the field team. Participants were enrolled in the same order in which they received diagnoses. Sealed and numbered opaque envelopes containing the treatment allocation were opened in strict numeric sequence. Blinding was unfeasible owing to the different drug administration modes.

Data management. Data were collected in purposely designed patient charts. Trial‐specific data were extracted onto case report forms. These data were double‐entered electronically with EpiData,

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version 3.0 (The EpiData Association), and were analyzed with Stata, version 9.0 (Stata). No statistical comparisons were performed, because interim analysis would alter the statistical power of the overall trial.

Result

Participants. Of 630 patients who had HAT diagnosed during the trial period, 261 had cases that were in the second stage, of which 103 met the entry criteria and were enrolled in the study (figure 1). The main reasons for ineligibility were relapsed status, young age, and/or low CSF leukocytes count. All enrolled patients were treated: 51 were treated with eflornithine, and 52 were treated with nifurtimox‐eflornithine.

Figure 1. Trial profile. E, eflornithine; HAT, human African trypanosomiasis; N+E, nifurtimox‐eflornithine. aRelapses were detected at 12 months in one patient and 18 months in the other. bRelapses were detected both at 18 months. cCondition was controlled at 12 months, with favorable evolution; patient moved away later. dOne patient had controlled disease at 3 and 6 months, with favorable evolution; the patient later died of cerebral malaria. The other patient had controlled disease at 3, 6, and 12 months, with favorable evolution; the patient later died of ovarian cancer.

Enrollment started in August 2003 and was closed in December 2004 because of a decrease in the disease prevalence in the area, resulting in a low

enrollment rate. Patient characteristics were similar in the 2 groups (table 1). Four patients (2 in each arm) had CSF leukocyte counts of 5–20 cells/μL and were wrongly enrolled. Because these 4 patients had trypanosomes in the CSF, they were kept in the study. All patients received complete treatment in accordance with the protocol.

Table 1. Baseline characteristics of trial participants, by treatment arm.

Characteristic Eflornithine arm(n = 51)

Nifurtimox‐eflornithine arm

(n = 52)

Demographic characteristics    

Female sex 23 (45.1) 26 (50.0)

Age, mean years (range) 36.1 (15–70) 33.1 (15–69)

Weight, mean kg ± SD 53.1 ± 7.2 51.7 ± 7.4

Mean body mass index a ± SD 19.7 ± 2.2 19.1 ± 2.0

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Body mass index a <18.5 15 (29.4) 22 (42.3)

Parasitologic findings    

Case detected by active screening 29 (56.9) 31 (59.6)

Presence of trypanosomes    

In lymph nodes 40 (78.4) 36 (69.2)

In blood 47 (92.2) 42 (80.8)

In CSF 37 (72.6) 30 (57.7)

Leukocyte count in CSF    

5–20 cells/μL 2 (3.9) 2 (3.9)

21–99 cells/μL 10 (19.6) 19 (36.5)

100 cells/μL 39 (76.5) 31 (59.6)

IgM titer in CSF >1:128 b 28 (57.1) 20 (40.8)

Clinical characteristics    

Mean Karnofsky score ± SD 84.6 ± 9.6 80.9 ± 15.9

Mean Glasgow coma score ± SD 15.0 ± 0.1 14.8 ± 0.8

Hemoglobin concentration, mean g/dL ± SD 12.7 ± 1.9 13.0 ± 1.8

Presence of malaria parasites 14 (27.5) 11 (21.2)

Lymphadenopathy 40 (78.4) 36 (69.2)

Hepatomegaly 2 (3.9) 5 (9.6)

Splenomegaly 14 (27.5) 11 (21.2)

Headache 35 (68.6) 38 (73.1)

Fever c 13 (25.5) 2 (3.8)

Pruritus 33 (64.7) 33 (63.5)

Daytime somnolence 35 (68.6) 32 (61.5)

Insomnia 18 (35.3) 16 (30.8)

History of seizures 5 (9.8) 4 (7.7)

Psychiatric signs 25 (49.0) 21 (40.4)

Speech disorder 8 (15.7) 9 (17.3)

Impotence or amenorrhea 15 (29.4) 19 (36.5)

Tremors 20 (39.2) 18 (34.6)

Anorexia 6 (11.8) 12 (23.1)

Duration of symptoms, mean months (range) 9.8 (1–72) 8.7 (0–24)

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Table 1. Baseline characteristics of trial participants, by treatment arm.

Outcomes and estimation. One patient died in temporal relation to the treatment regimen and was considered to have experienced treatment failure. The large majority of patients (98 [95.1%] of 103) completed the 18‐month follow‐up period or reached the study end point earlier, and the rest (5 [4.9%]) underwent partial follow‐up. Of the 5 patients who underwent partial follow‐up, all had demonstrated decreasing CSF leukocyte counts and IgM titers at their last follow‐up visit. One died of cerebral malaria (last follow‐up visit was at month 6), another died after ovarian cancer surgery (last follow‐up visit was at month 12), and 3 moved away after the month 12 follow‐up visits. There were 4 relapses in total (2 per arm). All 4 patients who experienced relapse had increasing CSF leukocyte counts without detected trypanosomes; 1 patient had relapse at 12 months, and 3 had relapse at 18 months. Cure rates were 94.1% (48 of 51 patients) in the eflornithine arm and 96.2% (50 of 52) in the nifurtimox ‐eflornithine arm. If patients with partial follow‐up are excluded from analysis, the cure rates are 93.3% (46 of 49 patients) in the eflornithine arm and 95.9% (45 of 49) in the nifurtimox‐eflornithine arm.

Paired before‐after treatment CSF IgM titers were available for 88 patients. Blood contamination of CSF (6% of specimens) and other constraints (1% of specimens) explain the missing measurements. Lumbar puncture was performed 12 h after the last dose of eflornithine and revealed that the titers decreased in 40 (46%) of 88 patients, stayed unchanged in 38 patients (43%), and increased in 10 patients (11%). The evolution of the IgM titer values during follow‐up ( ) showed a consistent decrease over time. Only in patients who experienced relapse did increases in IgM titers accompany increases in CSF leukocyte counts.

Drug reactions. Only 1 patient, who was in the eflornithine group, died within 30 days after the start of treatment; this death was attributed to septic shock following severe neutropenia. Of a total of 261 adverse events, 14 were classified as unrelated to treatment, and 247 were regarded as drug reactions (table 2). Noteworthy differences in overall toxicity included fever, hypertension, and diarrhea, all of which were less common in the nifurtimox‐eflornithine arm. On the other hand, the combination more frequently provoked nausea and vomiting, which tended to appear 1 h after

the simultaneous administration of both drugs (for the first daily dose) and less frequently when the drugs were administered at different times.

Table 2. Clinical and biological drug reactions during hospitalization.

Event No. (%) of events

Eflornithine arm(n = 51)

Nifurtimox‐eflornithine arm

(n = 52)

All Major All Major

Treatment‐related death 1 (2.0) … 0 (0.0) …

Neurologic reactions        

Seizures 2 (3.9) 2 4 (7.7) 4

Confusion 0 (0.0) … 1 (1.9) …

Anxiety and/or agitation 4 (7.8) … 2 (3.8) …

Dizziness 1 (2.0) … 1 (1.9) …

Insomnia 1 (2.0) … 2 (3.8) …

Gastrointestinal reactions        

Anorexia 0 (0.0) … 1 (1.9) …

Abdominal pain 11 (21.6) … 10 (19.2) …

Diarrhea 4 (7.8) … 0 (0.0) …

Constipation 0 (0.0) … 2 (3.8) …

Nausea and/or vomiting 8 (15.7) … 26 (50.0) …

Cardiovascular reactions        

Arrythmia 3 (5.9) … 1 (1.9) …

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Hypertension 8 (15.7) 1 1 (1.9) …

Edema 4 (7.8) … 0 (0.0) …

Infection        

Tissue infection 1 (2.0) … 1 (1.9) …

Septic shock (neutropenic) 1 (2.0) 1 0 (0.0) …

Other infection 2 (3.9) 1 3 (5.8) …

Other clinical events        

Fever a 13 (25.5) 2 5 (9.6) 1

Headache 15 (29.4) … 13 (25.0) …

Cough 3 (5.9) … 0 (0.0) …

Pruritus 7 (13.7) … 3 (5.8) …

Skin rash 3 (5.9) … 0 (0.0) …

Chest pain 3 (5.9) … 0 (0.0) …

Myalgia and/or arthralgia 3 (5.9) … 3 (5.8) …

Other 2 (3.9) … 4 (7.7) …

Biological reactions        

Anemia 11 (21.6) 1 4 (7.7) 0

Leukopenia 1 (2.0) 0 2 (3.8) 0

Neutropenia 29 (56.9) 6 11 (21.2) 1

Abnormal bilirubin levelb 1 (2.0) … 2 (3.8) …

Abnormal alanine aminotransferase levelc 0 (0.0) … 0 (0.0) …

Abnormal creatinine levelc 0 (0.0) … 1 (1.9) …

Weight loss 5% 0 (0.0) … 3 (5.8) …

Total no. of eventsd 141 14 106 6

No. (%) of patients who experienced major events … 13 (25.5) … 5 (9.6)

Treatment interruption 2 (3.9) … 1 (1.9) …

Treatment suspension 2 (3.9) … 1 (1.9) …

Treatment termination 0 (0.0) … 0 (0.0) …

Table 2. Clinical and biological drug reactions during hospitalization.

There were 14 major drug reactions (i.e., those of grades 3 and 4) in the eflornithine arm and 6 such reactions in the nifurtimox‐eflornithine arm, and the proportions of patients who experienced major reactions were 25.5% and 9.6% per arm, respectively ( table 2). Treatment was suspended because of severe complications for 2 patients in the eflornithine arm and 1 patient in the nifurtimox‐eflornithine arm. These interruptions were made as a

29

result of seizures (1 patient in each arm) and arrhythmia (1 in the eflornithine arm). The 6 major adverse events observed in the nifurtimox ‐eflornithine arm were seizures (4 patients), fever (1 patient), and neutropenia (1 patient), all of which resolved favorably.

Before‐and‐after treatment hematologic data were available for all patients. Neutropenia and anemia were the most common biological adverse events, appearing 3 times more frequently in the eflornithine arm. Grade 3 neutropenia (i.e., a neutrophil count <1000 cells/μL) developed in 6 patients in the eflornithine arm, compared with only 1 patient in the nifurtimox‐eflornithine arm. Abnormal biochemical values were rare and mild.

Discussion

The results obtained with the nifurtimox‐eflornithine combination are similar to those obtained with standard eflornithine treatment, even showing a trend of superiority in the safety parameters. These data confirm the observations made in our earlier studies from Uganda [18, 19]. With nearly complete follow‐up data for patients (100% reviewed at least 2 times and 95% with complete follow‐up), the high cure rates are reassuring.

The fact that 3 of the 4 relapses were detected at the month 18 visit, having been controlled at 12 months, suggests that the follow‐up in clinical studies should be no shorter than 18 months. The lower occurrence of major drug reactions in the nifurtimox‐eflornithine arm suggests a better safety profile of the combination.

Neutropenia occurred more frequently among recipients of eflornithine alone than among recipients of combination treatment, which contains half as much eflornithine. Patients with grade 3 neutropenia are vulnerable to bacterial infection, which explains the frequent infectious complications emerging during or after treatment, as well as the only death in our cohort. The combination clearly offers improved safety over melarsoprol, which causes reactive encephalopathy in 5%–10% of recipients, with a high fatality rate. There were no clinical data to support theoretical concerns about toxicity related to drug interactions with the combination therapy.

Nkayi presented unusual advantages in comparison to most other HAT foci, including sociopolitical stability, the supply of electricity, the availability of internet communication, daily flights to the capital, good hospital infrastructure, and qualified medical staff. The study did not achieve the planned sample size of 280 subjects in Nkayi because of bureaucratic delays that resulted in the bulk of patients being detected and treated by Médecins Sans Frontières before enrollment could begin. There were, however, some advantages to this situation: the smaller caseload facilitated good medical supervision and eased laboratory work. With a decreased prevalence of disease, the probability of reinfection during follow‐up was also minimized.

Limitations. As a result of the insufficient number of patients recruited at this site, we have not yet performed the noninferiority analysis designed in the protocol; this will only be possible when the studies from additional sites are completed. Therefore, these data should not be regarded as definitive proof.

Overall evidence. In the face of unsatisfactory therapeutic options for second‐stage sleeping sickness, the need to identify new, safe, feasible, and effective therapies is urgent [3]. Research is hampered by the fact that foci with high caseloads that can sustain good enrollment rates are often affected by conflict and located in isolated

and impoverished parts of Africa. Gathering the minimal elements for quality research projects (e.g., qualified personnel, infrastructure, communications, and stable and functional logistics) at such sites represents a challenge that very few researchers are willing to assume.

Even if, at this first study site, we did not attain the complete sample size, we believe that our data are of crucial interest because of the promising efficacy and safety results of the nifurtimox‐eflornithine combination, which was tested here for the first time with a simpler (twice‐daily) eflornithine administration schedule.

The short half‐life of eflornithine theoretically requires shorter administration intervals for effective therapy, and this was one of the key issues addressed in this study. We hypothesize that this adverse pharmacokinetic profile may be balanced by the long‐lasting pharmacodynamic effect on trypanosomes, explained by the long time (18–19 h) needed by T. brucei gambiense to replenish their ornithine decarboxylase after inhibition by eflornithine [26]. This would give sufficient opportunity for trypanocydal nifurtimox to eliminate the parasites. Our data seem to confirm that the 12‐h intervals are possible if the agent is combined with nifurtimox.

The simplified regimen in this nifurtimox‐eflornithine schedule, which involves 4‐fold fewer eflornithine infusions (i.e., 14 infusions instead of 56), represents an important advance in terms of access to safer and effective treatment. Most treatment centers are located near disease transmission foci, in remote areas where logistical means and trained staff are scarce, and most patients need treatment for second‐stage disease. The twice‐per‐day infusions are key because they fit well in the routine of rural health facilities. Another important advantage is the cost reduction coming from the shorter hospitalization durations and from the 4‐fold reduction in the number of intravenous infusions, requiring fewer infusion materials and logistics and less staffing.

A degree of protection against the development of drug resistance can also be expected from combination versus monotherapy, as is the case for drug combinations in use for other infectious diseases. In the context of increasing parasite resistance to melarsoprol, and because eflornithine is the only alternative agent for second‐stage HAT, the availability of a combination treatment regimen is urgent, to avert the development of eflornithine resistance as well.

Because these new data reinforce the evidence in favor of the nifurtimox‐eflornithine combination, timely completion of the additional sites and ensuring good follow‐up of patients are essential. It will also be necessary to organize field studies with simpler—but sound—methodology to assess the administration of treatment in more‐realistic situations. Studies of children will also be needed. For all this to be possible, the continued production and availability of

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nifurtimox must be ensured. If our findings are corroborated by ongoing studies from additional sites (i.e., from a multicenter extension of this study), the new nifurtimox‐eflornithine combination therapy will mark a major and multifaceted advance over current therapies.

Acknowledgments

We thank the Congolese Ministry of Health—in particular, Claude Manthelot and Damase Bodzongo, whose cooperation in setting up the study was invaluable. We acknowledge the clinical and laboratory field team members, international and local, whose hard work permitted this research to happen. The following individuals contributed to the protocol development in the scientific committee: Christian Burri, Cyrus Bacchi, Dominique Legros, François Chappuis, Marc Gastellu‐Etchegorry, Philippe Büscher, and Simon von Nieuwenhove. The following provided laboratory technical advice and training: Olivier Denis, Christian Kibonge, Barrie Rooney, Marina Pozzoli, Laurence Bonte, Philippe Büscher, and Veerle Lejon. Loretxu Pinoges kindly provided advice on statistical matters. We are indebted to the members of the Data and Safety Monitoring Board: Dr. Pieter De Raadt, Dr. Jean Dupouy‐Camet, Dr. Marleen Boelaert, Dr. Jacques Chandenier and Joris Menten. The Drugs for Neglected Diseases initiative and the Swiss Tropical Institute provided advice on the Good Clinical Practice guidelines.

Financial support. This study was funded by the Dutch section of Médecins Sans Frontières. The Médecins Sans Frontières International Nobel Prize Fund supported the original protocol development.

Potential conflicts of interest. All authors: no conflicts.

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CSE Global Theme Issue on Poverty and Human Development

Cited by

D. Steverding, X. Wang. (2009) Evaluation of anti-sleeping-sickness drugs and topoisomerase inhibitors in combination on Trypanosoma brucei. Journal of Antimicrobial Chemotherapy 63:6, 1293-1295

Online publication date: 1-Jul-2009.CrossRef D.  Mumba Ngoyi, V. Lejon, F.X. N’Siesi, M. Boelaert, P.  Büscher. (2009) Comparison of operational criteria for treatment outcome I n gambiense human African trypanosomiasis. Tropical Medicine & International Health 14:4, 438-444Online publication date: 1-May-2009.CrossRef Peter G. E. Kennedy. (2008) The continuing problem of human African trypanosomiasis (sleeping sickness). Annals of Neurology 64:2, 116-126Online publication date: 1-Sep-2008.CrossRef Veerle Lejon, Isabelle Roger, Dieudonné Mumba Ngoyi, Joris Menten, Jo Robays, Francois X. N’Siesi, Sylvie Bisser, Marleen Boelaert, and Philippe Büscher. (2008) Novel Markers for Treatment Outcome in Late‐Stage Trypanosoma brucei gambiense Trypanosomiasis. Clinical Infectious Diseases 47:1, 15-22Online publication date: 1-Jul-2008.

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Abstract-Full Text-PDF Version (402 kB)François ChappuisCSE Poverty and Human Development. (2007) Editorial Commentary: Melarsoprol‐Free Drug Combinations for Second‐Stage Gambian Sleeping Sickness: The Way to Go. Clinical Infectious Diseases 45:11, 1443-1445Online publication date: 1-Dec-2007.Citation-Full Text-PDF Version (162 kB)

Medical Research

Submitted to: Dr.Mazhar QayyumSubmitted by: Farthat Yasmeen

Date: 23/o6/2008

Trypanosoma brucei

Scientific Classification

Kingdom: Protista

Phylum: Euglenozoa

Subphylum: Mastigophora

Class: Kinetoplastoa

Order: Trypanosomastida

Genus: Trypanosoma

Species: Trypanosoma brucei

Description and Significance:

The genus Trypanosoma contains a large number of parasitic species, which infect wild and domesticated animals and humans in Africa. Commonly known as African sleeping sickness, human trypanosomiasis is caused by the species Trypanosoma brucei and is transmitted to human through either a vector or the blood of ingested animal. The most common vector of Trypanosoma brucei is the tsetse fly, which may spread the parasite to humans and animals through bites. Through the process of antigenic variation, some trypanosomes are able to evade the host’s immune system by modifying their surface membrane, essentially multiplying with every surface change. As the disease progress, Trypanosoma brucei gradually infiltrates the host’s central nervous system. Symptoms include headache, weakness, and joint pain in the initial stages; anemia, cardiovascular problems, and kidney disorders as the disease progresses; in its final stages, the disease may lead to extreme exhaustion and fatigue during the day, insomnia at night, coma, and ultimately death. Human trypanosomiasis affects as many as 66 million people in Sub-Saharan Africa. Trypanosomes are also found in the America in the form of Trypanosoma cruzi, which causes American human trypanosomiasis, or Chagas’ disease. This disease is found in two forms: as an amastigote in the cells, and as a trymastigote in the blood. The vectors for Trypanosome cruzi include

members of the order Hemiptera, such as assassin flies, whichingest the amastigote or trymastigote and carry them to animals or humans. The parasites enter the human host through mucus membranes in the nose, eye, or mouth upon release from the insect vectors. Left untreated, Chagas’ disease may cause dementia, megacolon and megaesophagus and damage to the heart muscle and may result in death.

The infection: Trypanosomiasis

The insect vector for T. brucei is the tsetse fly. The parasite lives in the midgut of the fly (procyclic form), whereupon it migrates to the salivary glands for injection to the mammalian host on biting. The parasite lives within the bloodstream (bloodstream form) where it can reinfect the fly vector after biting. Later during a T. brucei infection the parasite may migrate to other areas of the host. A T. brucei infection may be transferred human to human via bodily fluid exchange, primarily blood transfer.

There are three different sub-species of T. brucei, which cause different variants of trypanosomiasis.

T. brucei gambiense - Causes slow onset chronic trypanosomiasis in humans. Most common in central and western Africa, where humans are thought to be the primary reservoir.

T. brucei rhodesiense - Causes fast onset acute trypanosomiasis in humans. Most common in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir.

T. brucei brucei - Causes animal African trypanosomiasis, along with several other species of trypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by human apolipoprotein L1. However, as it shares many features with T. b. gambiense and T. b. rhodesiense (such as antigenic variation) it is used as a model for human infections in laboratory and animal studies.

The cell structure

The structure of the cell is typical of eukaryotes, see eukaryotic cell. All major organelles are seen, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus etc. Unusual features include the single large mitochondria with a condensed mitochondrial DNA structure, and its association with the basal body of the flagellum, unusually the cytoskeleton organization mechanism of the cell. The cell surface of the bloodstream form features a dense coat of variable surface glycoproteins (VSGs) which is replaced by an equally dense coat of procyclins when the parasite differentiates into the procylic in the tsetse fly midgut. Trypanosomatid cellular forms.

Trypanosomatids show specific cellular forms:

Amastigote - Basal body anterior of nucleus, with a short, essentially non-functional, flagellum.

Promastigote - Basal body anterior of nucleus, with a long detached flagellum.

Epimastigote - Basal body anterior of nucleus, with a long flagellum attached along the cell body.

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Trypomastigote - Basal body posterior of nucleus, with a long flagellum attached along the cell body.

These names are derived from the Greek mastig- meaning whip, referring to the trypanosome's whip-like flagellum.

T. brucei is found as a trypomastigote in the slender, stumpy, procyclic and metacyclic forms. The procylic form differentiates to the proliferitive epimastigote form in the salivary glands of the insect. Unlike Leishmania, the promastigote and the amastigote form does not form part of the T.brucei life cycle.

The genome

The genome of T. brucei is made up of:

11 pairs of large chromosomes of 1 to 6 megabase pairs. 3-5 intermediate chromosomes of 200 to 500 kilobase

pairs.

Around 100 mini chromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome.

The large chromosomes contain most genes, while the small chromosomes tend to carry genes involved in antigenic variation, including the VSG genes. The genome has been sequenced and is available online.

The mitochondrial genome is found condensed into the kinetoplast, an unusual feature unique to the kinetoplastea class. It and the basal body of the flagellum are strongly associated via a cytoskeleton structure.

The cytoskeleton

The cytoskeleton is predominantly made up of microtubules, forming a subpellicular corset. The microtubules lie parallel to each other along the long axis of the cell, with the number of microtubules at any point roughly proportional to the circumference of the cell at that point. As the cell grows (including for mitosis) additional microtubules grow between the existing tubules, leading to semiconservative inheritance of the cytoskeleton. The microtubules are orientated + at the posterior and - at the anterior. Microfilament and intermediate filaments also play an important role in the cytoskeleton, but these are generally overlooked.

Flagellar structure

The trypanosome flagellum has two main structures. It is made up of a typical flagellar axoneme, which lies parallel to the paraflagellar rod, a lattice structure of proteins unique to the kinetoplastida, euglenoids and dinoflagellates.

The microtubules of the flagellar axoneme lie in the normal 9+2 arrangement, orientated with the + at the anterior end and the - in the basal body. The cytoskeletal structure extends from the basal body to the kinetoplast. The flagellum is bound to the cytoskeleton of the main cell body by four specialised microtubules, which run parallel and in the same direction to the flagellar tubulin.

The flagellar function is twofold - locomotion via oscilations along the attached flagellum and cell body, and attachment to the fly gut during the procyclic phase.

The VSG coat

The surface of the trypanosome is covered by a dense coat of ~1x107 molecules of Variable Surface Glycoprotein (VSG).[4] This coat enables an infecting T. brucei population to persistently evade the host's immune system, allowing chronic infection. The two properties of the VSG coat that allow immune evasion are:

Shielding - the dense nature of the VSG coat prevents the immune system of the mammalian host from accessing the plasma membrane or any other invariant surface epitopes (such as ion channels, transporters, receptors etc.) of the parasite. The coat is uniform, made up of millions of copies of the same molecule; therefore the only parts of the trypanosome the immune system can 'see' are the N-terminal loops of the VSG that make up the coat.

Periodic antigenic variation - the VSG coat undergoes frequent stochastic genetic modification - 'switching' - allowing variants expressing a new VSG coat to escape the specific immune response raised against the previous coat.

Antigenic variation

Sequencing of the T. brucei genome has revealed a huge VSG gene archive, made up of thousands of different VSG genes. All but one of these is 'silent' VSGs, as each trypanosome expresses only one VSG gene at a time. VSG is highly immunogenic, and an immune response raised against a specific VSG will rapidly kill trypanosomes expressing this VSG. This can also be observed in vitro by a complement-mediated lysis assay. However, with each cell division there is a possibility that one or both of the progeny will switch expression to a silent VSG from the archive (see below). The frequency of such a switch has been measured to be approximately 1:100. This new VSG will likely not be recognised by the specific immune responses raised against previously expressed VSGs. It takes several days for an immune response against a specific to develop, giving trypanosomes, which have undergone VSG coat switching some time to reproduce (and undergo further VSG coat switching events) unhindered. Repetition of this process prevents extinction of the infecting trypanosome population, allowing chronic persistence of parasites in the host. The clinical effect of this cycle is successive 'waves' of parasitaemia (trypanosomes in the blood).

Trypanosome cell cycle

The mitotic division of T.brucei is unusual in terms of the cytoskeletal process. The basal body, unlike a centrosome of most eukaryotic cells, plays an important role in the organisation of the spindle.

Stages of mitosis:

6. The basal body replicates, both remaining associated with the kinetoplast.

7. The kinetoplast undergoes replication, and the daughter kinetoplasts are separated by the basal bodies.

8. The second flagellum grows while the nucleus undergoes replication.

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9. The mitochondrion divides, and cytokinesis progresses from the anterior to posterior end.

10. The division resolves. The daughter cells may stay connected for a significant length of time after cytokinesis is complete.

Prevention and Control

Infection by trypanosome species is acquired from the bite of an infected tsetse fly. Thus, preventing flies from biting with repellants or insect nets will reduce the transmission of the parasite. Control of the flies through insecticides and habitat alteration (removal of plant cover near the water sources) is possible, but has shown to be very difficult.

Case management and Treatment

There are three stages to case management:

Screening is the initial sorting of people who might be infected. This involves checking for clinical signs or the use of serological test.

Diagnosis shows whether the parasite is present. The only sign, one that has been known for centuries, is swollen cervical glands (Winterbottom sign)

Phase diagnosis shows the state of progression of the disease. It entail examination of CSF obtained by lumbar puncture and is used to determine the course of treatment

The long, asymptomatic first phase of T.b. gambiense sleeping sickness is one of the factors that make treatment difficult. Diagnosis must be made as early as possible in order to preclude the onset of irreversible neurological disorders and prevent transmission. Case detection is difficult and requires major human, technical and material sources. Since the disease is rife in rural areas among poor people with little access to health facilities, this problem is all the more difficult.

Treatment

If the disease is diagnosed early, the chances of cure are high. The type of treatment depends on the phase of disease; initial or neurological. Success in the latter phase depends on having a drug that can cross the blood-brain barrier to reach the parasite. Four drug have been used until now.

Suramine Pentamidine Melarsoprol Eflornithine

Drug Target and Drug Discovery

The genome of the parasite has been decoded and several proteins have been identified as potential targets for drug treatment. The decoded DNA also revealed the reason why generating a vaccine for this disease has been so difficult. Trypanosoma brucei has over 800 genes that manufacture proteins that the disease mixes and matches to evade immune system detection.

Recent findings indicate that the parasite is unable to survive in the bloodstream without its flagellum. This insight gives researchers a new angle with which to attack the parasite.

A new treatment based on a truncated version of apolipoprotein L-1 of high-density lipoprotein and a nanobody has recently been found to work in mice, but has not been tested to human.

New Scientist, 25 Aug.2007, pp. 35.7 ref

The cover story of the August 25,2006 issue of Cell journal describe an advance; Dr. Lee Soo Hee and colleagues, working at John Hopkins, have investigated the pathway by which the organism make myristate, a 14-carbon length fatty acid. Myristate is a component of the VSG, the molecule that makes trypanosome’s outer layer. This outer surface coat of VSG is vital to the trypanosome’s avoidance of immunological capture, Dr. Lee and Colleague discovered trypanosomes use a novel fatty acid synthesis pathway involving fatty acid elongases to make myristate and other fatty acids.

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