Dissertations / Theses on the topic 'Trypanosomiasis – Africa'

Cattle ticks and tick-borne infections (TBIs) undermine cattle health and productivity in the whole of sub-Saharan Africa (SSA) including Nigeria. In this West African country, two thirds of the cattle population are reared in the central-northern regions, kept under the traditional pastoral husbandry of Fulani herders. Under the Fulanis’ management, cattle are grazed extensively, being exposed to infestation by several tick genera (i.e. Amblyomma, Hyalomma, and Rhipicephalus spp., sub-genus Boophilus spp. included), vectors of the causative agents of the most important bovine TBIs in West Africa: anaplasmosis, babesiosis and ehrlichiosis (cowdriosis). Nevertheless, the Fulani pastoralists do not usually employ chemicals to control ticks in their cattle, merely relying on traditional methods (i.e. manual removal of the most conspicuous specimens). This approach, however, does not prevent cattle from being re-infested, leaving the animals challenged by a broad variety of other tick species, most of which are vectors of economically relevant TBIs. Knowledge of tick and TBIs occurrence is an essential pre-requisite to assist field diagnosis and devising effective control strategies for a given area. Existing information on tick infestation of cattle in Nigeria is rather out-dated, mostly derived from studies carried out in the south of the country. Similarly, all studies published to date on cattle TBIs in the country do not include any molecular analysis, being based instead on cytological and/or serological diagnostics. Therefore, the aim of the present thesis was to assess the presence of cattle ticks and TBIs occurring in an area of Central Nigeria (i.e. Plateau State). This is a densely populated area with traditionally managed cattle, where no acarides have historically been employed on livestock. The work undertaken herein firstly reviews the information available to date on ticks and TBIs known to be endemic in Nigerian cattle, identifying gaps present in the existing knowledge, leading to the rationale of this study. An initial survey was conducted documenting the tick species infesting cattle in Central Nigeria, in order to assess the infestation rate of surveyed animals at the time of the year when the tick load on the host is known to be most abundant (i.e. the wet season). The survey provided novel information on tick populations in cattle in Nigeria disclosing the presence of a broad variety of species, most of which are vectors of hazardous TBIs. In order to conduct a molecular diagnosis of the TBIs within the study area, a novel methodology was developed (i.e. reverse line blotting, RLB). The application of this approach was based on a thorough review of its application to the diagnosis of TBIs worldwide as well as in SSA. The optimisation of the RLB at the University of Edinburgh to enable the detection of a broad-spectrum of TBIs in Nigeria, caused by an array of five genera of microorganisms (i.e. Ehrlichia and Anaplasma, Theileria and Babesia, Rickettsia spp.) is presented. The assessment of the analytical sensitivity of this technique for the detection of Anaplasma marginale, a highly endemic tick-borne pathogen in SSA, demonstrated a detection threshold of ≥ 7 infected cells (keeping the limit of a natural infection). The occurrence of TBIs in cattle in the study area was assessed during a large-scale epidemiological survey through the application of the validated RLB. This study disclosed the occurrence of a high prevalence of several bovine TBIs in Central Nigeria, some of which are of great veterinary and zoonotic concern. The RLB enabled the detection of carrier status as well as of numerous multiple infections (69.5%, 95% CI: 65.5–73.6%). Based on the findings presented, endemic stability for highly prevalent haemoparasites (i.e. Theileria mutans, Theileria velifera, Theileria taurotragi, Anaplasma marginale, Ehrlichia species Omatjenne) is postulated, whereas a more instable epidemiological scenario is hypothesized for other microorganisms (i.e. Anaplasma centrale and Babesia bovis), which might be connected with outbreaks of clinically apparent disease, sporadically seen in the study area. The effect of a monthly tsetse-borne trypanosomiasis-focused control programme (based on the application 0.005% deltamethrin spray formulation, applied only to the lower quarters of cattle) on the kinetics of bovine TBIs was assessed at the village level. Longitudinal monitoring of control and treated cattle was conducted over the period of eleven months. Results generated provide input to the improvement of future control strategies to be rolled out across SSA, aiming to achieve an integrated control of both trypanosomiasis and TBIs. The present thesis contributes to a better understanding of the epidemiology of bovine TBIs in Nigeria as well as in the rest of West Africa, using a highly sensitive tool of wide applicability. These findings will be shared with the local pastoralist communities to further promote effective yet sustainable, vector control, in tune with the traditional long-established practices.

One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.

RESUME

La Trypanosomiase Humain Africaine (THA) demeure un problème de santé publique pour plusieurs pays en Afrique subsaharienne. Le contrôle de la THA est basé essentiellement sur la stratégie de dépistage actif suivi du traitement des personnes infectées. Le dépistage actif est réalisé par des unités mobiles spécialisées, bien que les services de santé fixes jouent un rôle important en détectant « passivement » des cas. Le dépistage reposait jadis sur la palpation ganglionnaire mais, depuis le développement du test d’agglutination sur carte (CATT), trois possibilités se sont offertes aux programmes de contrôle à savoir: i) continuer avec la palpation ganglionnaire ii) combiner la palpation ganglionnaire avec le CATT iii) recourir au CATT seul. Certains programmes comme celui de la République Démocratique du Congo (RDC) ont opté pour la combinaison en parallèle de la palpation ganglionnaire avec le CATT. Toute personne ayant une hypertrophie ganglionnaire cervicale et/ou un CATT positif est considéré comme suspecte de la THA. Elle sera soumise aux tests parasitologiques de confirmation à cause de la toxicité des médicaments anti-THA. Les tests parasitologiques classiques sont l’examen du suc ganglionnaire (PG), l’examen du sang à l’état frais (SF), la goutte épaisse colorée (GE). La sensibilité de cette séquence a été estimée insuffisante par plusieurs auteurs et serait à la base d’une grande perte de l’efficacité de la stratégie dépistage-traitement. D’autres techniques de concentration ont été développées comme la mini-Anion Exchange Concentration Technique (mAECT), la Centrifugation en Tube Capillaire (CTC) et le Quantitative Buffy Coat (QBC), mais ces techniques de concentration ne sont pas utilisées en routine.

En RDC, une interruption des activités de contrôle en 1990 a eu comme conséquence une réémergence importante de la maladie du sommeil. Depuis 1998 les activités de contrôle ont été refinancées de manière structurée.

Ce travail vise deux buts à savoir le plaidoyer pour la continuité des activités de contrôle et la rationalisation des stratégies de contrôle. Nous avons évalué l’évolution de la maladie du sommeil en rapport avec le financement, son impact sur les ménages ainsi que la communauté. L’exercice de rationalisation a porté sur les outils de dépistage et de confirmation. Nous avons d’abord évalué la validité des tests, leur faisabilité ainsi que les coûts et ensuite nous avons effectué une analyse décisionnelle formelle pour comparer les algorithmes de dépistage et pour les tests de confirmation.

Pendant la période de refinancement structurel de la lutte contre la THA en RDC (1998-2003), le budget alloué aux activités a été doublé lorsqu’on le compare à la période précédente (1993-1997). Le nombre des personnes examinées a aussi doublé mais par contre le nombre des nouveaux cas de THA est passé d’un pic de 26 000 cas en 1998 à 11 000 en 2003. Le coût par personne examinée a été de 1,5 US$ et celui d’un cas détecté et sauvé à 300 US$. Pendant cette période, les activités ont été financées par l’aide extérieure à plus de 95%. Cette subvention pourrait laisser supposer que l’impact de la THA au niveau des ménages et des communautés est réduit mais lorsque nous avons abordé cet aspect, il s’est avéré que le coût de la THA au niveau des ménages équivaut à un mois de leur revenu et que la THA fait perdre 2145 DALYs dans la communauté. L’intervention par la stratégie de dépistage-traitement a permis de sauver 1408 DALYs à un coût de 17 US$ par DALYs sauvé. Ce coût classe l’intervention comme « good value for money ».

Le recours au CATT seul s’est avéré comme la stratégie la plus efficiente pour le dépistage actif. Le gain marginal lorsque l’on ajoute la palpation ganglionnaire en parallèle est minime et n’est pas compensé par le coût élevé lié à un nombre important des suspects soumis aux tests parasitologiques. Les techniques de concentration ont une bonne sensibilité et leur faisabilité est acceptable. Leur ajout à l’arbre classique améliore la sensibilité de 29 % pour la CTC et de 42% pour la mAECT. Le coût de la CTC a été de 0,76 € et celui de la mAECT de 2,82 €. Le SF a été estimé très peu sensible. L’algorithme PG- GE-CTC-mAECT a été le plus efficient avec 277 € par vie sauvée et un ratio de coût-efficacité marginal de 125 € par unité de vie supplémentaire sauvée. L’algorithme PG-GE-CATT titration avec traitement des personnes avec une parasitologie négative mais un CATT positif à un seuil de 1/8 devient compétitif lorsque la prévalence de la THA est élevée.

Il est donc possible dans le contexte actuel de réduire la prévalence de la THA mais à condition que les activités ne soient pas interrompues. Le recours à un algorithme recourant au CATT dans le dépistage actif et à la séquence PG-GE-CTC-mAECT est le plus efficient et une efficacité de 80%. La faisabilité et l’efficacité peut être différent d’un endroit à l’autre à cause de la focalisation de la THA. Il est donc nécessaire de réévaluer cet algorithme dans un autre foyer de THA en étude pilote avant de décider d’un changement de politique. Le recours à cet algorithme implique un financement supplémentaire et une volonté politique.

SUMMARY

Human African Trypanosomiasis (HAT) remains a major public health problem affecting several countries in sub-Saharan Africa. HAT control is essentially based on active case finding conducted by specialized mobile teams. In the past the population screening was based on neck gland palpation, but since the development of the Card Agglutination Test for Trypanosomiasis (CATT) three control options are available to the control program: i) neck gland palpation ii) CATT iii) neck gland palpation and CATT done in parallel .Certain programs such as the one in DRC opted for the latter, combining CATT and neck gland palpation. All persons having hypertrophy of the neck gland and/or a positive CATT test are considered to be a HAT suspect. Confirmation tests are necessary because the screening algorithms are not 100 % specific and HAT drugs are very toxic. The classic parasitological confirmation tests are lymph node puncture (LNP), fresh blood examination (FBE) and thick blood film (TBF). The sensitivity of this combination is considered insufficient by several authors and causes important losses of efficacy of the screening-treatment strategy. More sensitive concentration methods were developed such as the mini Anion Exchange Concentration Techniques (mAECT), Capillary Tube Centrifugation (CTC) and the Quantitative Buffy Coat (QBC), but they are not used on a routine basis. Main reasons put forward are low feasibility, high cost and long time of execution.

In the Democratic Republic of Congo, HAT control activities were suddenly interrupted in 1990 and this led to an important re-emergence or the epidemic. Since 1998 onwards, control activities were financed again in a structured way.

This works aims to be both a plea for the continuation of HAT control as well as a contribution to the rationalization of the control strategies. We analyzed the evolution of sleeping sickness in the light of its financing, and we studied its impact on the household and the community. We aimed at a rationalization of the use of the screening and confirmation tools. We first evaluated the validity of the tests, their feasibility and the cost and we did a formal decision analysis to compare screening and confirmation algorithms.

The budget allocated to control activities was doubled during the period when structural aid funding was again granted (1998-2003) compared with the period before (1993-1997). The number of persons examined per year doubled as well but the number of cases found peaked at 26 000 in 1998 and dropped to 11 000 in the period afterwards. The cost per person examined was 1.5 US$ and per case detected and saved was 300 US$. The activities were financed for 95 % by external donors during this period. This subvention could give the impression that the impact of HAT on the household and the household was limited but when we took a closer look at this aspect we found that the cost at household level amounted to one month of income and that HAT caused the loss of 2145 DALYs in the community. The intervention consisting of active case finding and treatment allowed to save 1408 DALY’s at a cost of 17 US$ per DALY, putting the intervention in the class of “good value for money”.

The use of CATT alone as screening test emerged as the most efficient strategy for active case finding. The marginal gain when neck gland palpation is added is minor and is not compensated by the high cost of doing the parasitological confirmation test on a high number of suspected cases. The concentration methods have a good sensitivity and acceptable feasibility. Adding them to the classical tree improves its sensitivity with 29 % for CTC and with 42 % for mAECT. The cost of CTC was 0.76 US$ and of mAECT was 2.82 US$. Sensitivity of fresh blood examination was poor. The algorithm LNP-TBF-CTC-mAECT was the most efficient costing 277 Euro per life saved and a marginal cost effectiveness ratio of 125 Euro per supplementary life saved. The algorithm LNP-TBF-CATT titration with treatment of persons with a negative parasitology but a CATT positive at a dilution of 1/8 and more becomes competitive when HAT prevalence is high.

We conclude that it is possible in the current RDC context to reduce HAT prevalence on condition that control activities are not interrupted. Using an algorithm that includes CATT in active case finding and the combination LNP-TBF-CTC-mAECT is the most efficient with an efficacy of 80 %. Feasibility and efficacy may differ from one place to another because HAT is very focalized, so it is necessary to test this novel algorithm in another HAT focus on a pilot basis, before deciding on a policy change. Implementation of this algorithm will require additional financial resources and political commitment.

Doctorat en Sciences de la santé publique
info:eu-repo/semantics/nonPublished

This thesis started by reviewing the existing sleeping sickness historical records in Tanzania with the aim of exploring the evidence for the existence of Trypanosoma brucei gambiense in Tanzania. Findings from the available historical data did not provide sufficient evidence for the existence of T. b. gambiense sleeping sickness in Tanzania.
The thesis further estimated under-reporting of T. b. rhodesiense in endemic areas of Tanzania using an established model. Using data from a 2000-2004 outbreak of T. b. rhodesiense in Urambo, the model predicts 46% underreporting. All unreported cases were assumed to be undetected deaths as sleeping sickness is invariable fatal if left untreated. These underreporting findings were then used to recalibrate the burden of T. b. rhodesiense (using Disability-Adjusted Life Years – DALYs), as a metric. The burden imposed to rural communities by rhodesiense sleeping sickness is high. The costs of hospitalization are very high considering the long duration of hospital stay (26 days mean hospital stay) for sleeping sickness patients. Finally the thesis investigated spatial and behavioural risk factors for T. b. rhodesiense sleeping sickness in Urambo district, through a matched case control study both at the village and within village scales. Statistically significant cluster was observed at the village level (P = 0.001). However there was no significant spatial association in an individual village’s analysis. There was an increased risk of sleeping sickness in homesteads with a previous history of the disease (P < 0.001). Presence of wild animals in the villages (P<0.001) and forest visits (P = 0.001) were also significantly associated with sleeping sickness in the district.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

Poverty and disease are bound together in rural communities of sub-Saharan Africa (SSA) exacerbated by weak social services and conflict. The infectious disease burden in SSA combines the neglected tropical diseases (NTDs) and the 'big three' (malaria, HIV/AIDS and tuberculosis), so-called because they attract more global attention and hence funding. NTDs include human African trypanosomiasis (HAT or sleeping sickness), first noticed by the outside world during the slave trade era and later in the 2-th century by widespread epidemics of disease across the tsetse fly belt. HAT describes two diseases: i) Gambian HAT caused by Trypanosoma brucei gambiense is characteristically chronic with an infectious period lasting up to three years and ii) Rhodesian HAT caused by T.b. rhodesiense is an acute disease, killing its victim within weeks of infection. The two diseases are frequently considered together as both are transmitted by tsetse flies, the parasites are morphologically indistinguishable and the associated diseases are both fatal if left untreated. However, the two diseases are clinical, epidemiologically and geographical distinct, each requiring different control strategies. Under field conditions, where microscopy is the basic diagnostic tool, differentiation is simply by geographical location of the patient; the Great Rift Valley separates the Gambian disease present in West and Central Africa, from East and southern Africa's Rhodesian disease. Control strategies are also distinct; while the Belgian and French colonial strategies to control the disease were patient-centred, the British colonial powers in East Africa were motivated by the effect of tsetse borne diseases on animal health. Towards the end of the colonial ear, both types of disease were heading for elimination but during the immediate post-colonial era in the 1960s, political instability compromised the rigid HAT control programs that had been put in place. For zoonotic Rhodesian sleeping sickness, complex tsetse control programmes proved difficult to maintain and to justify economically; for Gambian sleeping sickness the generalised breakdown of medical services allowed the disease to return, sometimes to devastating levels. The millennium development goals (MDGs) set out in 2000, highlighted specific challenges and opportunities for national and global development. HAT impacts national health goals of national development plans and MDGs and impedes rural development of SSA. NTDs were not addressed directly by MDGs but the World Health Organization (WHO) has reaffirmed its commitment not only to control of HAT but also to eliminate it as a public health problem by 2020. Currently there are 25 countries reporting HAT to WHO, and while the overall prevalence of HAT across Africa continues to fall, epidemics have been recorded, particularly from central Africa, South Sudan and Uganda. Uganda is uniquely, the only country affected by both T.b. gambiense and T.b. rhodesiense and until the present study, there was no evidence to suggest that the two parasite species co-existed in Uganda. The development of a new control paradigm for T.b. rhodesiese in South East Uganda has lowered the incidence of human infections and, more importantly, halted the northerly spread of this parasite. However, recurring epidemics in several established and new disease foci in central Uganda highlight the difficulties involved in eliminating this disease. The present study assesses past and present HAT control strategies centred on Dokolo, Kaberamaido and Soroti Districts located at the centre of Uganda. These districts are highly endemic for T.b. rodesiense, they represent the region of concern for overlap with T.b. gambiense foci in central Uganda, and are the current focus of the Stamp out Sleeping sickness control initiative. The point prevalence of T. brucei s.1 in cattle reservoir from villages with (out) reported human disease located at specific distances to Otuboi, Chagwere and Ochero cattle markets, was evaluated before and six months after trypanocidal treatment, to assess the transferrable impact of zoonotic T.b. rhodesiense to the human population. Overall, the proportion of T. brucei s.1 in cattle dropped significantly from 22% at baseline to 9% six months after trypanocide treatment (P < 0.05, Chi-square + 17.92, 95% C.I. + 1.71 to 4.49). All villages located in sub-counties that received at least 80% treatment coverage had a drop in T. brucei s.1 prevalence from 30.4% (95%, C.I + 22.8 to 38.0) before treatment was done, to 12.9% (95%, C.I. + 7.4 to 18.4) six months after treatment. More specifically, impact on human infective T.b. rhodesiense was also halved. In fact only three cattle were detected with the parasite six months after treatment compared with six from those sampled as baseline. This study also utilises documented cases between 2009 and 2012 to assess the current HAT reporting system for monitoring and evaluating transmission dynamics of the disease. Using a questionnaire, capacity and preparedness of healthcare professionals to respond to disease epidemics was assessed. The point prevalence of sleeping sickness in the three districts in 2009 was determined by screening volunteers. Microscopic examinations detected trypanosomes in four volunteers (4/5311 or 0.075 %) while PCR detected significantly more infections (24, p < 0.001). Multiplex PCR showed that ten of the Trypanozoon infections were T.b. rhodesiense while nested PCR identified four infections as T.b. gamiense, indicating that the distribution of the two forms of sleeping sickness overlaps in Uganda. Second phase investigations followed up the PCR positive cases; these people were screened again, together with members of their homestead and the inhabitants of three neighbouring homes. Besides microscopy and PCR, study subjects were examined clinically for sleeping sickness and completed a questionnaire to assess community recognition of the disease. This extended screen revealed no new cases underlining the importance of stringent early screening that PCR techniques can provide. At local healthcare centres, 54% of reported sleeping sickness cases were diagnosed only at the late stage, indicating a weakness in early diagnosis and hence early reporting. Interviews with local health workers also revealed weaknesses in recognition of clinical signs and a gap in diagnostic capacity. While records at treating hospitals remain a useful indicator for targeting active foci of infection, improvement in capacity to diagnose HAT at an early stage should contribute both to rural health and disease control strategies and also towards WHO's 2020 target of elimination of HAT.

African trypanosomiasis is a disease caused by the Kinetoplastida parasites Trypanosoma brucei rhodesiense and T. b. gambiense. The distribution of the disease is split geographically with T. b. rhodesiense found in eastern sub-Saharan Africa and T. b. gambiense in the west of the continent. Current treatment for this fatal disease is wholly unsatisfactory with problems such as extreme toxicity, affordability and the emergence of resistance. The case for the generation of new potential chemotherapies is compelling and urgent. Phosphodiesterase (PDE) enzymes degrade the secondary signalling molecule cyclic adenosine monophosphate (cAMP) to AMP by hydrolysis, thereby modulating and regulating the signal transduction to the effector proteins. The phosphodiesterase enzymes in the PDEB family in T. brucei were shown to be essential to the host-infective bloodstream forms and validated as good drug targets using RNA-interference (Zoraghi, R. and Seebeck, T., 2002; Oberholzer, M., 2007). Prompted by these findings, two series of putative trypanosomal PDE inhibitors, from different sources, were thoroughly assessed in this project for their anti-trypanosomal activity and their intracellular effects on the trypanosome. The whole-cell in vitro efficacy for each compound, against T. brucei wildtype and the drug-resistant strain TbAT1 knockout, was established by the standard resazurin reduction assay. 25 compounds from Series 1 had EC50 values below 0.5 µM, with 7 under 100 nM and the most active having an EC50 value of 5.8 ± 3.4 nM. For the much smaller Series 2 (GJS Compounds), the most active compound was GJS-128 with an EC50 value of 79.4 ± 10.3 nM. This demonstrates that a number of compounds from both series have potent in vitro activity against trypanosomes that is better than or equal to the current chemotherapeutic compound diminazene, and some Series 1 compounds are on a par with pentamidine and melarsoprol. No major cross-resistance was displayed by the TbAT1 knockout strain to either Series 1 or the GJS series. Similarly, a panel of Series 1 compounds tested against the B48 strain (resistant to pentamidine and melaminophenyl arsenical drugs), and also against Trypanosoma equiperdum wildtype and diminazene resistant (PBR) strains, showed no major cross-resistance displayed by the other resistant strains. This suggests that there would also be little or no cross-resistance from refractory strains in the field, and also that the compounds are active against multiple Trypanosoma species. A small panel of Series 1 compounds were also tested for efficacy against trypanosomes in infected mice. 4 daily doses of 20 mg/kg bodyweight of Compound 48 significantly reduced parasitaemia by approximately 60% compared to untreated controls, however higher concentrations were not tolerated by the mice so a cure could not be demonstrated. A high-throughput method for monitoring the speed of action of test compounds on trypanosomes in real time was developed, based on the fluorescence of propidium iodide when bound with DNA. Optimisation of the protocol to 96-well plates and low cell densities provided higher resolution and accurate traces of the lysis of trypanosomes in a cell suspension compared to previously used methods, as well as a greatly increased capacity. The propidium iodide assay could also be converted to provide end-point EC50 values that were directly comparable to those established by the standard resazurin reduction assay. The majority of Series 1 compounds did not increase the intracellular concentration of cAMP on incubation with bloodstream form trypanosomes; those that did only induced a minor elevation of the intracellular concentration of the signalling molecule. Since genetic disruption to phosphodiesterase enzymes resulted in large increases in cAMP levels (Oberholzer, M. et al, 2007; Zoraghi, R. and Seebeck, T., 2002), the lack of increase in cAMP by the Series 1 compounds strongly suggest that they do not sufficiently inhibit the PDEs in live trypanosomes and kill the cells via an alternative pathway. In contrast, incubation with the GJS compounds did result in significant increases in intracellular cAMP concentration with the most active being GJS-128 recording an approximately 3-fold increase in cAMP over 3 hours at just 30 nM. The concentrations that begin to increase cAMP level are consistent with the EC50 values for trypanosomes cultured in vitro (this study), and is also in line with inhibition data of recombinant TbrPDEB enzymes (work conducted by Dr. Herrmann Tenor, ALTANA Pharma, and Prof. Thomas Seebeck, University of Bern). This gives a clear and consistent link between the cause of cAMP rise (inhibition of PDEB by GJS compounds) and the effect of that concentration increase on bloodstream form trypanosomes (cell death), demonstrating that the GJS series are inhibitors of trypanosomal PDEs and chemically validate PDEs as drug targets for potential new chemotherapies against African trypanosomiasis. The effect of PDE inhibition on the physiology of the bloodstream form trypanosomes was also investigated. Flow cytometry analysis and the assessment of DNA configuration by fluorescence microscopy after DAPI staining determined that PDE inhibition by GJS-128 resulted in a precise block of the cell cycle in cytokinesis. The replicating trypanosome synthesized and segregated its DNA into two nuclei and kinetoplasts as normal and proceeded to initiate the physical separation of mother and daughter cells. The cleavage furrow between the old and new flagella progressed normally until the point of abscission, at which point division was halted with only a small section of plasma membrane connecting the two almost separated cells. Both cells appeared viable and underwent subsequent rounds of DNA replication, segregation and attempted physical separation that was always blocked near completion. This indicates cAMP signalling plays an important role in the correct physical separation of the replicating bloodstream form trypanosomes. A trypanosome cell line resistant to GJS-128 was developed by chemical mutagenesis and continuous culture with gradually increasing, but sub-lethal concentrations of the PDE inhibitor. This cell line, termed R0.8, was >15-fold less sensitive to GJS-128 and displayed significant cross-resistance to the other GJS compounds, as well as to stable, membrane permeable cAMP analogues. The mode of resistance was investigated by comparing the cAMP profile of the R0.8 and parental wildtype strains on incubation with GJS-128. No major differences were observed suggesting that both the adenylyl cyclase and phosphodiesterase activities remained unchanged in the PDE inhibitor-resistant strain. In support of this, the sequencing of TbrPDEB1 and TbrPDEB2 in both strains, while uncovering the loss of heterozygosity in the R0.8 line, revealed no mutations that would impact on enzyme function or inhibitor binding in the resistant cell line. These data strongly suggest that the adaptation resulting in resistance to PDE inhibitors is located in the effector proteins downstream of the PDEs and adenylyl cyclases in the cAMP signalling pathway. Identifying a compound that inhibits phosphodiesterases in trypanosomes and elevates cAMP concentrations, along with the generation of a PDE inhibitor-resistant cell line will allow more detailed examination of all aspects of the cAMP signalling pathway in T. brucei and across the Kinetoplastida. Phosphodiesterases have also been demonstrated to be chemically inhibitable in trypanosomes and could prove to be the target of a new generation of chemotherapies against African trypanosomiasis.

In the absence of any vaccine, prophylactic drug and effective vector control, the fight against human African trypanosomiais (HAT) is based on the the combination of active case-finding and consequent drug treatment of identified positive cases. Unfortunately, low sensitivity and specificity of current diagnostic techniques often result in misdiagnosis, leaving infected patients without cure or exposing them to inappropriate chemotherapy protocols, which use dangerous and expensive drugs. The development of more efficient, simple, cheap and field-robust diagnostic tests is, therefore, urgently needed. In the field, direct observation by light microscopy of trypanosomes in human fluids (blood, lymph node aspirate, cerebrospinal fluid) is considered the ideal way of confirming HAT infection. However, in practice this approach is problematic, especially for the Gambian form of the disease, where patients may present with very low parasitaemia. Detection limits of parasitological techniques can be improved by adding a preliminary step of sample concentration, although this further increases the laboriousness of HAT diagnostic algorithm. Recent advances in fluorescence microscopy could be exploited to facilitate trypanosome detection. The introduction and implementation of fluorescence microscopy in HAT endemic countries would offer the advantages of an increased overall sensitivity of microscopical examination and a more rapid screening of the specimen. In contrast to traditional, expensive and fragile fluorescence microscopes, new LED-illuminated instruments are relatively cheap, very efficient and portable, lending themselves to utilisation in poorly equipped rural settings. In order to design a new diagnostic tool that exploits LED technology, however, selective and reliable fluorescent markers to label trypanosomes in human fluids are needed. The development of new tools to assist in the diagnosis of African trypanosomiasis by use of LED fluorescence microscopy was the overall objective of this project. The work was mainly focused on testing various fluorescent compounds for their ability to selectively stain trypanosomes. Fluorophores were otained from commercial and academic sources, or else directly synthesised during the project. An important requirement evaluated was the compounds’ compatibility with the currently available SMR LED Cytoscience fluorescence microscope, developed and kindly provided by our collaborator Prof. D. Jones (Philipps University, Marburg). The utility of a UV LED-driven microscope in performing the arsenical drug resistance test was also assessed. This assay, developed in our laboratory to detect trypanosome strains resistant to arsenical and diamidine compounds, could represent a useful tool for chemotherapeutic decision making in the field, where resistance to arsenical drugs is a rising problem.

Human African Trypanosomiasis (HAT) or African Sleeping Sickness is a disease prevalent in many parts of Sub-Saharan Africa. HAT is a parasitic infection caused by two species, Trypanosoma brucei gambiense and T. b. rhodesiense. Clinical diagnosis is not sufficient as symptoms from other endemic diseases, such as Malaria, are similar. Currently the diagnosis of T. b. gambiense infection mainly relies on the Card Agglutination Test for Trypanosomiasis (CATT), which has severe limitations. Other diagnostic tests for T. b. gambiense and T. b. rhodesiense infections require lab based equipment, trained personnel and have varying degrees of sensitivity and specificity. New approaches are needed, firstly to identify new diagnostic biomarkers, and secondly to find a more suitable platform for the test. Our aim was to develop a lateral flow test based on trypanosome antigens. We used sera from T. b. gambiense infected and non-infected patients to identify infection specific diagnostic trypanosome proteins. The trypanosome proteins identified were then cloned into E. coli for recombinant expression and purification. The recombinant proteins were then screened by ELISA against 145 patients’ sera from the WHO HAT specimen bank. Invariant Surface Glycoprotein (ISG) 65 and a soluble Variant Surface Glycoprotein (VSG) were selected for development into a lateral flow format and 80 randomised patients’ sera were used to evaluate these prototypes. Here we describe the results showing that un-optimised proto-type lateral flow tests match the reported CATT sensitivity and specificity scores.

The aims of the research presented in this thesis were concerned with investigating the effect of inducible nitric oxide (NO) synthase expression during Trypanosoma brucei infections on both host and parasite. NO was shown to exhibit a potent cytostatic effect on parasite proliferation. Oxyhaemoglobin is a potent scavenger of NO. The cytostatic effects of NO on the trypanosomes were completely prevented through the addition of erythrocytes to the cultures. This implies that in the host blood-stream, NO is unlikely to be involved in the eradication of the parasites. Through the adoptive transfer of suppressor macrophages from T.brucei-infected donor mice to naive recipients, it was demonstrated that NO mediates a suppressive effect on host lymphocyte responses in vivo. Furthermore, suppressor macrophages were shown to have a finite life-span and undergo NO-mediated apoptosis. Evidence also suggested that elevated NO production in the bone marrow of T.brucei -infected mice is likely to play a significant role in the anaemia resulting from T.brucei infection. Experiments demonstrated that a soluble lysate prepared from freeze-thawed blood-stream form T.brucei, activated interferon (IFN)-gamma primed macrophages to express high levels of NO synthase. Experiments also demonstrated that viable blood-stream forms, but not procyclic form trypanosomes, released a soluble factor which in combination with IFN-gamma induced NO synthase. The absolute requirement of IFN-gamma priming for NO synthase activation by T.brucei was studied using macrophages from mutant mice lacking functional IFN-gamma receptors (IFN-gamma R -/- mutant mice). In comparison to macrophages from wild-type mice, cells from IFN-gamma-R-/- mutant mice were unable to produce NO following stimulation in vitro or infection in vivo. Finally, utilising mice with specific immunodeficiencies it was demonstrated that natural killer cells and a/b T-lymphocytes were important sources of IFN-gamma during murine T.brucei infections.

Spatially-explicit information is essential for planning and implementing interventions against vector-borne diseases. This is also true for African trypanosomoses, a group of diseases of both humans and animals caused by protozoa of the Genus Trypanosoma, and transmitted by tsetse flies (Genus Glossina).

In this thesis the knowledge gaps and the requirements for an evidence-based decision making in the field of tsetse and trypanosomoses are identified, with a focus on georeferenced data and Geographic Information Systems (GIS). Datasets, tools and analyses are presented that aim to fill some of the identified knowledge gaps.

For the human form of the disease, also known as sleeping sickness, case detection and treatment are the mainstay of control, so that accurate knowledge of the geographic distribution of infections is paramount. In this study, an Atlas was developed that provides village-level information on the reported occurrence of sleeping sickness. The geodatabase underpinning the Atlas also includes the results of active screening activities, even when no cases were detected. The Atlas enables epidemiological maps to be generated at a range of scales, from local to global, thus providing evidence for strategic and technical decision making.

In the field of animal trypanosomosis control, also known as nagana, much emphasis has recently been placed on the vector. Accurate delineation of tsetse habitat appears as an essential component of ongoing and upcoming interventions against tsetse. The present study focused on land cover datasets and tsetse habitat. The suitability for tsetse of standardized land cover classes was explored at continental, regional and national level, using a combination of inductive and deductive approaches. The land cover classes most suitable for tsetse were identified and described, and tailored datasets were derived.

The suite of datasets, methodologies and tools presented in this thesis provides evidence for informed planning and implementation of interventions against African trypanosomoses at a range of spatial scales.
Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

Human African Trypanosomiasis (HAT) is a vector-borne disease transmitted by the bite of the tsetse fly that results in high human morbidity and mortality. The propagation of the disease has been linked to environmental factors, and understanding the vector’s habitat is vital to its control. There is no HAT vaccine, but biological control of the vector has been successful in reducing HAT incidence. However, in recent years the disease has re-emerged and spread. Due to insufficient knowledge of HAT endemic foci, the disease management remains challenging. To achieve effective deployment of control strategies, accurate knowledge of the spatial distribution of the HAT vector is vital. The current study is based in Nigeria, and looks at part of Delta State, and a part of Jigawa State, in which HAT has been identified. The work utilizes remote sensing satellite imaging and fuzzy logic to develop a HAT vector habitat classification scheme, to explore the dynamics of HAT propagation. The goal was to develop a surveillance methodology to identify factors that influence HAT epidemiology. Land cover and ancillary data were integrated to classify HAT vector habitat using geospatial-fuzzy multicriteria analysis. The work highlights the significance of geospatial techniques where epidemiological data are limited, for improving understanding of HAT. This study helped distinguish HAT vector habitat into different zones (breed, feed and rest), which allowed the direction and magnitude of HAT, a n d factors influencing propagation to be determined. This helped identify ‘HAT priority intervention areas’. The study findings suggested propagation of HAT resulted from suitability of water bodies, shrub and less-dense forest for the HAT vector, and continued exposure of human populations to these land cover classes. Overlapping of HAT vector habitat zones within built-up areas was also a cause. The study also found that HAT propagation was multidirectional, and that this may have been influenced by landscape characteristics. This novel approach can also be used in other part of Nigeria as well as adapted to investigate other diseases. In conclusion, the HAT vector habitat classification scheme is a transparent tool for policy makers for identifying vulnerable and at risk areas.

Human African trypanosomiasis (HAT) is a parasitic disease caused by the protozoan parasites T. b. rhodesiense and T. b. gambiense. The disease is currently endemic in 36 sub-Saharan countries with an estimated 60 million people at risk from the infection. The disease progresses through two stages; an early or haemolymphatic stage where the parasites are confined to the peripheral compartment and a late or encephalitic stage where the parasites penetrate the blood-brain barrier (BBB) and invade the CNS. Without treatment the disease is invariably fatal but at present chemotherapy is reliant on a small handful of drugs. Pentamidine and suramin are available for the treatment of the early stage of the disease while the CNS stage of the disease is treated with a combination of nifurtimox and eflornithine known as NECT therapy or melarsoprol. NECT therapy is only effective in the treatment of T. b. gambiense infections meaning treatment of T. b. rhodesiense infections is completely dependant on the trivalent arsenical melarsoprol. Melarsoprol is an extremely toxic compound, the administration of which is very painful and associated with numerous adverse reactions. The most series of which is a post treatment reactive encephalopy (PTRE). The PTRE occurs in up to 10% of all patients given melarsoprol of which 50% die as a result of the complication. This gives melarsoprol an overall fatality rate of 5% which is unacceptably high. There is therefore an urgent need for new trypanocides, which are safe and easily administrable. To improve the physiochemical and pharmacokinetic properties of melarsoprol the drug was complexed with two cyclodextrin molecules, hydroxypropyl-cyclodextrin (HPCD) and randomly methylated-cyclodextrin (RAMCD) to produce; mel/HPCD and mel/RAMCD. Cyclodextrins are cyclic oligosaccharides, widely used within the pharmaceutical industry to improve the solubility and oral bioavailability of poorly soluble lipophilic drugs. In this study, the trypanocidal activity of the melarsoprol cyclodextrin complexes was investigated in-vitro and in an in-vivo CNS stage model of T. b. brucei infection. The trypanocidal activity of melarsoprol is retained following its complexation with HPCD and RAMCD. The in-vitro trypanocidal activity of the melarsoprol cyclodextrin complexes against bloodstream T. b. brucei trypanosomes was comparable to that of contemporary melarsoprol. Furthermore, in an in-vivo murine model of CNS stage T. b. brucei the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD produced 100% cure rates when administered orally at a dose of 0.05mmol/kg, daily, for seven consecutive days. Contemporary melarsoprol when administered by the same route and schedule only cured 33.3% of the animals. The cyclodextrins HPCD and RAMCD thus increase the oral bioavailability of melarsoprol whilst retaining the compounds trypanocidal activity. An oral administrable, water soluble formulation of melarsoprol instantly eliminates the problems associated with the intravenous administration of conventional melarsoprol. Furthermore, an orally available formulation would be of great benefit in the resource poor, isolated settings in which HAT occurs, as patients would not require hospitalisation during treatment thus alleviating the pressure on local hospitals. In the current investigation quantitative taqman PCR was utilised to investigate the rate of parasite clearance from the CNS during complexed melarsoprol treatment. Both mel/HPCD and mel/RAMCD were rapidly trypanocidal. Twenty-four hours after administration of one dose the number of trypanosomes within the brain was reduced by greater than 80% and all trypanosomes were eliminated from the brain by twenty-four hours after administration of four doses of mel/HPCD and five doses of mel/RAMCD. The elimination of all trypanosomes from the CNS following four doses of mel/HPCD and five doses of mel/RAMCD, indicates that it may be possible to reduce the dosage schedule from seven daily doses to four daily doses of mel/HPCD and five doses of mel/RAMCD. A short, simple, easily administrable treatment protocol is an essential requirement of any new trypanocide as if the treatment schedule is prolonged and complicated patients are unlikely to comply. CNS stage trypanosome infection is associated with a breakdown of the blood-brain barrier (BBB). Ideally following successful chemotherapy BBB function should be restored. In this investigation the effect of curative mel/HPCD treatment on the BBB was investigated in a murine model of CNS T. b. brucei infection using small bore MRI analysis. Mel/HPCD treatment results in a rapid restoration of BBB function as by twenty-four hours after the completion of mel/HPCD therapy the integrity of the BBB was fully restored. However, a very mild neuroinflammatory reaction persisted in the brain for up to fifteen days after completion of chemotherapy. This suggests that the BBB damage observed in trypanosome infection may be due to either the parasites directly or their secretory products and not as a result of the ongoing neuroinflammatory reaction. Despite melarsoprol being in use for over 60 years its pharmacokinetics are poorly understood and a sensitive assay by which to quantify the concentration of arsenic reaching tissues following administration of the compound is not available. In this study a gas chromatography mass spectrometry (GC-MS) technique was developed to quantify the concentration of arsenic reaching the plasma and brain following oral and intravenous administration of the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD. The GC-MS assay had a limit of detection of 5ng/ml and a precision (expressed as the inter-day coefficient of variation) of 13.2%. The concentration of arsenic within the brain following the oral and intravenous administration of mel/HPCD was below the limit of quantification of the assay. The pharmacokinetics of mel/HPCD and mel/RAMCD could therefore not be determined in the present study. This study demonstrates that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are highly trypanocidal with no overt signs of toxicity and more importantly orally available. Following the oral administration of mel/HPCD or mel/RAMCD the melarsoprol is slowly released over a prolonged period of time from the cyclodextrin cavity. Patients are therefore not exposed to a ‘bolus’ of the drug as is the case in the intravenous administration of contemporary melarsoprol. The slow and sustained release of melarsoprol from the cyclodextrins should result in less adverse reactions and a decreased incidence of the PTRE. Furthermore, the complexed melarsoprol treatment protocol is shorter than the currently used 10 day concise melarsoprol treatment schedule therefore the total amount of melarsoprol administered to patients will be reduced. Patients should therefore experience fewer adverse reactions. In conclusion the results from this study demonstrate that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are promising oral candidates for the treatment of HAT.

Trypanosoma brucei is the vector of African trypanosomiasis in both domestic animals (nagana) and sleeping sickness in humans (Human African Trypanosomiasis). These protozoan parasites are transmitted by the bite of infected tsetse flies (Glossina sp.). African trypanosome infections cause parasite-induced anorexia (PIA) and cachexia in livestock, experimental animals and in humans, and are of economic, veterinary and medical importance in sub-Saharan Africa. The overall aim of this project was to characterise the phenomenon of inappetence in relation to overall energy budget in African trypanosome infection and to then identify potential causal factors and mechanisms. A mouse model of T.b. brucei infection was established with a reproducible time course for the development of inappetence and bodyweight loss. Following an initial parasitaemic peak on day 6 post-infection, a profound period of inappetence was observed from days 7 to 11, accompanied by a 10% loss of body mass. Metabolisable energy intake was reduced, while assimilation efficiency increased significantly but not enough to compensate for the severe reduction in food intake. During the course of T.b. brucei infection, both total energy expenditure and physical activity were reduced. Although physical activity was markedly declined in both light and dark phases, trypanosome infected mice maintained their circadian rhythm albeit at a lower amplitude, with most of the activity occurring at the start of the dark phase. Resting metabolic rate was unchanged in infection. Plasma concentrations of the inflammatory cytokines, IL-6 and TNF-α were increased in infected mice and were associated with inappetence. Reductions of leptin and insulin concentration corresponded to a loss in fat mass. The hypothalamic control of appetite appeared to be normal with increases in appetite stimulating AgRP, decreases in the appetite inhibiting POMC and MC4R. There has been no previous data published on the control of appetite and energy expenditure in African trypanosome infections thus the data presented here provides a novel insight into the pathophysiology of this serious disease, and may lead to new therapies to manage the clinical and veterinary consequences of trypanosome infection.

The epidemiological study of pathogens largely depends on three technologies, serology, microscopy and the polymerase chain reaction (PCR). Serological methods are unable to differentiate between current and past infections. Microscopy has historically been the mainstay of epidemiological study. In recent times the use of microscopy has been in decline, as it has been shown to have an inherent lack of sensitivity and specificity and produces many false negative results. PCR is now the method of choice for screening samples for the presence or absence of pathogens. Although PCR is widely regarded as an extremely sensitive technique, the fact that it assays a very small volume of sample is often overlooked. If the target pathogen is not present in the tiny aliquot of sample from an infected host, then a false negative results will occur. In endemic situations were the pathogen is present at low infection intensities, then the potential for false negatives results of this type is high. This intensity related false negative effect can lead to serious underestimation of diagnosed prevalence and incidence with consequent misinterpretation of the resulting data. This phenomenon has been reported in the literature for a range of pathogens and especially for epidemiological study of schistosomiasis. The extensive occurrence of false negatives during study of schistosomiasis samples was such an obstacle to epidemiological study it prompted the world health organisation to repeatedly call for quantitative methods to be employed to combat the problem. The main objectives of this thesis are to rationalise and simplify the methods of diagnosing African trypanosomes in epidemiological studies and to investigate the consequences of, and methods of dealing with infection intensity related false negative results that occur as a result of widespread sub-patent infections in the study population A new PCR assay was developed that was capable of analysing whole blood placed onto treated filter paper. The PCR assay was capable of differentiating between all the important African trypanosome species, producing a unique size of amplicon for each species of trypanosome. Initial results from repeated screening of human and cattle samples known to be parasitologically positive indicated that many false negative results occur. A more extensive analysis of thirty five bovine blood samples randomly chosen from a collection of field samples revealed that false negative results occurred regularly. The prevalence of infection after a single screening was 14.3% whereas the cumulative prevalence after over 100 repeated screenings rose to 85.7%. This showed that a severe underestimation of prevalence occurs from a single screening of the samples. In order to investigate the consequences of, and develop methods of dealing with this problem, computer based simulations were used to model the dynamics of screening samples with sub-patent infections. In order to construct the model the data obtained from repeat screening of the thirty-five bovine blood samples was fitted to a number of mathematical distributions. A negative binomial distribution best described the distribution of trypanosomes across the hosts. Exploration of the phenomenon with the resulting model showed the extensive underestimation of true prevalence that is possible. The simulations also showed that it is possible for populations with very different patterns of infection and true prevalence to all have the same diagnosed prevalence from a single screening per sample. Statistical comparison of these very different populations by diagnosed prevalence alone would conclude there was no significant difference between the populations. It was therefore concluded that the diagnosed prevalence from a single (or even multiple) screenings is an inadequate and potentially misleading measure of both infected hosts and parasite numbers. In order to deal with these problems new methods were evaluated for use in epidemiological studies. A simple method of producing quantitative measures of infection was advocated. The insensitivity of existing screening methods in detecting significant difference between populations was highlighted and a greatly improved methodology was shown. Finally, a method for inferring the true population prevalence from the data obtained from repeat screening of samples was suggested. Although some of these new methodologies have limitations, they represent a great improvement on the use of a single diagnostic test for each host. The work presented in this thesis highlights a serious potential limitation to our understanding of the epidemiology of pathogens that exist at sub-patent levels, and develops some possible methods of overcoming these limitations.

A growing problem with drug resistance in Human African Trypanosomiasis has necessitated the implementation of screening programmes to monitor for its spread. This thesis describes the study of several factors that can influence the selection and propagation of drug resistance in T. brucei. Human African Trypanosomiasis (HAT) is caused by T. brucei gambiense and T. brucei rhodesiense. The few drugs used for the treatment of the disease are either toxic, cause severe side effects or suffer from parasite resistance. The T. brucei P2 transporter, which is encoded by the gene TbAT1, mediates uptake of melaminophenyl arsenicals and diamidines. Reduced P2 uptake is associated with drug resistance. A number of point mutations found in a laboratory derived melarsoprol resistant T. brucei stock (STIB 777R) allowed development of a PCR/RFLP based molecular method to identify resistance alleles. By 1999, 20-30% of patients treated in Omugo, NW Uganda were failing to respond to melarsoprol. PCR/RFLP analysis indicated that mutant alleles accounted for 58.5% of those in circulation. Melarsoprol was withdrawn in 2001 and by 2003 mutant TbAT1 alleles accounted for only 14% of those in circulation in NW Uganda. The current study aimed to determine the incidence of the PCR/Sfa NI TbAT1 mutant alleles in 2006, some five years after melarsoprol had been withdrawn as first-line treatment. Successful molecular analysis of 91 of 132 (68.9%) T. b. gambiense field isolates from Omugo and Moyo in NW Uganda indicated the presence of only TbAT1 wild type alleles. Mutant alleles thus appear to have disappeared. This may be the result of parasite fitness cost following the withdrawal of melarsoprol as a stage II first-line drug from Omugo health centre, Arua, since 2001. This apparent instability of TbAT1 mutants in the field may be exploited for rational or alternating use of melarsoprol and eflornithine (DFMO) to ensure a longer life for eflornithine, delaying the onset of resistance. Insight into the overall population structure of the T. b. gambiense from Omugo, Arua (N=54) and Moyo (N=17) was obtained using mini/microsatellite marker analysis. Genetic diversity was observed to be more intra than inter regional. Multilocus genotype data analysis revealed the Omugo, Arua, population was genetically distinct from the Moyo population (Nei’s genetic distance=0.176). The evidence indicated surprisingly little genetic exchange with an excess in homozygosity (Fis >0) and alleles in linkage disequilibrium (P<0.05) within the Omugo, trypanosome population. This excess in homozygosity may be due to population sub-structuring, trypanosome inbreeding, or migration of patients. The latter is likely occurring from the neighbouring T. b. gambiense endemic disease focus in Southern Sudan. The findings suggested that the T. b. gambiense from Arua is not panmictic, clonal or epidemic but there is some level of genetic exchange. The possibility that T. b. gambiense can infect animals raises the prospect that wild or domestic animals may act as a reservoir and that a veterinary link to gambiense Human African Trypanosomiasis exists. Treatment of animals for babesiosis and trypanosomes with diminazene, uptake of which is mediated through TbAT1/P2 could select for P2-defective drug resistant trypanosomes, thereby threatening control of the human disease as well. Species detection by PCR for animal and human trypanosomes in dog isolates (N=190) from the tsetse fly endemic Jos Plataeu, Nigeria did not reveal T. b. gambiense, but multiple infections with T. brucei (95%), T. vivax (89%), and subspecies T. congolense forest (54%) and savannah (50%) were detected. The dogs were also infected with other parasites, including Babesia canis (22%) and Hepatozoon canis (16%). Multiple infections can make correct diagnosis difficult and the infections are likely to be missed by the less sensitive microscopy method. The trypanocidal action of the diamidine group of trypanocides, diminazene, pentamidine and furamidine (DB75) are principally mediated through the TbAT1/P2. In addition, pentamidine is taken up by two additional T. brucei transporters called High Affinity Pentamidine Transporter (HAPT1) and the Low Affinity Pentamidine Transporter (LAPT1). DB75 also has a secondary unknown route. Loss of TbAT1/P2 leads to significant resistance to DB75 and diminazene but not pentamidine. Identification of other markers of resistance is necessary to determine if other routes of drug entry do exist apart from P2 and whether these can be exploited for the delivery of new trypanocides into the trypanosomes. Adaptation of the T. brucei tbat1 knock-out cell line to higher concentrations of diminazene by in vitro selection for resistance led to loss of HAPT1. The resultant phenotype was similar to the previously characterised pentamidine resistant clone B48, but more resistant to diminazene and DB75. The adapted line was still capable of accumulating 1 µM radiolabelled diminazene suggesting both HAPT1 and LAPT1 as possible routes for diminazene uptake. Adaptation of the T. brucei tbat1 knock-out cell line to a high concentration of DB75 over the same 6 months period did not lead to increased resistance. Overall the project has confirmed an important role for tbat1/P2 in development of resistance to melarsoprol in the field. Importantly, it appears that removal of the selection pressure of melarsoprol leads to a loss of tbat1 alleles associated with resistance in a population of trypanosomes capable of genetic exchange in NW Uganda. Although evidence for a dog reservoir for T. b. gambiense in Nigeria was lacking in this study, a risk of selecting resistance in animals must remain high on any list of consideration. I have further shown that the diamidine drug, diminazene, used in veterinary medicine also appears to enter T. brucei via the HAPT1 transporter, as well as the P2 transporter. Loss of HAPT1 through selection with diminazene leads to high level pentamidine resistance, which could indicate a further risk in selection of human infectious trypanosomes also resistant to drugs like pentamidine.

African trypanosomiasis is a protozoan infection affecting tens of thousands of people and millions of livestock animals across sub-Saharan Africa. In humans the disease is fatal without chemotherapeutic intervention and in animals it causes a severe anaemia that greatly impairs productivity. Available drug compounds are difficult to administer and unacceptably toxic. A natural product, ascofuranone, inhibits a key trypanosome specific respiratory enzyme, trypanosome alternative oxidase, and was shown over a decade ago to be trypanocidal using both in vitro and in vivo experiments. The compound suffers from rapid metabolism and contains several functionalities undesirable in a drug compound. Despite the promising activity the lack of applicable synthetic methods available hampered the development of chemotherapeutics from ascofuranone. In this work, novel synthetic routes were completed to explore the lead compound. New synthetic methods were successfully developed using palladium catalysed Suzuki couplings and Lewis acid catalysed rearrangements. Ortho-lithiation approaches also afforded potent novel inhibitors. Of particular note is a benzisoxazole, which is expected to alleviate many of the metabolic issues associated with ascofuranone. Alternate heterocycle analogues were explored and an interesting indazole analogue obtained. Finally, chemical methods were developed towards the benzisoxazole and indazole motifs with carboxylic acids, amenable to diversification by amide coupling. A preliminary range of novel amide containing 5, 6-heterocycles were synthesized to begin SAR exploration of these structures.

The protozoan parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT) and Nagana disease in mammals. These diseases present a major socioeconomic burden to large areas of sub-Saharan Africa. Current therapeutics involve complex and toxic regimens which can lead to fatal side-effects. In addition, there is evidence for drug resistance emerging in the field. Hence, there is a desperate need for novel therapies. Benzoxaboroles are a novel class of boron-containing compounds under development for use against a wide spectrum of diseases. AN5568 is a lead compound for the treatment of HAT, which has demonstrated effective clearance of both early- and late-stage trypanosomiasis in a murine model, and is currently undergoing clinical trials. However, the mechanism by which AN5568 kills T. brucei is elusive. In this study we sought to use 'omics'-based techniques to investigate the mode of action of AN5568 in a laboratory strain of Trypanosoma brucei brucei. Cells treated with the benzoxaborole showed significant perturbations in methionine metabolism. In particular, there were increases in S-adenosyl-L-methionine, an essential methyl group donor involved in methyltransferase reactions. These changes were similar to those elicited by the nonspecific methyltransferase inhibitor sinefungin. Changes were also observed in lipid metabolism, sugar nucleotide metabolism and glycophosphatidylinositol biosynthesis. Further analyses were carried out to investigate the effect of AN5568 on cellular stress responses and cell morphology. To further probe the mechanics of AN5568-treatment, a drug-resistant cell line was generated. This cell line showed cross-resistance with sinefungin, further supporting similar modes of action for these two drugs. Interestingly, the AN5568-resistant cell line exhibited upregulation of procyclic form-specific genes, as well as downregulation of blood-stream form-specific genes, which led to the hypothesis that the cell line had undergone a differentiation event. However, microscopy analysis showed that overall morphology of the cells still resembled those of bloodstream forms, despite them having acquired a procyclic-like metabolic physiology. A secondary aim of this project was to elucidate the metabolic changes that lead to increased growth rates in T. brucei cells undergoing loss-of-heterozygosity on chromosome 10. This phenomenon, whereby a significant portion of the chromosome is lost, has been observed independently on multiple occasions in lab adapted T. brucei strains, yet how this alteration affects intracellular metabolism was hitherto unknown. Using two procyclic T. brucei cell lines, this study was able to show that the increased growth rates are glucose-dependent with a potential intracellular alteration in succinate and acetate production. These data have important implications for the field, where LOH has been observed in the clonally expanding T.b. gambiense type I.

A tripanosomose Africana, também conhecida como Doença do Sono, causada pelo protozoário Trypanosoma brucei, é uma doença tropical negligenciada. Esta doença pode ser controlada, tal como foi provado no passado; no entanto, o crescente número de pessoas afectadas e em risco torna o desenvolvimento de uma vacina uma prioridade. T. brucei é capaz de evadir constantemente o sistema imunitário do hospedeiro, devido ao seu extraordinário mecanismo de defesa, que lhe proporciona uma grande variação antigénica. Devido a este mecanismo de defesa tem sido muito difícil de desenvolver uma vacina eficaz. Contudo, têm sido procuradas novas técnicas, entre elas, uma estratégia de vacinação com DNA plasmídico que têm revelado resultados promissores. Tendo em conta estes resultados, este trabalho tem como objectivo o uso de três estratégias de imunização: a primeira, recorrendo a vacinas de DNA, usando dois plasmídeos que codificam candidatos antigénicos de Trypanosoma brucei; a segunda, usando estes candidatos antigénicos conjugados com uma anoformulação; e a terceira, usando VLPs (Vírus-Like Particles). Os três modelos usados no desenvolvimento de vacinas de DNA contra T. brucei recorreram ao uso de duas importantes proteínas do parasita: a MSP (Major Surface Protease) e a PLC (Phospholipase C). A MSP é uma metaloprotease de zinco de superfície, que se acredita ser responsável pela libertação de um fragmento de VSG (Variable Surface Glycoprotein). A PLC é uma fosfolipase, ancorada a um resíduo de GPI (Glycosylphosphatidylinositol), que cliva integralmente uma proteína de VSG da superfície da célula. Como se pode ver, ambas as proteínas são responsáveis pela libertação das VSG, pela normal diferenciação da forma procíclica para a forma de corrente, e participam de forma sinérgica para a perda de VSG durante a diferenciação. Após a imunização com as duas primeiras estratégias, apesar de em baixos níveis, os murganhos produziram anticorpos anti-Trypanosoma brucei brucei. Os que apresentaram melhor resposta imunológica foram os imunizados com a mistura de plasmídeos conjugados com a nano-formulação. Em relação ao terceiro modelo de imunização, o desenho das VLPs foi efectuado, e o próximo passo é a avaliação biológica das mesmas.
African Trypanosomiasis, also known as sleeping sickness, caused by the protozoan Trypanosoma brucei, is a neglected tropical disease. This disease can be successfully controlled, as has been proven in the past; nevertheless, the growing number of people affected and at risk makes the development of a vaccine a priority. T. brucei is capable of constantly evading the host immune system, due to a remarkable mechanism of defense, which provides a great antigenic variation. Due to this mechanism it has been very difficult to develop an effective vaccine. However, new approaches have been pursued, one of which, the vaccination strategy with plasmid DNA has revealed some promising results. Based on this, this work aims to use three immunization strategies: the first one, DNA vaccination, using two plasmids DNA, encoding antigenic candidates from Trypanosoma brucei; the second one, using these antigenic candidates together with a nanoformulation; and the third one, using VLPs (Virus-Like Particles). The three models used in the development of DNA vaccines against T. brucei use two important proteins of the parasite: MSP (Major Surface Protease) and PLC (Phospholipase C). MSP is a surface zinc metalloprotease that is believed to be responsible by the release of a VSG (Variable Surface Glycoprotein) fragment. PLC is a phospholipase anchored to a GPI (Glycosylphosphatidylinositol) residue that cleaves a full-length VSG protein from the cell surface. As we can see, both proteins are responsible by the VSG release, by the normal differentiation from bloodstream to procyclic form, and they participate synergistically in VSG loss during differentiation. After immunization with the first two strategies, although the titres were low, mice produced antibodies anti-Trypanosoma brucei brucei. The animals that presented a better immune response were the ones immunized with the mix of plasmids together with the nanoformulation. Regarding the third model of immunization, the design of the VLPs was made, and the next step is evaluating them biologically.

Drug resistance together with difficulties involved in the development of new trypanocides are a major problem in the present control of African trypanosomiasis. DNA based diagnostics for drug resistance would overcome problems in the identification of drug-resistant populations and contribute to effective control measures. However, this requires a detailed knowledge of the mode of action and the mechanisms by which trypanosomes can overcome the toxic effects of trypanocides. In this study, a search for molecular differences between a multidrug-resistant isolate of Trypanosoma brucei brucei, CP 547, and a reference drug-sensitive population, ILTat 1.4, led to the identification of a 6.6 kbp extrachromosomal element in the multidrug-resistant population. In light of the involvement of extrachromosomal elements in drug resistance in Leishmana spp. and cancer cells, the identification of the 6.6 kbp element warranted its characterisation. Several different approaches sere attempted before a sequence which hybridised to the 6.6 kbp element its eventually isolated. This sequence is represented by a 108 bp repeat sequence which forms long arrays of tandem repeats. Since N/a III is the sole restriction enzyme that cuts within the repeat, it has been referred to as an N/a III repeal The repeat is flanked by a 5 bp spacer sequence. However, a unique 5 bp direct repeat flanking two complete, and one partial copy of the N/a III repeat may signify the transposition of these sequences. Hybridisation with the N/a III repeat revealed the presence of 'higher' hybridising elements which also appear to be predominantly composed of long tandem arrays of the N/a Ill repeal Through exploitation of the p01) merase chain reaction using arbitrary primers (AP-PCR), additional sequences were identified which are associated with some of the 6.6 kbp and 'higher' hybridising elements. The 6.6 kbp element and some of the 'higher' hybridising elements display features of circular DNA molecules. The 6.6 kbp element also displays some level of size and sequence heterogeneity within different populations of the same trypanosome isolate. The copy number of the 6.6 kbp element is also not stable and appears to be directly affected by the application of selective drug pressure, but a direct association between the presence of the element and the expression of multidrug resistance could not be determined. The N/a III repeat family represents a newly identified repetitive family specific to members of the Trypanozoon subgenus. This repeat family, representing about 5% of the parasite genome, is dispersed through all size classes of chromosomes, in addition to its presence on the extrachromosomal elements. Transcriptional studies of the N/a III repeats have revealed that their transcription is developmentally regulated, since heterogeneous transcripts ranging from greater than 10 kb to smaller than 300 bp are present in the actively dividing long slender bloodstream and insect stage procyclic forms of the parasite but not nondividing, stumpy bloodstream forms. Lastly, the N/a III repeat lacks an open reading frame and transcripts do not appear to have a spliced leader sequence at the 5' end. Furthermore, there is almost an equal representation of polyadenylatcd and non-polyadenlyated transcripts.

The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A ~50° kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.

The two human pathogenic sub-species T.b.gambiense and T.b.rhodesiense can be distinguished from the morphologically identical T.b.brucei by their ability to infect humans, enabling them to cause sleeping sickness. This is because they are resistant to lysis by the lytic factor (APOL-I) present in normal human serum (NHS). In T.b.rhodesiense resistance to this lytic factor is due to a truncated VSG gene termed SRA which blocks lysis by interacting with APOL-I in the lysosome. SRA does not exist in T.b.gambiense. The search for a similar truncated VSG gene lead to the identification of a T.b.gambiense specific glycoprotein termed TGSGP. TGSGP transfected alone into the sensitive T.b.brucei is unable to confer resistance to this sub-species. This is either due to incorrect processing of this gene is this sub-species or because TGSGP requires a partner to confer resistance.

In the search for a partner, the genomic locus of TGSGP was cloned and sequenced. We found that TGSGP is linked to a truncated gene homologous to the S.cerevisiae AUT1 gene, a gene implicated in autophagy and more specifically in membrane expansion. Southern blot hybridization and PCR analysis on genomic DNA from several isolates demonstrated that this feature was a specific to T.b.gambiense. In addition, we observed a correlation between the aut1 allele size and the geographical origin of the isolate.

Since in trypanosomes lysis by NHS is due to an uncontrolled expansion of the lysosome, we speculated that the truncation of the aut1 allele could be implication in the resistance to human serum. We characterized the genomic organisation of the AUT1 locus. T.b.brucei possesses two native AUT1 alleles whilst T.b.gambiense possesses a truncated aut1 allele, as well as a native AUT1 allele. We showed that in the T.b.gambiense LiTAR isolate (aut1/AUT1), despite the presence of a wild-type allele this gene is no longer expressed at the mRNA and protein level. Our complimentary results by run-on transcription assay showed that the AUT1 region is transcribed but that the messenger is unstable. LiTAR is a functional knock-out for AUT1, but Northern blot analysis on several T.b.gambiense isolates showed that this is not a generalised T.b.gambiense characteristic.

We explored the role of AUT1 in trypanosomes by invalidation of the AUT1 gene in T.b.brucei and by the over-expression of the AUT1 and aut1 alleles in T.b.brucei. By functional analysis of AUT1 knocked-down cells we showed that AUT1 is not essential in trypanosomes. By recreating in T.b.brucei the T.b.gambiense AUT1/aut1 genotype we were able to show that the expression of the aut1 UTR down-regulated the expression of the wild-type AUT1 allele. We speculated that this may be due to a natural RNAi mechanism. Par northern blot, using probes covering the potential target region of AUT1, we detected a 50nt small RNA specific to T.b.gambiense. In addition, we showed that in a LiTAR strain in which the RNAi pathway was abolished AUT1 expression is restored.

We continued to investigate TGSGP’s role in the resistance to human serum by invalidation of TGSGP in T.b.gambiense and by expressing TGSGP in the NHS-sensitive T.b.brucei. Because T.b.gambiense cannot be cultured in vitro we established a new in vivo transfection technique and as the knock-out of TGSGP is most probably lethal, we created an inducible RNAi T.b.gambiense cell strain. These indispensable tools will be used to test whether invalidation TGSGP is sufficient to confer resistance to NHS. Many strategies were tested in order to correctly expressing TGSGP in T.b.brucei; in none of these transfectants was TGSGP correctly located in the flagellar pocket as is the case in T.b.gambiense and only partial resistance was ever obtained. In order to identify the factors in human serum that could interacts with TGSGP, we subjected NHS to affinity chromatography using TGSGP as bait. We showed that TGSGP interacts with APOA-I, a major component of HDLs.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

All the compounds were assayed for their ability to inhibit adenosine uptake by the P2 transporter. In vitro toxicity against intact bloodstream form trypomastigotes of T. b. brucei and T. b. rhodesiense was also measured. Compound 6 and compound 54 showed IC 50 against T. b. rhodesience line of 25 nM and 18 nM respectively. Two compounds retained their trypanocidal effect in mice curing all the mice infected with a STIB 795 T. b. brucei model of infection. One compound cured also 1 mouse of 4 infected with the more stringent model STIB 900 T. b. rhodesiense . The comet assay showed that the compound is not genotoxic at the doses tested, indicating that this is a good drug lead against HAT.

Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.