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Home , African trypanosomiasis, Visceral leishmaniasis

NEUROLOGICAL PROGRESS The Continuing Problem of Human African (Sleeping Sickness)

Peter G. E. Kennedy, MD, PhD, DSc

Human , also known as sleeping sickness, is a neglected , and it continues to pose a major threat to 60 million people in 36 countries in sub-Saharan . Transmitted by the bite of the , the disease is caused by protozoan parasites of the genus and comes in two types: East African human African trypanosomiasis caused by rhodesiense and the West African form caused by Trypanosoma brucei gambiense. There is an early or hemo- lymphatic stage and a late or encephalitic stage, when the parasites cross the blood–brain barrier to invade the . Two critical current issues are disease staging and drug therapy, especially for late-stage disease. to analyze will remain the only method of disease staging until reliable noninvasive methods are developed, but there is no widespread consensus as to what exactly defines biologically central nervous system disease or what specific cerebro- spinal fluid findings should justify drug therapy for late-stage involvement. All four main drugs used for human African trypano- somiasis are toxic, and , the only drug that is effective for both types of central nervous system disease, is so toxic that it kills 5% of patients who receive it. , alone or combined with , is being used increasingly as first-line therapy for gambiense disease. There is a pressing need for an effective, safe oral drug for both stages of the disease, but this will require a significant increase in investment for new drug discovery from Western governments and the pharmaceutical industry. Ann Neurol 2008;64:116–127

Human African trypanosomiasis (HAT), which is also tracellular form in the host. There are two forms of the known as sleeping sickness, is one of the “neglected human disease, the East African variant caused by ,” a group that includes visceral , Trypanosoma brucei rhodesiense and the West African , and Chagas’ disease.1 Although these form caused by Trypanosoma brucei gambiense3 (Fig 1). diseases kill or disable hundreds of thousands of people If untreated, the disease is always fatal. of in underdeveloped tropical regions, current treatment the disease in both humans and cattle is by the bite of for them is often antiquated, highly toxic, and fre- the blood-sucking tsetse fly of the Glossina species.4 In- quently ineffective. The pharmaceutical industry and festation by the tsetse fly covers 10 million square ki- Western governments have until recently shown little lometers, one third of Africa’s landmass, which is an interest in developing new drugs for these diseases be- area slightly larger than the United States.1 African an- cause this is associated with little or no prospect of imal trypanosomiasis, important in domestic livestock generating significant short- or long-term financial such as cattle that suffer from a wasting disease called gain. Although there has been an entirely understand- nagana, as well as wild animals, was first shown to be able emphasis in recent times on combating such caused by Trypanosoma brucei by David Bruce in 1899 global killers as , acquired immune deficiency while investigating a major outbreak of nagana in Zu- syndrome, and , it should be appreciated luland.5,6 Subsequent work by Aldo Castellani enabled that HAT is a major threat to the health of 60 million the identification of trypanosomes in the blood and ce- people in 36 countries in sub-Saharan Africa.2 More- rebrospinal fluid (CSF) in human patients with HAT over, HAT is the world’s third most important para- in 1903,6,7 and parasites causing the two human dis- sitic disease affecting human health after malaria and ease variants were identified during the period 1902 to schistosomiasis, as defined by the global burden of par- 1910.6 Both animals and humans can act as reservoirs asitic disease, calculated as the disability adjusted life of parasites capable of causing the human disease, but years lost.1 the detailed mechanisms by which this occurs are not HAT is caused by protozoan parasites of the genus fully understood. Animal trypanosomiasis has a major Trypanosoma, single-celled organisms that remain in ex- human and economic impact because it adversely af-

From the Department of Neurology, Division of Clinical Neuro- Published online in Wiley InterScience (www.interscience.wiley.com). sciences, Faculty of Medicine, University of Glasgow Institute of DOI: 10.1002/ana.21429 Neurological Sciences, Southern General Hospital, Glasgow, GS1, 4TF, Scotland, UK. Address correspondence to Dr Kennedy, Southern General Hospi- tal, Glasgow G51 4TF, United Kingdom. Received Mar 11, 2008, and in revised form Apr 23. Accepted for publication May 1, 2008. E-mail: [email protected]

116 Published 2008 by Wiley-Liss, Inc., through Wiley Subscription Services Outline of Parasite-Host Biology Details of parasite-host biology have been given else- where.3,4 Although animals are the main reservoir for T.b. rhodesiense parasites, humans are the main reser- voir for T.b. gambiense parasites.1,4 In brief, the tsetse fly feeds on an infected animal or human, after which the ingested trypanosomes undergo a number of biochemical and structural alterations in the fly’s mid- gut. Infective forms of the trypanosome then reach the fly’s salivary glands from which they are transmitted to the human host through biting. A fly remains infective for life, and the whole infective cycle is probably com- pleted successfully in only 1 in 10 flies.4 Approximately 5 to 15 days after infection, a painful skin lesion called a trypanosomal chancre may develop at the site of the bite.4 The parasites spread in the host bloodstream 1 to 3 weeks after the initial bite, and invade the lymph nodes and systemic organs including the , , heart, endocrine system, and eyes in what is termed the early, stage 1, or hemolymphatic stage.3,4 If untreated, within a few weeks in the case of rhodesiense infection, Fig 1. Distribution of East and West African sleeping sickness or many months in the case of gambiense infection, the in sub-Saharan Africa. Green areas represent Trypanosoma parasites will cross the blood–brain barrier (BBB) and brucei gambiense infection; brown areas represent Trypano- enter the central nervous system (CNS), which marks 4,13 soma brucei rhodesiense infection. (Modified from Atouguia the late, stage 2, or encephalitic stage of the disease. and Kennedy.) The entire tempo of the disease is faster in the more aggressive rhodesiense infection compared with the fects livestock production and farming. During the first chronic gambiense infection, probably as a result of the half of the twentieth century, HAT caused by T.b. greater adaptation of the latter parasite to the host.1,3 gambiense decimated entire communities in central Af- Much is known about the molecular biology of the rica,8 but then the disease was almost brought under trypanosome, and the entire T. brucei genome was se- control during the 1960s primarily as a result of highly quenced in 2005.14 It has about 9,000 genes, about effective surveillance programs. But HAT then quickly 10% of which are variable surface glycoprotein (VSG) reemerged with a progressive increase in the numbers genes encoding the VSG that are distributed on the of new cases and deaths. The World Health Organiza- entire surface of the trypanosome.3 During infection, tion (WHO) provided estimates during the period the trypanosome is able to rapidly switch the expres- 1986 to 2004 about the disease that have been widely sion of the VSG genes in and out of the expression quoted, with an annual prevalence of 300,000 to site, the result of which is antigenic variation in which 500,000 cases.2,9,10 Factors causing this increase were the surface VSG genes change so fast that the parasite primarily war and famine, which resulted in severe dis- is able to constantly evade the host’s immune re- ruption of disease surveillance and treatment, especially sponse.15 For this reason, it has not been possible, so in , Angola, , and the Congos where the far, to develop a vaccine for HAT. disease occurred in epidemics.11 Although it is still dif- ficult to provide accurate estimates of disease incidence Clinical Features of the Disease and prevalence, more recent WHO estimates have sug- There is seldom a clear clinical distinction between the gested that as a result of more efficient surveillance, a early and late stages of HAT that may appear to run significant improvement has occurred with currently as into each other. Patients in the early, or hemolym- low as 70,000 existing cases, mainly infected with T.b- phatic, stage may report nonspecific symptoms such as .gambiense.10 However, HAT has already demonstrated malaise, headache, weight loss, arthralgia, and , its ability to recur even after it had been virtually and also have episodes of accompanied by rigors brought under control. About 50 cases of HAT occur and vomiting, which may be misdiagnosed as malar- annually outside of Africa,12 usually as a result of ia.13,16 There may also be generalized lymphadenopa- Western travelers returning to North America or Eu- thy, and enlargement of posterior cervical lymph nodes rope from the East African game reserves; therefore, all is typical of gambiense disease (“Winterbottom’s sign”). physicians need to be aware of the key features of the Other symptoms and signs may correspond to partic- disease and its most appropriate drug therapy. ular involvement. Thus, there may be several dif-

Kennedy: Human African Trypanosomiasis 117 ferent kinds of skin rash, as well as pruritus, especially Table 1. Neurological Features of Human African in European patients in whom a macular, irregular, ev- Trypanosomiasis anescent rash has been described as occurring in the 17 shoulders, trunk, and upper legs. Derangement of Psychiatric and mental features liver function, , a mainly hemolytic ane- Lassitude and mental disturbances mia, and cardiac dysfunction such as and irritability tachycardia, myocarditis, pericarditis, and congestive Behavior disturbances (eg, violence, suicidal cardiac failure have been described.16,17 Many types of tendencies) endocrine dysfunction may occur such as loss of libido Uncontrolled sexual impulses and impotence, problems with menstrual function and , fertility (abortion, premature births or stillbirths, steril- ity), hair loss, gynecomastia, orchitis, and testicular at- disturbances rophy.4,16 Parasite invasion of the eye may result in Reversal of normal sleep/wake cycle iritis, conjunctivitis, iridocyclitis, keratitis, and choroi- Daytime dal atrophy.4,16 Patients are also prone to facial edema. Nocturnal The neurological features of late- or encephalitic- Uncontrollable urges to sleep stage HAT are also protean and are summarized in Ta- Alteration of sleep structure with of REM sleep ble 1. The various psychiatric and mental symptoms such as anxiety, lassitude and indifference, agitation, ir- Motor disturbances ritability, , sexual hyperactivity, suicidal tenden- Pyramidal weakness cies, and hallucinations could all be misdiagnosed ei- Extrapyramidal features ( and abnormal ther as only early-stage disease or as manifestations of a movements) primary psychiatric illness, but they are unlikely to oc- Myelopathy and myelitis cur in isolation. The neurological symptoms and signs, Muscle fasciculation which may develop insidiously, have been described Slurred speech 4,13,16,17 previously and are summarized here. A large Cerebellar number of motor features may occur in late-stage HAT Peripheral motor neuropathy with virtually every motor system at risk. Tremors in Pout reflex the hands and tongue are common, choreiform move- Palmarmental reflexes ments of the head, limbs, and trunk may occur, as may pyramidal weakness of the limbs.4 Lower limb Sensory disturbances may also occur as a result of spinal cord involvement Pruritus (myelopathy or myelitis) or peripheral motor neuropa- Deep hyperesthesia thy. Patients may also show cerebellar ataxia with walk- Also anesthesia, paraesthesia ing difficulties and slurred speech. Muscle fasciculation may also be a feature.4 The most characteristic sensory Visual involvement feature of late-stage HAT is a painful limb hyperesthe- Diplopia sia that, when it has a deep quality, is called Kerandel’s Optic neuritis sign and is common in European patients, a quarter of Papilledema whom experience it, but is unusual in African suffer- Optic atrophy 17 ers. Abnormal reflexes indicating frontal lobe involve- Drug induced ment such as pout reflex and palmomental reflexes may Peripheral neuropathy also be present. The visual system may also be affected Posttreatment reactive by late-stage disease producing diplopia, optic neuritis, 13,16 Multifocal inflammatory syndrome papilledema, and subsequent optic atrophy. Seizures The characteristic sleep disturbances that occur in Modified from Atouguia JLM, Kennedy PGE. Neurological the encephalitic stage give the disease its common aspects of human African trypanosomiasis. In: Davis LE, name. There is a disruption of the normal sleep/wake Kennedy PGE, eds. Infectious diseases of the nervous system. cycle so the patient sleeps during the day but has noc- Oxford: Butterworth-Heinemann, 2000:321-372, by turnal insomnia.18 Uncontrollable urges to sleep occur permission. without warning, and this becomes continuous in the final stages. Recently, it has been shown using poly- resolve with effective treatment. If untreated, or unsuc- somnography that there is an alteration of sleep struc- cessfully treated, the natural course of HAT is for the ture in these patients with the frequent onset of sleep patient to progressively deteriorate with increasing onset of rapid eye movements.18 Normally, REM sleep sleep disturbances, cerebral edema, incontinence, men- occurs at the end of stage 4 sleep; these abnormalities tal deterioration, seizures, and finally, death. The entire

118 Annals of Neurology Vol 64 No 2 August 2008 course of the disease to death takes several weeks in peripheral blood or lymph-node aspirates (Fig 2). This rhodesiense and several months or even years in gambi- is easier in rhodesiense infection because of the persis- ense disease. Because of the relative paucity of detailed tently high parasitemia that generally occurs than in systematic studies of the neurological features of HAT, gambiense disease, where the parasitemia tends to be it is difficult to say precisely how frequently the various low. In the latter case, parasitological confirmation us- symptoms and signs occur individually or in combina- ing concentration techniques is usually preceded by se- tion. However, an early insight into this issue was pro- rological suspicion established using the card agglutina- vided by the classic study of Duggan and Hutching- tion trypanosomiasis test, which is simple, quick, and ton17 of 109 cases of HAT in Europeans. They found easy to perform.20 Problems in management may arise, that in patients with both early- and late-stage disease, however, when the card agglutination trypanosomiasis the percentage of patients showing somnolence was test is equivocally positive. Recently, new molecular di- 37.8%, headache was 24.5%, hyperesthesia was 26.6%, agnostic techniques for diagnosing HAT have been and abnormal movements was 25.7%, psychi- tested. Thus, DNA amplification techniques such as atric symptoms was 20.1%, ataxia was 16.6%, and polymerase chain reaction21 and loop-mediated ampli- slurred speech was 10.6%. Blum and colleagues19 re- fication22 have been used to detect trypanosomes in cently provided considerable clarification of neurologi- patients’ peripheral blood, CSF, or both. Polymerase cal feature frequency in HAT. In what is the most ex- chain reaction may have a high sensitivity rate, but it is tensive analysis performed to date, these authors also associated with problems with test reproducibility, defined the frequency of specific neurological involve- an issue that appears to be less of a problem with loop- ment in a total of 2,541 patients with late-stage HAT mediated amplification.22 over a period of 3 years. They reported that the per- One of the most important issues in HAT is the centage of patients showing a was, as correct staging of the disease so that the early and late would be expected, high at 74.4%, motor weakness stages can be distinguished reliably. This is absolutely was 35.4%, gait disturbance was 22%, tremor was critical because the current treatment of late-stage dis- 21.2%, headache was 78.7%, behavior disturbance was ease, when the parasites have invaded the CNS, is so 25%, speech impairment was 14.2%, and abnormal toxic23 (see later). Because there are no reliable markers movements was 10.7%.19 As the authors pointed out, of early-stage disease, all patients who are suspected of the reasons for the high variability of neurological having CNS involvement, including all those with a symptoms and signs in different geographic areas and positive card agglutination trypanosomiasis test, must in the previous publications of HAT have yet to be undergo a lumbar puncture to examine the CSF. The clarified. physician dealing with such a case might expect a typ- ical late-stage HAT CSF to show a pleocytosis, mainly Diagnostic Considerations lymphocytes, with a white blood cell (WBC) count be- The correct diagnosis of HAT can be suspected in the tween 0 and 300/␮l, and at times even as high as appropriate context that occurs when an individual de- 1,000/␮l or more, a moderate increase in protein con- velops a fever and suggestive symptoms in a HAT- centration between 40 and 200mg/100ml, and an in- endemic or epidemic region. The most important crease in the immunoglobulin concentration, especially differential diagnosis is malaria, especially when inap- IgM caused by the strong IgM intrathecal synthesis.4,24 propriate antimalarial treatment is given to a patient To give some idea of the most frequently encountered who actually has HAT and who then shows a tempo- CSF findings, in a major study of 181 patients with rary reduction of an .4,13 This dan- gerous situation may be complicated by the fact that HAT and malaria may occur together in the same pa- tient. Other infectious diseases that may enter into the differential include human immunodeficiency virus in- fection, tuberculosis, toxoplasmosis, viral encephalitis, brucellosis, lymphoma, typhoid fever, and hookworm.4 The physician confronted with a patient with HAT would expect to find a number of nonspecific periph- eral blood abnormalities such as a mainly hemolytic , increased erythrocyte sedimentation rate, Fig 2. Giemsa-stained light photomicrograph showing the pres- thrombocytopenia, abnormal liver function tests, in- ence of Trypanosoma brucei parasites (original magnification creased IgM , and possibly a range of auto- ϫ1,000), which were found in a thin film blood smear. 4 antibodies resulting from autoimmune responses. The (Courtesy Centers for Disease Control and Prevention/Dr Mae definitive method of establishing a diagnosis of HAT is Melvin, Centers for Disease Control Public Health Image by demonstrating the presence of trypanosomes in the Library)

Kennedy: Human African Trypanosomiasis 119 late second stage gambiense disease, the median CSF WBC count was 93/␮l, with an interquartile range of 22 to 266/␮l and a maximum of 1,430/␮l.25 In the same study, the median CSF protein was 78.7mg/ 100ml, with an interquartile range of 45.4 to 106.5mg/100ml, and the greatest value was 203.8mg/ 100ml.25 But there is not a universal agreement as to what criteria define CNS involvement (apart from the detection of trypanosomes in the CSF, which is seldom easy). Moreover, there is also some disagreement as to which CSF criteria actually determine whether the pa- tient should be treated with late-stage–specific drugs.3,24 The WHO criteria are the most commonly used and define late-stage HAT as the presence of try- Fig 3. Magnetic resonance imaging scan of a 13-year-old pa- panosomes in the CSF and/or more than five ␮ 9 tient with central nervous system human African trypanosomi- WBCs/ l in the CSF. However, some clinicians in asis 3 years after successful completion of multiple treatments West Africa use a higher cutoff WBC count of more for numerous relapses. The scan shows ventricular enlargement 25 than 20, and others have suggested, reasonably in my (especially of the frontal horns) and diffuse white matter view, a midway figure of 10 WBCs/␮l.26 The CSF changes, which are prominent in the right frontal horn (ar- IgM has also been shown to be a useful indicator of row) and periventricular regions. (Reprinted from Atouguia CNS involvement.25 There is an urgent need for a and Kennedy.) more reliable diagnostic test for late-stage HAT that is cheap, reliable, easy to perform, field adaptable, and with a high sensitivity and specificity.27 A particular intermittent high-voltage delta bursts between periods of lower-voltage delta activity.4 problem with introducing a new test is that there is currently no “gold standard” test with which to com- Current Treatment of Early- and Late-Stage pare it.27 Currently, there is no noninvasive test that Disease can reliably distinguish early- from late-stage disease. Current drug therapy for both early- and late-stage There have been a few case reports of the use of HAT is unsatisfactory with a heavy reliance on four magnetic resonance imaging (MRI) in CNS HAT, al- main highly toxic drugs, most of which would have though these have been, unsurprisingly, in patients re- probably failed current rigorous safety standards.33 turning or managed in Western hospitals. MRI find- Three of the drugs, , and melar- ings are nonspecific, although consistent findings have soprol, were developed during the first half of the included diffuse hyperintensities in the basal ganglia, twentieth century, and the last drug to be registered for internal and external capsules, asymmetric white matter HAT, namely, eflornithine (also known as DFMO), abnormalities (Fig 3), and ventricular enlarge- 1,3,23 4,28,29 was registered in 1981. Another drug named ni- ment. Based on understandably limited data, furtimox is registered for Chagas’ disease and is cur- MRI appears to be more sensitive than computed to- rently under close evaluation in promising trials of mography in detecting HAT-associated abnormali- 28 combination . This depressing paucity of ties, although computed tomography has been re- safe and effective drugs for HAT has recently attracted ported as showing such changes as focal low densities increasing attention from nongovernmental organiza- in the internal capsules and centrum semiovale and ce- tions and other funding bodies. 4,28 rebral edema. Recent reports on two patients in the The standard treatment for early-stage rhodesiense American neurological literature have indicated that disease is intravenous suramin given as five injections MRI may be useful in the diagnosis of HAT in pa- over 3 weeks. Important adverse effects include ana- tients returning from visits to Africa, and may also help phylactic shock, renal failure, and skin lesions. Early- in distinguishing different CNS syndromes seen in stage gambiense disease is treated with intramuscular CNS disease.30–32 As expected, patients with late-stage pentamidine given as daily injections over 7 to 10 days. HAT have an abnormal electroencephalogram, with Important adverse effects of pentamidine include hypo- three different types of nonspecific abnormalities that glycemia, hyperglycemia, and hypotension.3,4 These both mirror the severity of the disease and improve drugs are usually effective if treatment is started early. markedly with successful treatment.4 These electroen- The treatment of late-stage HAT is even more prob- cephalographic types are a sustained low- voltage back- lematic because the only drug that is effective for both ground similar to that seen during light sleep, paroxys- types of the disease is the highly toxic arsenical melar- mal waves, or various types of and rapid soprol (Mel B), which was first used for such patients

120 Annals of Neurology Vol 64 No 2 August 2008 in 1949.3,4,23 Until recently, the standard regimen was either alone or in combination with other drugs. Ef- two to four courses of intravenous injections (three in- lornithine, which is expensive, became an orphan jections per course, with 1-week intervals between drug even after it had been shown to be effective them), but a more recent 10-day continuous melarso- against gambiense disease in 1981.1,3 But after an in- prol regimen has been introduced for treatment of novative partnership among Mede´cin Sans Frontie`res, gambiense disease34 and is now favored in many cen- WHO, and the pharmaceutical industry, the drug was ters. Although melarsoprol is usually effective, it is fol- again made available for HAT use in sub-Saharan Af- lowed by a severe posttreatment reactive encephalopa- rica1 and is being used increasingly as first-line ther- thy (PTRE) in about 10% of patients, half of whom apy and alternative therapy. It does, however, have to die of it. This overall mortality rate of 5% of all pa- be given by daily intravenous injection over at least tients receiving melarsoprol is one of the greatest prob- 14 days, and its adverse effects include lems in this field and highlights the extreme impor- toxicity, seizures, and gastrointestinal symptoms.3,4,23 tance of correct staging of the disease. Although Recent clinical trials have tested various combinations treating patients who do not, in fact, have CNS disease of drugs for late-stage gambiense disease, and the with melarsoprol will lead to an unnecessary 5% risk emerging results are increasingly favoring the use of a for death, not treating a patient who does actually have nifurtimox-eflornithine regimen as the most promis- CNS disease will inevitably lead to death because of ing first-line therapy.38,39 The only new drug on the the 100% fatality rate of HAT if untreated.1,3,13,35 Pa- immediate horizon for HAT is the diamidine deriva- tients who acquire PTRE (usually after the first treat- tive DB 289, a promising development funded by the ment course or near the end of the continuous treat- Bill and Melinda Gates Foundation.3,13 This drug is ment course) may experience development of deep given orally, can treat only early-stage disease, and has coma with seizures, convulsive status epilepticus, or been under recent evaluation in a Phase 3 clinical rapid progressive coma in the absence of seizures or trial. However, clinical trials with this drug (manu- cerebral edema.4,13 Patients with the PTRE are treated factured by Immtech Pharmaceuticals, New York, with intravenous corticosteroids, anticonvulsants, and NY) have recently been put on hold because of pos- intensive general medical support, and those who sur- sible liver toxicity, and further development of this vive need to continue the melarsoprol course. The pos- drug has unfortunately been discontinued. A sum- sible role of corticosteroid pretreatment to prevent mary of current drug therapy for HAT is shown in and/or ameliorate the PTRE is currently unclear,3,13 Table 2. although the author would probably choose this option After successful treatment, all patients need to be for himself. Recently, it has been shown that a combi- followed up with blood and CSF analyses at 6-month nation regimen of melarsoprol and nifurtimox for gam- intervals for 2 years, after which the patient is con- biense disease is more effective than standard melarso- sidered cured if these tests are normal.4,13 Treatment prol monotherapy regimens.36 Melarsoprol may also failures do occur, however, and it has recently been produce cardiac arrhythmias, skin lesions, agranulocy- shown that the presence of intrathecal IgM synthesis tosis, peripheral neuropathy, and as recently described, and increased CSF IgM IL-10 concentrations are sig- a steroid-responsive multifocal inflammatory ill- nificantly associated with the failure of treatment in ness.3,30,31 early-stage gambiense disease.40 Analysis of sleep struc- There are alternative treatment options for late- ture with may also prove useful in stage gambiense disease, although not for rhodesiense detecting patients with relapses. Follow-up of patients disease for which melarsoprol remains the only effec- in the field is problematic, and as has recently been tive drug. Treatment failure with melarsoprol, how- pointed out, counting all patients who never turn up ever, is well recognized.37 The drug eflornithine is be- for follow-up CSF examination can hardly be a good ing increasingly used for CNS gambiense disease, measure of cure rates, especially when half the pa-

Table 2. Summary of Drug Therapy in Sleeping Sickness Disease First-Line Therapy Alternative Therapy

Early-stage Trypanosoma brucei rhodesiense Suramin None Early-stage Trypanosoma brucei gambiense Pentamidine Suramin Late-stage T.b. rhodesiense Melarsoprol None Late-stage T.b. gambiense Melarsoprol Eflornithine Ϯ nifurtimoxa aIt is likely that eflornithine with or without nifurtimox may soon be the preferred first-line therapy for treating late-stage gambiense disease.

Kennedy: Human African Trypanosomiasis 121 tients never show up.8 Patients who have been suc- cessfully treated for late-stage disease may still experi- ence development of long-term neurological sequelae such as weakness, ataxia, cognitive impairment, epi- lepsy, and psychiatric disorders.4

Neuropathogenesis Pathological data in HAT have been obtained from a relatively small number of autopsy studies. The key findings in CNS HAT are an extensive meningoen- cephalitis, widespread infiltration of white matter with inflammatory cells such as , lymphocytes

Fig 4. Brain pathology in central nervous system (CNS) hu- man African trypanosomiasis. (A) Late-stage disease in a pa- tient dying 3 to 5 months after first injection of melarsoprol. Many large astrocytes are located in white matter. Stained for glial fibrillary acidic protein by immunoperoxidase. Original magnification ϫ400. (B) Morular (Mott) cells (arrows) ob- served in the brain of a patient with CNS trypanosomiasis who had not received melarsoprol. Morular cells are plasma cells filled with immunoglobulin. Hematoxylin and eosin (HE) stain. Original magnification ϫ400. (C) Posttreatment reac- tive encephalopathy (PTRE) in a patient 9 days after receiving melarsoprol. Ischaemic cell changes (arrows) are seen in neu- rons in the hippocampus. HE stain. Original magnification ϫ250. (D) PTRE with hemorrhagic leukoencephalopa- thy in a patient 9 days after receiving melarsoprol. There is fibrinoid necrosis in an arteriole (arrow) and focal hemor- rhage in the pons. Martius scarlet blue stain. Original magni- Fig 5. A schematic of the sleeping sickness mouse model of cen- fication ϫ250. (Reprinted from Adams and colleagues,41 by tral nervous system (CNS) disease. PTRE ϭ posttreatment reac- permission.) tive encephalopathy. (Reprinted from Kennedy,1 by permission.)

122 Annals of Neurology Vol 64 No 2 August 2008 and plasma cells, extensive perivascular cuffing, astro- has been derived from animal models, in particular, a cyte and activation, and a small amount of highly reproducible mouse model of HAT that closely demyelination.41–43 A pathognomonic finding in the mimics human late-stage disease and also allows the white matter is the presence of morular or Mott cells, identification of new therapeutic targets44,45,48 (Fig which are plasma cells containing IgM eosinophilic in- 5). Subcurative therapy of infected mice with the clusions41,42 (Fig 4). drug berenil leads to an exacerbation of CNS disease The neuropathogenesis of HAT has been described with neuropathological features that show strong sim- in considerable detail elsewhere,3,42–45 and only few ilarities with the human PTRE.49 This approach has key aspects are mentioned here. Not surprisingly, allowed the identification of key players in the gener- only limited data have been obtained from patients in ation of the neuroinflammatory response such as the the African field, primarily from analysis of immune roles of astrocyte activation, the neuropeptide sub- factors in blood and CSF where cause and effect can stance P, and the balance of proinflammatory and be difficult to establish. Recently, it was shown in counterinflammatory , including tumor ne- two sites in Uganda that a greater plasma interferon-␥ crosis factor-␣ and IL-10, respectively50–53 (summa- level correlated with a greater severity of neurological rized in Fig 6). Knock-out mice lacking specific in- impairment in rhodesiense HAT patients, indicating a flammatory factors have also been used in several pathological role of this .46 Patients with late- laboratories.54,55 It appears likely that there is a com- stage rhodesiense disease have been shown to have sig- plex array of CNS-cytokine interactions that ulti- nificantly increased levels of the counterinflammatory mately determine the CNS damage in HAT.3,45 cytokine IL-10 in the plasma and CSF, declining to Moreover, several drugs and novel drug combinations normal levels after treatment.47 It is possible that it is have been tested in the mouse model.56–58 Rodent the balance of cytokines that is a key determinant of models have also provided insights into the mecha- the outcome of CNS disease. nism of BBB traversal by trypanosomes, an area of Much of our mechanistic knowledge in this area crucial importance. For example, trypanosomes show

Fig 6. Schematic representation of possible immunopathological pathways leading to brain dysfunction in human African trypanoso- miasis, based on data and concepts from both human and animal data. Cytokines shown in red probably have important roles in neuropathogenesis. The schematic emphasizes the central importance of early astrocyte activation, cytokine responses, and macrophage activation. There are likely to be multiple factors acting together to produce and also multiple potential sources of different cytokines. IFN ϭ interferon; MHC ϭ major histocompatibility complex; NK ϭ natural killer; NO ϭ nitric oxide; Tltf ϭ trypanosome-derived lymphocyte triggering factor; TNF ϭ tumor necrosis factor; VSG ϭ variable surface glycoprotein. (Re- printed from Kennedy,3 by permission.).

Kennedy: Human African Trypanosomiasis 123 early invasion in brain areas that lack a BBB, such as therapy. Regrettably, progress in both of these areas the pineal gland and median eminence.59 A seminal continues to be modest. In the absence of a noninva- study showed that in interferon-␥ knock-out mice, sive method of staging, it is inevitable that distinction parasites accumulated in the perivascular compart- between early- and late-stage HAT will continue to ment, confined between the endothelial and paren- rely entirely on CSF examination. But not only is chymal basement membranes, thus confirming the there disagreement as to what biologically constitutes critical role of interferon-␥ in parasite entry into the CNS disease, there is also an absence of a consensus CNS.60 The same group has shown that, in as to what are the correct grounds for making thera- trypanosome-infected rats, there is immunological peutic choices. Current drug therapy, especially for and biochemical disruption of the suprachiasmatic late-stage HAT, is unacceptably toxic, and remark- nuclei of the hypothalamus with resultant disorgani- ably, there are no new drugs at all on the horizon for zation of normal circadian rhythms61,62 (Fig 7). treating CNS disease. If there were available a non- These findings strongly suggest a credible neuro- toxic oral drug for late-stage HAT, then most of the pathological basis for the altered sleep/wake cycles staging problems would be immediately obviated. seen in HAT. These difficult problems are a direct result of many decades of underinvestment in this neglected disease, Conclusions a trend that is beginning to be addressed. However, HAT continues to be a major health problem they must be seen in the context of the wider efforts throughout sub-Saharan Africa and is likely to be so to control the disease at the level of the tsetse fly vec- for the foreseeable future. The two key related issues tor1,63 because disruption of the man/tsetse fly con- in disease management are disease staging and drug tact through various technologies probably holds the

Fig 7. (A, B) Images of the hypothalamic suprachiasmatic nucleus (which plays a role of circadian pacemaker in the mammalian brain) in sections processed for glial fibrillary acidic protein (GFAP) immunoreactivity. (A) Control noninfected rat. (B) Rat in- fected with Trypanosoma brucei brucei; note the astrocytic activation (shown by hypertrophy and increased immunoreactivity of astrocytes) in the infected animal. (C–E) Images of the cingulate cortex of a rat infected with Trypanosoma brucei brucei (pro- cessed for double immunohistochemistry to show GFAP-immunolabeled astrocytes (red: C) and the proinflammatory cytokine tumor necrosis factor (TNF)-␣ (green: D); (E) merging of two images (yellow) and, therefore, the colocalization of the two markers. (C) Note the activation of astrocytes, (D) the induction of TNF-␣ expression in cells of the brain parenchyma, and (E) that TNF-␣ is expressed in astrocytes. Scale bars ϭ 100␮m (A, B); 25␮m (C–E). oc ϭ optic chiasm; 3v ϭ third ventricle. (Courtesy Maria Pal- omba, Gigliola Grassi-Zucconi and Marina Bentivoglio, University of Verona, Verona, Italy)

124 Annals of Neurology Vol 64 No 2 August 2008 key to the potential ultimate control of sleeping sick- 19. Blum J, Schmid C, Burri C. Clinical aspects of 2541 patients ness. with second stage human African trypanosomiasis. Acta Trop 2006;97:55–64. 20. Truc P, Lejon V, Magnus E, et al. Evaluation of the micro- CATT, CATT/Trypanosoma brucei gambiense, and LATEX/ The authors research is supported by grants from the Wellcome T.b. gambiense methods for serodiagnosis and surveillance of Trust and the Medical Research Council. human African trypanosomiasis in West and Central Africa. I thank Drs J. Ndung’u and V. Lejon for their helpful comments on Bull World Health Organ 2002;80:882–886. the manuscript, and Dr J. Atouguia for advice. I am grateful to Prof 21. Jamonneau V, Solano P, Garcia A, et al. Stage determination M. Bentivoglio for generously providing the unpublished Figure 7. and therapeutic decision in human African trypanosomiasis: This article is dedicated to the memory of my recently departed value of polymerase chain reaction and immunoglobulin M father, Philip Kennedy, a man of great compassion, outstanding in- quantification on the cerebrospinal fluid of sleeping sickness pa- telligence, and unquenchable humor. tients in Coˆte d’Ivoire. Trop Med Int Health 2003;8:589–594. 22. Njiru ZK, Mikosza ASJ, Armstrong T, et al. Loop-mediated isothermal amplification (LAMP) method for rapid detection of References Trypanosoma brucei rhodesiense. PLos Negl Trop Dis 2008;2: 1. Kennedy PGE. The fatal sleep. Edinburgh, United Kingdom: 147–154. Luath Press Limited, 2007. 23. Fairlamb AH. Chemotherapy of human African trypanosomiasis: 2. Epidemiology and control of African trypanosomiasis. Report current and future prospects. Trends Parasitol 2003;19:488–494. of a WHO expert committee. 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