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Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. We hypothesised that recent findings of neurological features and parasite brain infiltration occurring at much earlier stages in HAT than previously thought could be explained by early activation of host genetic programmes controlling CNS disease. Accordingly, a transcriptomal analysis was performed on brain tissue at 0, 7, 14, 21 and 28dpi from the HAT CD1/GVR35 mouse model. Up to 21dpi, most parasites are restricted to the blood and lymphatic system. Thereafter the trypanosomes enter the brain initiating the encephalitic stage. Analysis of ten different time point Comparison pairings, revealed a dynamic transcriptome comprising four message populations. All 7dpi Comparisons had by far more differentially expressed genes compared to all others. Prior to invasion of the parenchyma, by 7dpi, ~2,000 genes were up-regulated, denoted [7dpi↑] in contrast to a down regulated population [7dpi↓] also numbering ~2,000. However, by 14dpi both patterns had returned to around the pre-infected levels. The third, [28dpi↑] featured over three hundred transcripts which had increased modestly up to14dpi, thereafter were significantly up-regulated and peaked at 28dpi. The fourth, a minor population, [7dpi↑-28dpi↑], had similar elevated levels at 7dpi and 28dpi. KEGG and GO enrichment analysis predicted a diverse phenotype by 7dpi with changes to innate and adaptive immunity, a Type I interferon response, neurotransmission, synaptic plasticity, pleiotropic signalling, circadian activity and vascular permeability without disruption of the blood brain barrier. This key observation is consistent with recent rodent model neuroinvasion studies and clinical reports of Stage 1 HAT patients exhibiting CNS symptoms. Together, these findings challenge the strict Stage1/Stage2 phenotypic demarcation in HAT and show that that significant neurological, and immune changes can be detected prior to the onset of CNS disease. Author summary: Human African Trypanosomiasis, (sleeping sickness), transmitted by the endemic tsetse fly, remains a public health and economic blight for around 70 million inhabitants of sub-Saharan Africa. After a variable period in the haemo-lymphatic system (Stage 1), the trypanosomes cross the blood brain barrier and enter the CNS initiating the encephalitic (Stage 2) of the disease defined by the host's strong immune response with attendant neuropsychiatric debilitating symptoms including sleeping sickness, the hallmark symptom. If not treated, death invariably follows. Our understanding of the contribution of the host's genetics to these two neuropathogenic events is poorly understood. Using a mouse trypanosomiasis model spanning 28 days, we compared the gene expression profiles of trypanosome infected and control mice brains over several time points. By day 7, the activity of over 4,000 genes were altered and genetic programmes, responsible for the development of the Stage 2 encephalitic symptoms were activated prior to any significant breakdown of the blood brain barrier. These findings challenge this traditional Stage1/Stage 2 phenotypic demarcation and accords with reports of Stage 1 patients presenting with Stage 2 encephalitic symptoms and the diagnosis of asymptomatic patients reflects the complex interaction between host and parasite genetics that impact on disease progression. [ABSTRACT FROM AUTHOR]

Background: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging. [ABSTRACT FROM AUTHOR]

Invasion of the central nervous system (CNS) by African trypanosomes represents a critical step in the development of human African trypanosomiasis. In both clinical cases and experimental mouse infections it has been demonstrated that predisposition to CNS invasion is associated with a type 1 systemic inflammatory response. Using the Trypanosoma brucei brucei GVR35 experimental infection model, we demonstrate that systemic delivery of the counter-inflammatory cytokine IL-10 lowers plasma IFN-γ and TNF-α concentrations, CNS parasitosis and ameliorates neuro-inflammatory pathology and clinical symptoms of disease. The results provide evidence that CNS invasion may be susceptible to immunological attenuation. [ABSTRACT FROM AUTHOR]

Background: Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance. Methodology/Principal Findings: This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF. Conclusions: Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value. Author Summary: Human African trypanosomiasis, caused by the parasites Trypanosoma brucei rhodesiense and T.b.gambiense, is clinically defined by two diagnostic stages, an early stage where the parasites appear to be localised to the blood and lymphatic systems, and a late stage where the parasites are also localised in the central nervous system (CNS) and cause a meningoencephalitis, which is fatal if untreated. We studied the progression between these stages of infection in T.b.rhodesiense infections in Uganda. Progression from early to late stage was associated with an increase in inflammatory responses in the CNS as measured by analysis of the cerebrospinal fluid. However, contrary to predictions based on experimental model studies, neither disease stage progression nor CNS inflammatory responses were directly associated with development of neurological symptoms. Our results suggest that biological basis of the boundary between the two diagnostic stages in this infection may not be clear cut, with implications for therapeutic decision making. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis (HAT), or sleeping sickness, results from infection with the protozoan parasites Trypanosoma brucei (T.b.) gambiense or T.b.rhodesiense and is invariably fatal if untreated. There are 60 million people at risk from the disease throughout sub-Saharan Africa. The infection progresses from the haemolymphatic stage where parasites invade the blood, lymphatics and peripheral organs, to the late encephalitic stage where they enter the central nervous system (CNS) to cause serious neurological disease. The trivalent arsenical drug melarsoprol (Arsobal) is the only currently available treatment for CNS-stage T.b.rhodesiense infection. However, it must be administered intravenously due to the presence of propylene glycol solvent and is associated with numerous adverse reactions. A severe post-treatment reactive encephalopathy occurs in about 10% of treated patients, half of whom die. Thus melarsoprol kills 5% of all patients receiving it. Cyclodextrins have been used to improve the solubility and reduce the toxicity of a wide variety of drugs. We therefore investigated two melarsoprol cyclodextrin inclusion complexes; melarsoprol hydroxypropyl-β-cyclodextrin and melarsoprol randomly-methylated-β-cyclodextrin. We found that these compounds retain trypanocidal properties in vitro and cure CNS-stage murine infections when delivered orally, once per day for 7-days, at a dosage of 0.05 mmol/kg. No overt signs of toxicity were detected. Parasite load within the brain was rapidly reduced following treatment onset and magnetic resonance imaging showed restoration of normal blood-brain barrier integrity on completion of chemotherapy. These findings strongly suggest that complexed melarsoprol could be employed as an oral treatment for CNS-stage HAT, delivering considerable improvements over current parenteral chemotherapy. Author Summary: Human African trypanosomiasis (HAT) is caused by infection with either Trypanosoma brucei gambiense or T.b.rhodesiense and is fatal if untreated. In the late stages of the disease the parasites enter the brain, producing severe neurological symptoms. The arsenical drug melarsoprol is the only treatment available for rhodesiense disease once it has reached the brain. Melarsoprol is very poorly soluble in water which severely limits its oral bioavailability. Currently melarsoprol is solubilised in propylene glycol which restricts its administration to the intravenous route and treatment schedules are protracted and extremely painful. Further, this formulation of melarsoprol is toxic and kills 5% of all treated patients through the induction of a severe post-treatment reactive encephalopathy. In this study we show that combining melarsoprol with cyclodextrin molecules increases the oral bioavailability of the drug. In a murine model of late stage HAT oral treatment regimens employing melarsoprol cyclodextrin inclusion complexes rapidly cleared parasites from the brain, restored blood-brain barrier function and reduced the severity of the neuropathological response induced by the infection. If complexed melarsoprol is equally efficacious in patients with HAT this would not only have significant positive socio-economic impact but also constitute a major therapeutic advance in the field. [ABSTRACT FROM AUTHOR]

Background: Diverse clinical features have been reported in human African trypanosomiasis (HAT) foci caused by Trypanosoma brucei rhodesiense (T.b.rhodesiense) giving rise to the hypothesis that HAT manifests as a chronic disease in South-East African countries and increased in virulence towards the North. Such variation in disease severity suggests there are differences in host susceptibility to trypanosome infection and/or genetic variation in trypanosome virulence. Our molecular tools allow us to study the role of host and parasite genotypes, but obtaining matched extensive clinical data from a large cohort of HAT patients has previously proved problematic. Methods/Principal Findings: We present a retrospective cohort study providing detailed clinical profiles of 275 HAT patients recruited in two northern foci (Uganda) and one southern focus (Malawi) in East Africa. Characteristic clinical signs and symptoms of T.b.rhodesiense infection were recorded and the degree of neurological dysfunction determined on admission. Clinical observations were mapped by patient estimated post-infection time. We have identified common presenting symptoms in T.b.rhodesiense infection; however, marked differences in disease progression and severity were identified between foci. HAT was characterised as a chronic haemo-lymphatic stage infection in Malawi, and as an acute disease with marked neurological impairment in Uganda. Within Uganda, a more rapid progression to meningo-encephaltic stage of infection was observed in one focus (Soroti) where HAT was characterised by early onset neurodysfunction; however, severe neuropathology was more frequently observed in patients in a second focus (Tororo). Conclusions/Significance: We have established focus-specific HAT clinical phenotypes showing dramatic variations in disease severity and rate of stage progression both between northern and southern East African foci and between Ugandan foci. Understanding the contribution of host and parasite factors in causing such clinical diversity in T.b.rhodesiense HAT has much relevance for both improvement of disease management and the identification of new drug therapy. Author Summary: T. b. gambiense and T. b. rhodesiense cause human African trypanosomiasis (HAT). These parasite subspecies differ in their rate of progression to central nervous system (CNS) infection, and consequently to coma and death. Variation in disease progression and severity has also been documented between northern and southern East African T.b. rhodesiense HAT. However, it is unknown if this is caused by differences in patient susceptibility to infection, genetic variation in parasite virulence, or both, as despite the existence of good molecular tools, previous studies have involved limited numbers of HAT cases. In this paper we present extensive clinical data on T. b. rhodesiense cases and describe robust clinical profiles for three disease foci. Common presenting symptoms were identified. We also describe marked differences in disease progression and severity both between Ugandan and Malawi foci, and between two Ugandan foci (Tororo and Soroti) giving rise to three foci-specific clinical phenotypes ranging from a chronic haemo-lymphatic stage infection in Malawi, to rapid disease progression and neurological dysfunction in Soroti and severe neuropathology in Tororo cases. Most importantly, we have now established clinical focus-specific phenotypes that will be available to supplement host and parasite genetics studies to determine their contribution to HAT disease virulence. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasites Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, and is a major cause of systemic and neurological disability throughout sub-Saharan Africa. Following early-stage disease, the trypanosomes cross the blood–brain barrier to invade the central nervous system leading to the encephalitic, or late stage, infection. Treatment of human African trypanosomiasis currently relies on a limited number of highly toxic drugs, but untreated, is invariably fatal. Melarsoprol, a trivalent arsenical, is the only drug that can be used to cure both forms of the infection once the central nervous system has become involved, but unfortunately, this drug induces an extremely severe post-treatment reactive encephalopathy (PTRE) in up to 10% of treated patients, half of whom die from this complication. Since it is unlikely that any new and less toxic drug will be developed for treatment of human African trypanosomiasis in the near future, increasing attention is now being focussed on the potential use of existing compounds, either alone or in combination chemotherapy, for improved efficacy and safety. The kynurenine pathway is the major pathway in the metabolism of tryptophan. A number of the catabolites produced along this pathway show neurotoxic or neuroprotective activities, and their role in the generation of central nervous system inflammation is well documented. In the current study, Ro-61-8048, a high affinity kynurenine-3-monooxygenase inhibitor, was used to determine the effect of manipulating the kynurenine pathway in a highly reproducible mouse model of human African trypanosomiasis. It was found that Ro-61-8048 treatment had no significant effect (P = 0.4445) on the severity of the neuroinflammatory pathology in mice during the early central nervous system stage of the disease when only a low level of inflammation was present. However, a significant (P = 0.0284) reduction in the severity of the neuroinflammatory response was detected when the inhibitor was administered in animals exhibiting the more severe, late central nervous system stage, of the infection. In vitro assays showed that Ro-61-8048 had no direct effect on trypanosome proliferation suggesting that the anti-inflammatory action is due to a direct effect of the inhibitor on the host cells and not a secondary response to parasite destruction. These findings demonstrate that kynurenine pathway catabolites are involved in the generation of the more severe inflammatory reaction associated with the late central nervous system stages of the disease and suggest that Ro-61-8048 or a similar drug may prove to be beneficial in preventing or ameliorating the PTRE when administered as an adjunct to conventional trypanocidal chemotherapy. [ABSTRACT FROM AUTHOR]

The neurological complications of human African trypanosomiasis (HAT) in man caused by the unicellular protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense are a consequence of the penetration of the blood-brain barrier (BBB) by trypanosomes that enter the central nervous system (CNS). Yet the mechanisms by which African trypanosomes cross the true BBB comprised of brain microvascular endothelial cells (BMECs) remain unclear. Human BBB models used to determine how African trypanosomes initially interact in vitro with the human BBB proper suggest that parasites cross the human BBB in part by generating Ca2+ activation signals in human BMECs through the activity of parasite cysteine proteases. In vivo murine models of HAT have suggested additional mechanisms of BBB traversal by trypanosomes, with recent compelling evidence for the important role of interferon-γ in facilitating this process. A clear understanding of how trypanosomes enter the CNS is critical for both understanding the neuropathogenesis of HAT and in developing more effective drug therapies for late-stage disease. [ABSTRACT FROM AUTHOR]

We describe 2 spatially distinct foci of human African trypansomiasis in eastern Uganda. The Tororo and Soroti foci of Trypanosoma brucei rhodesiense infection were genetically distinct as characterized by 6 microsatellite and 1 minisatellite polymorphic markers and were characterized by differences in disease progression and hostimmune response. In particular, infections with the Tororo genotype exhibited an increased frequency of progression to and severity of the meningoencephalitic stage and higher plasma interferon (IFN)-γ concentration, compared with those with the Soroti genotype. We propose that the magnitude of the systemic IFN-γ response determines the time at which infected individuals develop central nervous system infection and that this is consistent with the recently described role of IFN-γ in facilitating blood-brain barrier transmigration of trypanosomes in an experimental model of infection. The identification of trypanosome isolates with differing disease progression phenotypes provides the first field-based genetic evidence for virulence variants in T. brucei rhodesiense. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. Current therapy with melarsoprol for CNS HAT has unacceptable side-effects with an overall mortality of 5%. This review discusses the issues of diagnosis and staging of CNS disease, its neuropathogenesis, and the possibility of new therapies for treating late-stage disease. [ABSTRACT FROM AUTHOR]