The Continuing Problem of Human African Trypanosomiasis (Sleeping Sickness)

Transcription

1 NEUROLOGICAL PROGRESS The Continuing Problem of Human African Trypanosomiasis (Sleeping Sickness) Peter G. E. Kennedy, MD, PhD, DSc Human African trypanosomiasis, also known as sleeping sickness, is a neglected disease, and it continues to pose a major threat to 60 million people in 36 countries in sub-saharan Africa. Transmitted by the bite of the tsetse fly, the disease is caused by protozoan parasites of the genus Trypanosoma and comes in two types: East African human African trypanosomiasis caused by Trypanosoma brucei rhodesiense and the West African form caused by Trypanosoma brucei gambiense. There is an early or hemolymphatic stage and a late or encephalitic stage, when the parasites cross the blood brain barrier to invade the central nervous system. Two critical current issues are disease staging and drug therapy, especially for late-stage disease. Lumbar puncture to analyze cerebrospinal fluid 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 cerebrospinal fluid findings should justify drug therapy for late-stage involvement. All four main drugs used for human African trypanosomiasis are toxic, and melarsoprol, 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. Eflornithine, alone or combined with nifurtimox, 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: Human African trypanosomiasis (HAT), which is also known as sleeping sickness, is one of the neglected diseases, a group that includes visceral leishmaniasis, schistosomiasis, and Chagas disease. 1 Although these diseases kill or disable hundreds of thousands of people in underdeveloped tropical regions, current treatment for them is often antiquated, highly toxic, and frequently ineffective. The pharmaceutical industry and Western governments have until recently shown little interest in developing new drugs for these diseases because this is associated with little or no prospect of generating significant short- or long-term financial gain. Although there has been an entirely understandable emphasis in recent times on combating such global killers as malaria, acquired immune deficiency syndrome, and tuberculosis, it should be appreciated that HAT is a major threat to the health of 60 million people in 36 countries in sub-saharan Africa. 2 Moreover, HAT is the world s third most important parasitic disease affecting human health after malaria and schistosomiasis, as defined by the global burden of parasitic disease, calculated as the disability adjusted life years lost. 1 HAT is caused by protozoan parasites of the genus Trypanosoma, single-celled organisms that remain in extracellular form in the host. There are two forms of the human disease, the East African variant caused by Trypanosoma brucei rhodesiense and the West African form caused by Trypanosoma brucei gambiense 3 (Fig 1). If untreated, the disease is always fatal. Transmission of the disease in both humans and cattle is by the bite of the blood-sucking tsetse fly of the Glossina species. 4 Infestation by the tsetse fly covers 10 million square kilometers, one third of Africa s landmass, which is an area slightly larger than the United States. 1 African animal trypanosomiasis, important in domestic livestock such as cattle that suffer from a wasting disease called nagana, as well as wild animals, was first shown to be caused by Trypanosoma brucei by David Bruce in 1899 while investigating a major outbreak of nagana in Zululand. 5,6 Subsequent work by Aldo Castellani enabled the identification of trypanosomes in the blood and cerebrospinal fluid (CSF) in human patients with HAT in 1903, 6,7 and parasites causing the two human disease variants were identified during the period 1902 to Both animals and humans can act as reservoirs of parasites capable of causing the human disease, but the detailed mechanisms by which this occurs are not fully understood. Animal trypanosomiasis has a major human and economic impact because it adversely af- From the Department of Neurology, Division of Clinical Neurosciences, Faculty of Medicine, University of Glasgow Institute of Neurological Sciences, Southern General Hospital, Glasgow, GS1, 4TF, Scotland, UK. Received Mar 11, 2008, and in revised form Apr 23. Accepted for publication May 1, Published online in Wiley InterScience ( DOI: /ana Address correspondence to Dr Kennedy, Southern General Hospital, Glasgow G51 4TF, United Kingdom. p.g.kennedy@clinmed.gla.ac.uk 116 Published 2008 by Wiley-Liss, Inc., through Wiley Subscription Services

2 Fig 1. Distribution of East and West African sleeping sickness in sub-saharan Africa. Green areas represent Trypanosoma brucei gambiense infection; brown areas represent Trypanosoma brucei rhodesiense infection. (Modified from Atouguia and Kennedy.) fects livestock production and farming. During the first half of the twentieth century, HAT caused by T.b. gambiense decimated entire communities in central Africa, 8 but then the disease was almost brought under control during the 1960s primarily as a result of highly effective surveillance programs. But HAT then quickly reemerged with a progressive increase in the numbers of new cases and deaths. The World Health Organization (WHO) provided estimates during the period 1986 to 2004 about the disease that have been widely quoted, with an annual prevalence of 300,000 to 500,000 cases. 2,9,10 Factors causing this increase were primarily war and famine, which resulted in severe disruption of disease surveillance and treatment, especially in Uganda, Angola, Sudan, and the Congos where the disease occurred in epidemics. 11 Although it is still difficult to provide accurate estimates of disease incidence and prevalence, more recent WHO estimates have suggested that as a result of more efficient surveillance, a significant improvement has occurred with currently as low as 70,000 existing cases, mainly infected with T.b-.gambiense. 10 However, HAT has already demonstrated its ability to recur even after it had been virtually brought under control. About 50 cases of HAT occur annually outside of Africa, 12 usually as a result of Western travelers returning to North America or Europe from the East African game reserves; therefore, all physicians need to be aware of the key features of the disease and its most appropriate drug therapy. Outline of Parasite-Host Biology Details of parasite-host biology have been given elsewhere. 3,4 Although animals are the main reservoir for T.b. rhodesiense parasites, humans are the main reservoir for T.b. gambiense parasites. 1,4 In brief, the tsetse fly vector feeds on an infected animal or human, after which the ingested trypanosomes undergo a number of biochemical and structural alterations in the fly s midgut. 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 completed 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 liver, spleen, 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, or many months in the case of gambiense infection, the parasites will cross the blood brain barrier (BBB) and enter the central nervous system (CNS), which marks the late, stage 2, or encephalitic stage of the disease. 4,13 The entire tempo of the disease is faster in the more aggressive rhodesiense infection compared with the chronic gambiense infection, probably as a result of the greater adaptation of the latter parasite to the host. 1,3 Much is known about the molecular biology of the trypanosome, and the entire T. brucei genome was sequenced in It has about 9,000 genes, about 10% of which are variable surface glycoprotein (VSG) genes encoding the VSG that are distributed on the entire surface of the trypanosome. 3 During infection, the trypanosome is able to rapidly switch the expression of the VSG genes in and out of the expression site, the result of which is antigenic variation in which the surface VSG genes change so fast that the parasite is able to constantly evade the host s immune response. 15 For this reason, it has not been possible, so far, to develop a vaccine for HAT. Clinical Features of the Disease There is seldom a clear clinical distinction between the early and late stages of HAT that may appear to run into each other. Patients in the early, or hemolymphatic, stage may report nonspecific symptoms such as malaise, headache, weight loss, arthralgia, and fatigue, and also have episodes of fever accompanied by rigors and vomiting, which may be misdiagnosed as malaria. 13,16 There may also be generalized lymphadenopathy, and enlargement of posterior cervical lymph nodes is typical of gambiense disease ( Winterbottom s sign ). Other symptoms and signs may correspond to particular organ involvement. Thus, there may be several dif- Kennedy: Human African Trypanosomiasis 117

3 ferent kinds of skin rash, as well as pruritus, especially in European patients in whom a macular, irregular, evanescent rash has been described as occurring in the shoulders, trunk, and upper legs. 17 Derangement of liver function, hepatomegaly, a mainly hemolytic anemia, splenomegaly and cardiac dysfunction such as tachycardia, myocarditis, pericarditis, and congestive cardiac failure have been described. 16,17 Many types of endocrine dysfunction may occur such as loss of libido and impotence, problems with menstrual function and fertility (abortion, premature births or stillbirths, sterility), hair loss, gynecomastia, orchitis, and testicular atrophy. 4,16 Parasite invasion of the eye may result in iritis, conjunctivitis, iridocyclitis, keratitis, and choroidal atrophy. 4,16 Patients are also prone to facial edema. The neurological features of late- or encephaliticstage HAT are also protean and are summarized in Table 1. The various psychiatric and mental symptoms such as anxiety, lassitude and indifference, agitation, irritability, mania, sexual hyperactivity, suicidal tendencies, and hallucinations could all be misdiagnosed either as only early-stage disease or as manifestations of a primary psychiatric illness, but they are unlikely to occur in isolation. The neurological symptoms and signs, which may develop insidiously, have been described previously 4,13,16,17 and are summarized here. A large number of motor features may occur in late-stage HAT with virtually every motor system at risk. Tremors in the hands and tongue are common, choreiform movements of the head, limbs, and trunk may occur, as may pyramidal weakness of the limbs. 4 Lower limb paralysis may also occur as a result of spinal cord involvement (myelopathy or myelitis) or peripheral motor neuropathy. Patients may also show cerebellar ataxia with walking difficulties and slurred speech. Muscle fasciculation may also be a feature. 4 The most characteristic sensory feature of late-stage HAT is a painful limb hyperesthesia that, when it has a deep quality, is called Kerandel s sign and is common in European patients, a quarter of whom experience it, but is unusual in African sufferers. 17 Abnormal reflexes indicating frontal lobe involvement such as pout reflex and palmomental reflexes may also be present. The visual system may also be affected by late-stage disease producing diplopia, optic neuritis, papilledema, and subsequent optic atrophy. 13,16 The characteristic sleep disturbances that occur in the encephalitic stage give the disease its common name. There is a disruption of the normal sleep/wake cycle so the patient sleeps during the day but has nocturnal insomnia. 18 Uncontrollable urges to sleep occur without warning, and this becomes continuous in the final stages. Recently, it has been shown using polysomnography that there is an alteration of sleep structure in these patients with the frequent onset of sleep onset of rapid eye movements. 18 Normally, REM sleep occurs at the end of stage 4 sleep; these abnormalities Table 1. Neurological Features of Human African Trypanosomiasis Psychiatric and mental features Lassitude and mental disturbances Anxiety and irritability Behavior disturbances (eg, violence, suicidal tendencies) Uncontrolled sexual impulses Hallucinations, delirium Sleep disturbances Reversal of normal sleep/wake cycle Daytime somnolence Nocturnal insomnia Uncontrollable urges to sleep Alteration of sleep structure with sleep onset of REM sleep Motor disturbances Pyramidal weakness Extrapyramidal features (tremors and abnormal movements) Myelopathy and myelitis Muscle fasciculation Slurred speech Cerebellar ataxia Peripheral motor neuropathy Pout reflex Palmarmental reflexes Sensory disturbances Pruritus Deep hyperesthesia Also anesthesia, paraesthesia Visual involvement Diplopia Optic neuritis Papilledema Optic atrophy Drug induced Peripheral neuropathy Posttreatment reactive encephalopathy Multifocal inflammatory syndrome Seizures Modified from Atouguia JLM, Kennedy PGE. Neurological aspects of human African trypanosomiasis. In: Davis LE, Kennedy PGE, eds. Infectious diseases of the nervous system. Oxford: Butterworth-Heinemann, 2000: , by permission. resolve with effective treatment. If untreated, or unsuccessfully treated, the natural course of HAT is for the patient to progressively deteriorate with increasing sleep disturbances, cerebral edema, incontinence, mental deterioration, seizures, and finally, death. The entire 118 Annals of Neurology Vol 64 No 2 August 2008

4 course of the disease to death takes several weeks in rhodesiense and several months or even years in gambiense disease. Because of the relative paucity of detailed systematic studies of the neurological features of HAT, it is difficult to say precisely how frequently the various symptoms and signs occur individually or in combination. However, an early insight into this issue was provided by the classic study of Duggan and Hutchington 17 of 109 cases of HAT in Europeans. They found that in patients with both early- and late-stage disease, the percentage of patients showing somnolence was 37.8%, headache was 24.5%, hyperesthesia was 26.6%, tremor and abnormal movements was 25.7%, psychiatric symptoms was 20.1%, ataxia was 16.6%, and slurred speech was 10.6%. Blum and colleagues 19 recently provided considerable clarification of neurological feature frequency in HAT. In what is the most extensive analysis performed to date, these authors defined the frequency of specific neurological involvement in a total of 2,541 patients with late-stage HAT over a period of 3 years. They reported that the percentage of patients showing a sleep disorder was, as would be expected, high at 74.4%, motor weakness was 35.4%, gait disturbance was 22%, tremor was 21.2%, headache was 78.7%, behavior disturbance was 25%, speech impairment was 14.2%, and abnormal movements was 10.7%. 19 As the authors pointed out, the reasons for the high variability of neurological symptoms and signs in different geographic areas and in the previous publications of HAT have yet to be clarified. Diagnostic Considerations The correct diagnosis of HAT can be suspected in the appropriate context that occurs when an individual develops a fever and suggestive symptoms in a HATendemic or epidemic region. The most important differential diagnosis is malaria, especially when inappropriate antimalarial treatment is given to a patient who actually has HAT and who then shows a temporary reduction of an intermittent fever. 4,13 This dangerous situation may be complicated by the fact that HAT and malaria may occur together in the same patient. Other infectious diseases that may enter into the differential include human immunodeficiency virus infection, 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 peripheral blood abnormalities such as a mainly hemolytic anemia, increased erythrocyte sedimentation rate, thrombocytopenia, abnormal liver function tests, increased IgM antibodies, and possibly a range of autoantibodies resulting from autoimmune responses. 4 The definitive method of establishing a diagnosis of HAT is by demonstrating the presence of trypanosomes in the peripheral blood or lymph-node aspirates (Fig 2). This is easier in rhodesiense infection because of the persistently high parasitemia that generally occurs than in gambiense disease, where the parasitemia tends to be low. In the latter case, parasitological confirmation using concentration techniques is usually preceded by serological suspicion established using the card agglutination trypanosomiasis test, which is simple, quick, and easy to perform. 20 Problems in management may arise, however, when the card agglutination trypanosomiasis test is equivocally positive. Recently, new molecular diagnostic techniques for diagnosing HAT have been tested. Thus, DNA amplification techniques such as polymerase chain reaction 21 and loop-mediated amplification 22 have been used to detect trypanosomes in patients peripheral blood, CSF, or both. Polymerase chain reaction may have a high sensitivity rate, but it is also associated with problems with test reproducibility, an issue that appears to be less of a problem with loopmediated amplification. 22 One of the most important issues in HAT is the correct staging of the disease so that the early and late stages can be distinguished reliably. This is absolutely critical because the current treatment of late-stage disease, when the parasites have invaded the CNS, is so toxic 23 (see later). Because there are no reliable markers of early-stage disease, all patients who are suspected of having CNS involvement, including all those with a positive card agglutination trypanosomiasis test, must undergo a lumbar puncture to examine the CSF. The physician dealing with such a case might expect a typical late-stage HAT CSF to show a pleocytosis, mainly lymphocytes, with a white blood cell (WBC) count between 0 and 300/ l, and at times even as high as 1,000/ l or more, a moderate increase in protein concentration between 40 and 200mg/100ml, and an increase in the immunoglobulin concentration, especially IgM caused by the strong IgM intrathecal synthesis. 4,24 To give some idea of the most frequently encountered CSF findings, in a major study of 181 patients with Fig 2. Giemsa-stained light photomicrograph showing the presence of Trypanosoma brucei parasites (original magnification 1,000), which were found in a thin film blood smear. (Courtesy Centers for Disease Control and Prevention/Dr Mae Melvin, Centers for Disease Control Public Health Image Library) Kennedy: Human African Trypanosomiasis 119

5 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 patient 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 trypanosomes in the CSF and/or more than five WBCs/ l in the CSF. 9 However, some clinicians in West Africa use a higher cutoff WBC count of more than 20, 25 and others have suggested, reasonably in my view, a midway figure of 10 WBCs/ l. 26 The CSF IgM has also been shown to be a useful indicator of CNS involvement. 25 There is an urgent need for a 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 problem with introducing a new test is that there is currently no gold standard test with which to compare it. 27 Currently, there is no noninvasive test that can reliably distinguish early- from late-stage disease. There have been a few case reports of the use of magnetic resonance imaging (MRI) in CNS HAT, although these have been, unsurprisingly, in patients returning or managed in Western hospitals. MRI findings are nonspecific, although consistent findings have included diffuse hyperintensities in the basal ganglia, internal and external capsules, asymmetric white matter abnormalities (Fig 3), and ventricular enlargement. 4,28,29 Based on understandably limited data, MRI appears to be more sensitive than computed tomography in detecting HAT-associated abnormalities, 28 although computed tomography has been reported as showing such changes as focal low densities in the internal capsules and centrum semiovale and cerebral edema. 4,28 Recent reports on two patients in the American neurological literature have indicated that MRI may be useful in the diagnosis of HAT in patients returning from visits to Africa, and may also help in distinguishing different CNS syndromes seen in CNS disease As expected, patients with late-stage HAT have an abnormal electroencephalogram, with three different types of nonspecific abnormalities that both mirror the severity of the disease and improve markedly with successful treatment. 4 These electroencephalographic types are a sustained low- voltage background similar to that seen during light sleep, paroxysmal waves, or various types of delta wave and rapid Fig 3. Magnetic resonance imaging scan of a 13-year-old patient with central nervous system human African trypanosomiasis 3 years after successful completion of multiple treatments for numerous relapses. The scan shows ventricular enlargement (especially of the frontal horns) and diffuse white matter changes, which are prominent in the right frontal horn (arrow) and periventricular regions. (Reprinted from Atouguia and Kennedy.) intermittent high-voltage delta bursts between periods of lower-voltage delta activity. 4 Current Treatment of Early- and Late-Stage Disease Current drug therapy for both early- and late-stage HAT is unsatisfactory with a heavy reliance on four main highly toxic drugs, most of which would have probably failed current rigorous safety standards. 33 Three of the drugs, suramin, pentamidine and melarsoprol, were developed during the first half of the twentieth century, and the last drug to be registered for HAT, namely, eflornithine (also known as DFMO), was registered in ,3,23 Another drug named nifurtimox is registered for Chagas disease and is currently under close evaluation in promising trials of combination chemotherapy. This depressing paucity of safe and effective drugs for HAT has recently attracted increasing attention from nongovernmental organizations and other funding bodies. The standard treatment for early-stage rhodesiense disease is intravenous suramin given as five injections over 3 weeks. Important adverse effects include anaphylactic shock, renal failure, and skin lesions. Earlystage gambiense disease is treated with intramuscular pentamidine given as daily injections over 7 to 10 days. Important adverse effects of pentamidine include hypoglycemia, hyperglycemia, and hypotension. 3,4 These drugs are usually effective if treatment is started early. The treatment of late-stage HAT is even more problematic because the only drug that is effective for both types of the disease is the highly toxic arsenical melarsoprol (Mel B), which was first used for such patients 120 Annals of Neurology Vol 64 No 2 August 2008

6 in ,4,23 Until recently, the standard regimen was two to four courses of intravenous injections (three injections per course, with 1-week intervals between them), but a more recent 10-day continuous melarsoprol regimen has been introduced for treatment of gambiense disease 34 and is now favored in many centers. Although melarsoprol is usually effective, it is followed by a severe posttreatment reactive encephalopathy (PTRE) in about 10% of patients, half of whom die of it. This overall mortality rate of 5% of all patients receiving melarsoprol is one of the greatest problems in this field and highlights the extreme importance of correct staging of the disease. Although treating patients who do not, in fact, have CNS disease with melarsoprol will lead to an unnecessary 5% risk for death, not treating a patient who does actually have CNS disease will inevitably lead to death because of the 100% fatality rate of HAT if untreated. 1,3,13,35 Patients who acquire PTRE (usually after the first treatment course or near the end of the continuous treatment course) may experience development of deep coma with seizures, convulsive status epilepticus, or rapid progressive coma in the absence of seizures or cerebral edema. 4,13 Patients with the PTRE are treated with intravenous corticosteroids, anticonvulsants, and intensive general medical support, and those who survive need to continue the melarsoprol course. The possible role of corticosteroid pretreatment to prevent and/or ameliorate the PTRE is currently unclear, 3,13 although the author would probably choose this option for himself. Recently, it has been shown that a combination regimen of melarsoprol and nifurtimox for gambiense disease is more effective than standard melarsoprol monotherapy regimens. 36 Melarsoprol may also produce cardiac arrhythmias, skin lesions, agranulocytosis, peripheral neuropathy, and as recently described, a steroid-responsive multifocal inflammatory illness. 3,30,31 There are alternative treatment options for latestage gambiense disease, although not for rhodesiense disease for which melarsoprol remains the only effective drug. Treatment failure with melarsoprol, however, is well recognized. 37 The drug eflornithine is being increasingly used for CNS gambiense disease, either alone or in combination with other drugs. Eflornithine, which is expensive, became an orphan drug even after it had been shown to be effective against gambiense disease in ,3 But after an innovative partnership among Medécin Sans Frontières, WHO, and the pharmaceutical industry, the drug was again made available for HAT use in sub-saharan Africa 1 and is being used increasingly as first-line therapy and alternative therapy. It does, however, have to be given by daily intravenous injection over at least 14 days, and its adverse effects include bone marrow toxicity, seizures, and gastrointestinal symptoms. 3,4,23 Recent clinical trials have tested various combinations of drugs for late-stage gambiense disease, and the emerging results are increasingly favoring the use of a nifurtimox-eflornithine regimen as the most promising first-line therapy. 38,39 The only new drug on the immediate horizon for HAT is the diamidine derivative DB 289, a promising development funded by the Bill and Melinda Gates Foundation. 3,13 This drug is given orally, can treat only early-stage disease, and has been under recent evaluation in a Phase 3 clinical trial. However, clinical trials with this drug (manufactured by Immtech Pharmaceuticals, New York, NY) have recently been put on hold because of possible liver toxicity, and further development of this drug has unfortunately been discontinued. A summary of current drug therapy for HAT is shown in Table 2. After successful treatment, all patients need to be followed up with blood and CSF analyses at 6-month intervals for 2 years, after which the patient is considered cured if these tests are normal. 4,13 Treatment failures do occur, however, and it has recently been shown that the presence of intrathecal IgM synthesis and increased CSF IgM IL-10 concentrations are significantly associated with the failure of treatment in early-stage gambiense disease. 40 Analysis of sleep structure with polysomnography may also prove useful in detecting patients with relapses. Follow-up of patients in the field is problematic, and as has recently been pointed out, counting all patients who never turn up for follow-up CSF examination can hardly be a good 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 nifurtimox a a It 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

7 tients never show up. 8 Patients who have been successfully treated for late-stage disease may still experience development of long-term neurological sequelae such as weakness, ataxia, cognitive impairment, epilepsy, 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 meningoencephalitis, widespread infiltration of white matter with inflammatory cells such as macrophages, lymphocytes Fig 4. Brain pathology in central nervous system (CNS) human African trypanosomiasis. (A) Late-stage disease in a patient 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 400. (B) Morular (Mott) cells (arrows) observed 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 400. (C) Posttreatment reactive encephalopathy (PTRE) in a patient 9 days after receiving melarsoprol. Ischaemic cell changes (arrows) are seen in neurons in the hippocampus. HE stain. Original magnification 250. (D) PTRE with acute hemorrhagic leukoencephalopathy in a patient 9 days after receiving melarsoprol. There is fibrinoid necrosis in an arteriole (arrow) and focal hemorrhage in the pons. Martius scarlet blue stain. Original magnification 250. (Reprinted from Adams and colleagues, 41 by permission.) Fig 5. A schematic of the sleeping sickness mouse model of central nervous system (CNS) disease. PTRE posttreatment reactive encephalopathy. (Reprinted from Kennedy, 1 by permission.) 122 Annals of Neurology Vol 64 No 2 August 2008

8 and plasma cells, extensive perivascular cuffing, astrocyte and macrophage activation, and a small amount of demyelination A pathognomonic finding in the white matter is the presence of morular or Mott cells, which are plasma cells containing IgM eosinophilic inclusions 41,42 (Fig 4). The neuropathogenesis of HAT has been described in considerable detail elsewhere, 3,42 45 and only few key aspects are mentioned here. Not surprisingly, only limited data have been obtained from patients in the African field, primarily from analysis of immune factors in blood and CSF where cause and effect can be difficult to establish. Recently, it was shown in two sites in Uganda that a greater plasma interferon- level correlated with a greater severity of neurological impairment in rhodesiense HAT patients, indicating a pathological role of this cytokine. 46 Patients with latestage rhodesiense disease have been shown to have significantly increased levels of the counterinflammatory cytokine IL-10 in the plasma and CSF, declining to normal levels after treatment. 47 It is possible that it is the balance of cytokines that is a key determinant of the outcome of CNS disease. Much of our mechanistic knowledge in this area has been derived from animal models, in particular, a highly reproducible mouse model of HAT that closely mimics human late-stage disease and also allows the identification of new therapeutic targets 44,45,48 (Fig 5). Subcurative therapy of infected mice with the drug berenil leads to an exacerbation of CNS disease with neuropathological features that show strong similarities with the human PTRE. 49 This approach has allowed the identification of key players in the generation of the neuroinflammatory response such as the roles of astrocyte activation, the neuropeptide substance P, and the balance of proinflammatory and counterinflammatory cytokines, including tumor necrosis factor- and IL-10, respectively (summarized in Fig 6). Knock-out mice lacking specific inflammatory factors have also been used in several laboratories. 54,55 It appears likely that there is a complex array of CNS-cytokine interactions that ultimately determine the CNS damage in HAT. 3,45 Moreover, several drugs and novel drug combinations have been tested in the mouse model Rodent models have also provided insights into the mechanism of BBB traversal by trypanosomes, an area of crucial importance. For example, trypanosomes show Fig 6. Schematic representation of possible immunopathological pathways leading to brain dysfunction in human African trypanosomiasis, 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 brain damage and also multiple potential sources of different cytokines. IFN interferon; MHC major histocompatibility complex; NK natural killer; NO nitric oxide; Tltf trypanosome-derived lymphocyte triggering factor; TNF tumor necrosis factor; VSG variable surface glycoprotein. (Reprinted from Kennedy, 3 by permission.). Kennedy: Human African Trypanosomiasis 123

9 early invasion in brain areas that lack a BBB, such as the pineal gland and median eminence.59 A seminal study showed that in interferon- knock-out mice, parasites accumulated in the perivascular compartment, confined between the endothelial and parenchymal basement membranes, thus confirming the critical role of interferon- in parasite entry into the CNS.60 The same group has shown that, in trypanosome-infected rats, there is immunological and biochemical disruption of the suprachiasmatic nuclei of the hypothalamus with resultant disorganization of normal circadian rhythms61,62 (Fig 7). These findings strongly suggest a credible neuropathological basis for the altered sleep/wake cycles seen in HAT. Conclusions HAT continues to be a major throughout sub-saharan Africa and for the foreseeable future. The two in disease management are disease health problem is likely to be so key related issues staging and drug therapy. Regrettably, progress in both of these areas continues to be modest. In the absence of a noninvasive method of staging, it is inevitable that distinction between early- and late-stage HAT will continue to rely entirely on CSF examination. But not only is there disagreement as to what biologically constitutes CNS disease, there is also an absence of a consensus as to what are the correct grounds for making therapeutic choices. Current drug therapy, especially for late-stage HAT, is unacceptably toxic, and remarkably, there are no new drugs at all on the horizon for treating CNS disease. If there were available a nontoxic oral drug for late-stage HAT, then most of the staging problems would be immediately obviated. These difficult problems are a direct result of many decades of underinvestment in this neglected disease, a trend that is beginning to be addressed. However, they must be seen in the context of the wider efforts to control the disease at the level of the tsetse fly vector1,63 because disruption of the man/tsetse fly contact 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 infected 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 (processed 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 100 m (A, B); 25 m (C E). oc optic chiasm; 3v third ventricle. (Courtesy Maria Palomba, Gigliola Grassi-Zucconi and Marina Bentivoglio, University of Verona, Verona, Italy) 124 Annals of Neurology Vol 64 No 2 August 2008

10 key to the potential ultimate control of sleeping sickness. The authors research is supported by grants from the Wellcome Trust and the Medical Research Council. I thank Drs J. Ndung u and V. Lejon for their helpful comments on the manuscript, and Dr J. Atouguia for advice. I am grateful to Prof M. Bentivoglio for generously providing the unpublished Figure 7. This article is dedicated to the memory of my recently departed father, Philip Kennedy, a man of great compassion, outstanding intelligence, and unquenchable humor. References 1. Kennedy PGE. The fatal sleep. Edinburgh, United Kingdom: Luath Press Limited, Epidemiology and control of African trypanosomiasis. Report of a WHO expert committee. Geneva, Switzerland: World Health Organization Technical Report Series, 1986; Kennedy PGE. Human African trypanosomiasis of the CNS: current issues and challenges. J Clin Invest 2004;113: Atouguia JLM, Kennedy PGE. Neurological aspects of human African trypanosomiasis. 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11 38. Priotto G, Kasparian S, Ngouama D, et al. Nifurtimox- Eflornithine combination therapy for second-stage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Congo. Clin Infect Dis 2007;45: Checchi F, Piola P, Ayikoru H, et al. Nifurtimox plus Eflornithine for late-stage sleeping sickess in Uganda: a case series. PLos Negl Trop Dis 2007;1: Lejon V, Robays J, N Siesi FX, et al. Treatment failure related to intrathecal immunoglobulin M (IgM) synthesis, cerebrospinal fluid IgM, and interleukin-10 in patients with hemolymphatic-stage sleeping sickness. Clin Vaccine Immunol 2007;14: Adams JH, Haller L, Boa FY, et al. Human African trypanosomiasis (T./b.gambiense): a study of 16 fatal cases of sleeping sickness with some observations on acute reactive arsenical encephalopathy. Neuropathol Appl Neurobiol 1986;12: Pentreath VW, Kennedy PGE. Pathogenesis of human African trypanosomiasis. In: Maudlin I, Holmes PH, Miles MA, eds. The Trypanosomiases. Oxford: CAB International, 2004: Cook GC. Protozoan and helminthic infections. In: Lambert HP, ed. Infections of the central nervous system. Philadelphia: BC Dekker, 1991: Kennedy PGE. The pathogenesis and modulation of the posttreatment reactive encephalopathy in a mouse model of human African trypanosomiasis. J Neuroimmunol 1999;100: Kennedy PGE. Diagnostic and neuropathogenesis issues in human African trypanosomiasis. Intl J Parasitol 2006;36: MacLean L, Odiit M, MacLeod A, et al. Spatially and genetically distinct African trypanosome virulence variants defined by host interferon-y response. J Infect Dis 2007;196: MacLean L, Odiit M, Sternberg JM. Nitric oxide and cytokine synthesis in human African trypanosomiasis. J Infect Dis 2001; 184: Kennedy PGE. Animal models of human African trypanosomiasis very useful or too far removed? Roy Soc Trop Med Hyg 2007;101: Hunter CA, Jennings FW, Adams JH, et al. Sub-curative chemotherapy and fatal post-treatment reactive encephalopathies in African trypanosomiasis. Lancet 1992;339: Hunter CA, Jennings FW, Kennedy PGE, Murray M. Astrocyte activation correlates with cytokine production in central nervous system of Trypanosoma brucei brucei-infected mice. Lab Invest 1992;67: Kennedy PGE, Rodgers J, Jennings FW, et al. A Substance P antagonist, RP-67,580 ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei. Proc Natl Acad Sci USA 1997;94: Hunter CA, Gow JW, Kennedy PGE, et al. Immunopathology of experimental African sleeping sickness: detection of cytokine mrna in the brains of Trypanosoma brucei brucei-infected mice. Infect Immun 1991;5: Sternberg JM, Rodgers J, Bradley B, et al. Meningoencephalitic African trypanosomiasis: brain IL-10 and IL-6 are associated with protection from neuroinflammatory pathology. J Neuroimmunol 2005;167: Kennedy PGE, Rodgers J, Bradley B, et al. Clinical and neuroinflammatory responses to meningoencephalitis in substance P receptor knockout mice. Brain 2003;126: Barkhulzen M, Magez S, Atkinson RA, Brombacher F. Interleukin-12p70-dependent interferon-gamma production is crucial for resistance in African trypanosomiasis. Infect Dis 2007;196: Hunter CA, Jennings FW, Kennedy PGE, Murray M. The use of azathioprine to ameliorate post-treatment encephalopathy associated with African trypanosomiasis. Neuropathol Appl Neurobiol 1992;18: Jennings FW, Gichuki CW, Kennedy PGE, et al. The role of the polyamine inhibitor eflornithine in the neuropathogenesis of experimental murine African trypanosomiasis. Neuropathol Appl Neurobiol 1997;23: Jennings FW, Rodgers J, Bradley B, et al. Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen. Parasitol Intl 2002;51: Schultzberg M, Ambatsis M, Samuelsson EB, et al. Spread of trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia. J Neurosci Res 1988;21: Masocha W, Robertson B, Rottenberg ME, et al. Cerebral vessel laminins and IFN- define trypanosoma brucei brucei penetration of the blood-brain barrier. J Clin Invest 2004;114: Bentivoglio M, Grassi-Zucconi G, Olsson T, Kristensson K. Trypanosoma brucei and the nervous system. Trends Neurosci 1994;17: Bentivoglio M, Kristensson K. Neural-immune interactions in disorders of sleep-wakefulness organization. Trends Neurosci 2007;30: Barrett MP, Burchmore RJS, Stich A, et al. The trypanosomiases. Lancet 2003;362: Annals of Neurology Vol 64 No 2 August 2008