Stage Human African Trypanosomiasis Research Articles

Chemotherapy of Second Stage Human African Trypanosomiasis: Comparison between the Parenteral Diamidine DB829 and Its Oral Prodrug DB868 in Vervet Monkeys

Chemotherapy of Second Stage Human African Trypanosomiasis: Comparison between the Parenteral Diamidine DB829 and Its Oral Prodrug DB868 in Vervet Monkeys

Increased acute immune response during the meningo-encephalitic stage of Trypanosoma brucei rhodesiense sleeping sickness compared to Trypanosoma brucei gambiense

The host central nervous system (CNS) response to infection with Trypanosoma brucei (T. b.) gambiense or T. b. rhodesiense parasites, the causing agent of human African trypanosomiasis (HAT), is a poorly explored area. The two parasites are responsible for respectively a chronic and an acute form of HAT. In both cases, however, the disease progresses from a haemolymphatic first stage (S1) to a meningo-encephalitic second stage (S2) due to the penetration of parasites into the CNS.In the present study, we investigated and compared the cerebrospinal fluid (CSF) from S2 patients affected by either T. b. gambiense or T. b. rhodesiense HAT, using a mass spectrometry quantitative proteomics approach. Gene ontology and pathway analyses on the 222 quantified human proteins revealed a predominant activation of the innate immune and the acute phase responses in rhodesiense HAT patients. These results were further confirmed through the verification of the over-expression of two proteins involved in these mechanisms, C-reactive protein (CRP) and orosomucoid 1 (ORM1), in 126 S2 HAT patients suffering from either the chronic or the acute form of HAT. Both proteins were significantly increased (p<0.0001) in the CSF of rhodesiense HAT patients.These findings contribute in better understanding the pathophysiological mechanisms of late stage HAT caused by T. b. gambiense or T. b. rhodesiense and pave the way for further investigations on the clinical significance of CRP and ORM1.Mass spectrometry data are available via ProteomeXchange (identifier PXD001082).

Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.

Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS) is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs) has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general ‘ATP-engaging’ binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.

Determination of an Optimal Dosing Regimen for Fexinidazole, a Novel Oral Drug for the Treatment of Human African Trypanosomiasis: First-in-Human Studies

Background and ObjectivesFexinidazole is a 5-nitroimidazole recently included in a clinical efficacy trial as an oral drug for the treatment of human African trypanosomiasis (HAT). Preclinical studies showed it acts as a pharmacologically active pro-drug with two key active metabolites: sulfoxide and sulfone (the most active metabolite). The present studies aimed to determine the best dose regimen for the treatment of stage 2 sleeping sickness patients, which could eventually also treat stage 1 patients.MethodsFexinidazole was assessed in 154 healthy adult male subjects of sub-Saharan African origin. Three initial first-in-human studies and two additional studies assessed a single ascending dose and multiple ascending doses (both under fasted conditions), tablet versus suspension formulation and food effect (fasted vs. high-fat meal and field-adapted food), and multiple ascending doses with a loading dose regimen under fed conditions.ResultsFexinidazole was well-tolerated in a single dose from 100 to 3,600 mg, with quick absorption of the parent drug and rapid metabolism into sulfoxide [time to maximum concentration (t max) 2–5 h] and sulfone (t max 18–24 h). The tablet formulation was approximately 25 % less bioavailable than the suspension, and food intake increased drug absorption and plasma concentrations of fexinidazole and its two metabolites by approximately 200 %. Fourteen-day multiple ascending dosing administered up to 3,600 mg/day in fasted conditions showed that fexinidazole was generally well-tolerated (mild to moderate, spontaneously reversible drug-related adverse events). Following the high-fat food effect finding, another study was conducted to evaluate the impact of a low-fat regimen closer to that of the target population, showing that the type of meal does not influence fexinidazole absorption. The last study showed that a loading dose of 1,800 mg/day for 4 days followed by a 1,200 mg/day regimen for 6 days with a normal meal provided the desired exposure of fexinidazole and its metabolites, particularly sulfone, with good tolerability. Based on preclinical evidence from a chronic infection mouse model, systemic drug concentrations obtained are expected to be clinically effective in stage 2 HAT.ConclusionsThese studies show that fexinidazole can be safely assessed in patients as a potential oral cure for both stages of HAT.Electronic supplementary materialThe online version of this article (doi:10.1007/s40262-014-0136-3) contains supplementary material, which is available to authorized users.

Stage determination in sleeping sickness: comparison of two cell counting and two parasite detection techniques

Diagnosis of the neurological stage of human African trypanosomiasis is performed by examination of cerebrospinal fluid (CSF) for the presence of trypanosomes and numbers of white blood cells (WBC). Both CSF parameters are also used to assess treatment outcome during follow-up. In view of the importance of CSF examination, and the practical problems encountered with it, we compared the sensitivity of two trypanosome concentration techniques and the repeatability of two cell counting methods, as well as occurrence of systematic differences between them. Patients were recruited at Dipumba hospital, in Mbuji-Mayi in the Democratic Republic of the Congo. In 94 CSF samples, trypanosome detection was performed with modified single centrifugation (MSC) and double centrifugation (DC). On 189 CSF samples with ≤30cells/μl, cell counting was performed in duplicate in a Fuchs-Rosenthal counting chamber and in a disposable Uriglass counting chamber. Modified single centrifugation detected trypanosomes in significantly (P<0.0001) more patients (85) than DC (46). Cell counts did not differ systematically in the two methods. Variability in the differences between duplicate cell counts was significantly higher (P=0.002) in Uriglass (SD of differences 2.03) than in Fuchs-Rosenthal (SD of differences 1.62). For analysis of CSF in the context of sleeping sickness stage determination and follow-up after treatment, we strongly recommend the MSC for parasite detection and the application of disposable counting chambers. When the first cell count is ≤20cells/μl, we recommend repeating the counting procedure on the same CSF specimen and taking the average of both countings.

Management of trypanosomiasis and leishmaniasis.

BackgroundThe current treatments for human African trypanosomiasis (HAT), Chagas disease and leishmaniasis (collectively referred to as the kinetoplastid diseases) are far from ideal but, for some, there has been significant recent progress. For HAT the only advances in treatment over the past two decades have been the introduction of an eflornithine/nifurtimox co-administration and a shorter regime of the old standard melarsoprol.Sources of dataPubMed.Areas of AgreementThere is a need for new safe, oral drugs for cost-effective treatment of patients and use in control programmes for all the trypanosomatid diseases.Areas of controversyCutaneous leishmaniasis is not on the agenda and treatments are lagging behind.Growing pointsThere are three compounds in development for the treatment of the CNS stage of HAT: fexinidazole, currently due to entry into phase II clinical studies, a benzoxaborole (SCYX-7158) in phase I trials and a diamidine derivative (CPD-0802), in advanced pre-clinical development. For Chagas disease, two anti-fungal triazoles are now in clinical trial. In addition, clinical studies with benznidazole, a drug previously recommended only for acute stage treatment, are close to completion to determine the effectiveness in the treatment of early chronic and indeterminate Chagas disease. For visceral leishmaniasis new formulations, therapeutic switching, in particular AmBisome, and the potential for combinations of established drugs have significantly improved the opportunities for the treatment in the Indian subcontinent, but not in East Africa.Areas timely for developing researchImproved diagnostic tools are needed to support treatment, for test of cure in clinical trials and for monitoring/surveillance of populations in control programmes.

New Pentamidine Analogues in Medicinal Chemistry

Sixty years after its introduction, 1,5-bis(4-amidinophenoxy)pentane (Pentamidine) is still one of the most used drugs for the treatment of the first stage of Human African trypanosomiasis and other neglected diseases such as malaria and leishmaniasis. These protozoan infections are prevalent in the poorest world areas such as sub-saharian and developing countries, however the increasing immigration from these countries to the richest part of the world and the overlap of HIV with parasitic infections result in a growing number of cases in developed nations. A great effort has been made to develop new generations of diamidines for the treatment of these infections transmitted by insects. This review summarises the synthesis and evaluation of pentamidine analogues reported in the last years in the effort to find new drugs with better pharmaceutical activity, higher lipophilicity and lower citotoxycicty.

Monitoring the use of nifurtimox-eflornithine combination therapy (NECT) in the treatment of second stage gambiense human African trypanosomiasis.

After inclusion of the nifurtimox-eflornithine combination therapy (NECT) in the Model List of Essential Medicines for the treatment of second-stage gambiense human African trypanosomiasis (HAT), the World Health Organization, in collaboration with National Sleeping Sickness Control Programs and nongovernmental organizations set up a pharmacovigilance system to assess the safety and efficacy of NECT during its routine use. Data were collected for 1735 patients treated with NECT in nine disease endemic countries during 2010-2011. At least one adverse event (AE) was described in 1043 patients (60.1%) and a total of 3060 AE were reported. Serious adverse events (SAE) were reported for 19 patients (1.1% of treated), leading to nine deaths (case fatality rate of 0.5%). The most frequent AE were gastrointestinal disorders (vomiting/nausea and abdominal pain), followed by headache, musculoskeletal pains, and vertigo. The most frequent SAE and cause of death were convulsions, fever, and coma that were considered as reactive encephalopathy. Two hundred and sixty-two children below 15 years old were treated. The characteristics of AE were similar to adults, but the major AE were less frequent in children with only one SAE and no deaths registered in this group. Gastrointestinal problems (vomiting and abdominal pain) were more frequent than in adults, but musculoskeletal pains, vertigo, asthenia, neuropsychiatric troubles (headaches, seizures, tremors, hallucinations, insomnia) were less frequent in children. Patient follow-up after treatment is continuing, but initial data could suggest that NECT is effective as only a low number of relapses have so far been reported (19 cases). However, additional monitoring is required to assess the efficacy of the treatment, particularly in children. NECT has given satisfactory results of safety in the usual conditions where HAT patients are managed and it is currently the best option for treatment of second stage of gambiense HAT.

Pharmacology of DB844, an orally active aza analogue of pafuramidine, in a monkey model of second stage human African trypanosomiasis.

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-yl]-nicotinamide (DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.

Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

State of the art in African trypanosome drug discovery.

African sleeping sickness is endemic in sub-Saharan Africa where the WHO estimates that 60 million people are at risk for the disease. Human African trypanosomiasis (HAT) is 100% fatal if untreated and the current drug therapies have significant limitations due to toxicity and difficult treatment regimes. No new chemical agents have been approved since eflornithine in 1990. The pentamidine analog DB289, which was in late stage clinical trials for the treatment of early stage HAT recently failed due to toxicity issues. A new protocol for the treatment of late-stage T. brucei gambiense that uses combination nifurtomox/eflornithine (NECT) was recently shown to have better safety and efficacy than eflornithine alone, while being easier to administer. This breakthrough represents the only new therapy for HAT since the approval of eflornithine. A number of research programs are on going to exploit the unusual biochemical pathways in the parasite to identify new targets for target based drug discovery programs. HTS efforts are also underway to discover new chemical entities through whole organism screening approaches. A number of inhibitors with anti-trypanosomal activity have been identified by both approaches, but none of the programs are yet at the stage of identifying a preclinical candidate. This dire situation underscores the need for continued effort to identify new chemical agents for the treatment of HAT.

Polysomnography as a diagnosis and post-treatment follow-up tool in human African trypanosomiasis: A case study in an infant

Gambian ( Trypanosoma brucei gambiense) human African trypanosomiasis (HAT) evolves from the hemolymphatic stage 1, treated with pentamidine, to the meningoencephalitic stage 2, often treated with melarsoprol. This arseniate may provoke a deadly reactive encephalopathy. It is therefore crucial to diagnose precisely the stages of HAT, especially when clinical and biological examinations are doubtful. We present here the case of a 30-month old girl (E20 KOLNG) diagnosed with stage 1 HAT during a field survey in June 2007 in Congo. She was followed-up every six months for 18 months in a village dispensary facility at Mpouya. Her health status deteriorated in December 2008, although cerebrospinal fluid (CSF) white blood cell (WBC) count was normal. The child was hospitalized at Brazzaville and a daytime polysomnographic recording (electroencephalogram, electrooculogram, and electromyogram) was performed (Temec Vitaport 3® portable recorder) to avoid a new lumbar puncture. The child presented a complete polysomnographic syndrome of HAT with a major disturbance of the distribution of sleep and wake episodes and the occurrence of sleep onset REM periods (SOREMPs). The relapse at stage 2 was confirmed by a new CSF examination that showed an elevated WBC count (23 cells·μL − 1 ) with the presence of B lymphocytes. Melarsoprol treatment was undertaken. A post-treatment recording was immediately performed, showing the resolution of sleepwake pattern abnormalities. Another polysomnography, taken four months later, confirmed the normalization of sleep–wake patterns indicating healing. We therefore propose that polysomnography, being a non-invasive technique, should be used in children to alleviate burden caused by HAT staging procedures, especially regarding lumbar punctures in remote African villages.

The distribution of nifurtimox across the healthy and trypanosome-infected murine blood-brain and blood-cerebrospinal fluid barriers.

Nifurtimox, an antiparasitic drug, is used to treat American trypanosomiasis (Chagas disease) and has shown promise in treating central nervous system (CNS)-stage human African trypanosomiasis (HAT; sleeping sickness). In combination with other antiparasitic drugs, the efficacy of nifurtimox against HAT improves, although why this happens is unclear. Studying how nifurtimox crosses the blood-brain barrier (BBB) and reaches the CNS may clarify this issue and is the focus of this study. To study the interaction of nifurtimox with the blood-CNS interfaces, we used the in situ brain/choroid plexus perfusion technique in healthy and trypanosome-infected mice and the isolated incubated choroid plexus. Results revealed that nifurtimox could cross the healthy and infected blood-brain and blood-cerebrospinal fluid (CSF) barriers (K(in) brain parenchyma was 50.8 ± 9.0 μl · min(-1) · g(-1)). In fact, the loss of barrier integrity associated with trypanosome infection failed to change the distribution of [(3)H]nifurtimox to any significant extent, suggesting there is not an effective paracellular barrier for [(3)H]nifurtimox entry into the CNS. Our studies also indicate that [(3)H]nifurtimox is not a substrate for P-glycoprotein, an efflux transporter expressed on the luminal membrane of the BBB. However, there was evidence of [(3)H]nifurtimox interaction with transporters at both the blood-brain and blood-CSF barriers as demonstrated by cross-competition studies with the other antitrypanosomal agents, eflornithine, suramin, melarsoprol, and pentamidine. Consequently, CNS efficacy may be improved with nifurtimox-pentamidine combinations, but over time may be reduced when nifurtimox is combined with eflornithine, suramin, or melarsoprol.

Matrix metalloproteinase‐9 and intercellular adhesion molecule 1 are powerful staging markers for human African trypanosomiasis

A critical step before treatment of human African trypanosomiasis (HAT) is the correct staging of the disease. As late stage is established when trypanosomes cross the blood-brain barrier and invade the central nervous system, we hypothesized that matrix metalloproteinases and cell adhesion molecules could indicate, alone or in combination, the disease progression from the first to the second stage of HAT. We measured the levels of MMP-2, MMP-9, ICAM-1, VCAM-1 and E-selectin in the cerebrospinal fluid (CSF) of 63 Trypanosoma brucei gambiense-infected patients (15 stage 1 and 48 stage 2). Staging was based on counting of white blood cells (WBC) and/or parasite detection in CSF. Concentrations were obtained either by ELISA or multiplex bead suspension assays, and results were compared with three known HAT staging markers (CXCL10, CXCL8 and H-FABP). ICAM-1 and MMP-9 accurately discriminated between stage 1 and stage 2 patients with HAT with 95% sensitivity (SE) for 100% specificity (SP), which was better than CXCL10 (93% SE for 100% SP), one of the most promising known markers. Combination of ICAM-1 and MMP-9 with H-FABP provided a panel that resulted in 100% of SE and SP for staging HAT. ICAM-1 and MMP-9, alone or in combination, appeared as powerful CSF staging markers of HAT. Final validation of all newly discovered staging markers on a large multi-centric cohort including both forms of the disease as well as patients with others infections should be performed.

A fluorescence-based assay for the uptake of CPD0801 (DB829) by African trypanosomes

Drug therapies currently used for second stage Human African Trypanosomiasis (HAT) exhibit problems with toxicity, difficulty of administration, and resistance linked to the loss of transporter function. Key to the development of new drugs for HAT is a better understanding of the transport properties of candidate compounds. Standard methods for studying transport utilize radio-labelled permeant or HPLC–MS, however the natural fluorescence of many trypanocidal compounds can be exploited.Here we present a fluorescence-based assay for measuring uptake, by trypanosomes, of CPD0801, a drug candidate for second stage HAT. Sample fluorescence is measured in a 96-well format using a benchtop fluorimeter. Our method is directly applicable to the study of other diamidines with similar fluorescent properties and readily adapted for use with other cell types or fluorescent molecules as we demonstrate for the veterinary trypanocide ethidium.

Identification of Stage Biomarkers for Human African Trypanosomiasis

Human African trypanosomiasis (HAT), caused by infection with sub-species of Trypanosoma brucei (T. b.), manifests as a hemolymphatic stage followed by an encephalitic stage. The distinction of the two stages needs improvement as drugs used for the late stage are highly toxic. Transcripts encoding 16 secreted proteins differentially expressed in the brains of mice at late stage T. b. brucei infection when the early stage drug suramin is no longer effective and different to immunoglobulins, chemokines, and cytokines, were selected by microarray analysis. Lipocalin 2 and secretory leukocyte peptidase inhibitor (SLPI) mRNA showed the highest differential expression in mice. These transcripts were also upregulated in brains from infected rats. Lipocalin 2 was increased in cerebrospinal fluid (CSF) from rats during late stage T. b. brucei infection. Protein levels of lipocalin 2, SLPI, and the chemokine CXCL10 were found increased in CSF from Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense late stage HAT compared to early stage.

Antitrypanosomal Activity of 1,2-Dihydroquinolin-6-ols and Their Ester Derivatives

The current chemotherapy for second stage human African trypanosomiasis is unsatisfactory. A synthetic optimization study based on the lead antitrypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364, 1a) was undertaken in an attempt to discover new trypanocides with potent in vivo activity. While 6-ether derivatives were less active than the lead compound, several N1-substituted derivatives displayed nanomolar IC(50) values against T. b. rhodesiense STIB900 in vitro, with selectivity indexes up to >18000. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (10a) displayed an IC(50) value of 0.014 microM against these parasites and a selectivity index of 1700. Intraperitoneal administration of 10a at 50 (mg/kg)/day for 4 days caused a promising prolongation of lifespan in T. b. brucei STIB795-infected mice (>14 days vs 7.75 days for untreated controls). Reactive oxygen species were produced when T. b. brucei were exposed to 10a in vitro, implicating oxidative stress in the trypanocidal mode of action of these 1,2-dihydroquinoline derivatives.

Expression and Role of CXCL10 during the Encephalitic Stage of Experimental and Clinical African Trypanosomiasis

Human African trypanosomiasis, caused by Trypanosoma brucei, involves an early hemolymphatic stage followed by a late encephalitic stage. We studied the expression of chemokines with use of microarray and enzyme-linked immunosorbent assay in T. brucei brucei-infected mice and in patients with human African trypanosomiasis and examined their role in controlling brain accumulation of T cells and parasites. The messenger RNAs (mRNAs) encoding CXCR3 ligands CXCL9 and CXCL10 demonstrated the greatest increases among chemokines in brain specimens of infected mice, as determined by microarray. CXCL9 and CXCL10 mRNA accumulation was interferon (IFN)-gamma-dependent. Expression of CXCL10 was predominantly observed in astrocytes. Weight loss was registered in wild-type but not in CXCL10(-/-) and CXCR3(-/-) infected mice. Infected CXCL10(-/-) or CXCR3(-/-) mice demonstrated reduced accumulation of trypanosomes and T cells in the brain parenchyma but similar parasitemia levels, compared with wild-type mice. CXCL10 and IFN-gamma levels were increased in the cerebrospinal fluid of patients with late stage but not early stage human African trypanosomiasis. Levels of CXCL10 in patients with late stage human African trypanosomiasis were associated with somnolence, low body weight, and trypanosomes in the cerebrospinal fluid. IFN-gamma-dependent CXCL10 is critical for accumulation of T cells and trypanosomes in the brain during experimental African trypanosomiasis. Data suggest CXCL10 as a candidate marker for late stage human African trypanosomiasis.

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

BackgroundThere is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).Methodology/Principal FindingsCordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.Conclusions/SignificanceAltogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

The epidemiology of trypanosomiasis in Rumphi district, Malawi: a ten year retrospective study

Human African Trypanosomiasis (HAT) is caused by two species of the tsetse fly vectored protozoan hemoflagellates belonging to Trypanosma brucei, namely T.b gambiense which predominates in Western Africa and follows a chronic disease course and T.b rhodensiense which is more prevalent in Southern and Eastern Africa, Malawi included, and follows a more acute and aggressive disease course. Previous studies in the Democratic Republic of Congo, Angola, Uganda and Sudan have demonstrated that the prevalence rates of T.b rhodensiense infection have reached epidemic proportions. To describe the epidemiology of Trypanosomiasis in Rumphi District over the past ten years. A total of 163 records from January 2000 to December 2006 were retrospectively studied. There were more males than females (121 vs. 40) with the 20 - 29 years age bracket having the highest number of cases (26.3%, n = 160). Stage 2 HAT was the commonest stage at presentation (58.2%, n = 158) with the patients in the same being 3.5 times more likely to die than those with stage 1 HAT. Case fatality rates for late and early stage disease were 21.5% (n = 92) and 7.2% (n = 66) respectively with 84.6% having been cured (n = 162). Convulsions were associated with fatal disease outcome and the majority of cases (97.2%, n = 103) lived within 5 kilometres of the Vwaza game reserve boundary. More men have been infected than women, with a high involvement in the 20 - 29 age brackets. A dramatic increase with active case finding indicates a high under-detection of the disease with late stage HAT being predominant at presentation. Though it has been found that cases with late stage disease have an increased likelihood of dying compared to those in early stage HAT, the high proportion of successful treatment indicates that the disease still carries a high degree of favourable outcome with treatment. It has also been demonstrated in this study that more than 95% of trypanosomiasis cases live within 5 km of game reserve boundary. Disease interventions should be implemented in areas within 5 km of marshland game reserve boundary as priority areas.