Dissertations / Theses on the topic 'Trypanosomiase Humaine africaine'

La Trypanosomose Humaine Africaine (THA)à T. B. Gambiense est caractérisée par une atteinte du système nerveux central (phase II) dont la pathogénie, encore indéterminée, pourrait relever de phénomènes auto-immuns. L'étude du répertoire immunitaire de patients atteints de THA en phase lymphatico-sanguine (phase I) et en phase II, nous a permis de mettre en évidence dans le sérum et dans le LCR de ces patients la présence conjointe d'autoanticorps dirigés contre deux protéines du cytosquelette cellulaire. Il s'agit d'anticorps dirigés contre des protéines de filaments intermédiaires spécifiques des neurones, les sous-unités de 160 et 200 kDa de neurofilaments (NF). Ces anticorps étaient associés à d'autres qui reconnaissaient des protéines ubiquitaires appartenant aux microtubules, les tubulines α et ß. Ces deux types d'autoanticorps étaient significativement associés à la phase II. Chez les patients en phase I et chez les malades en phase II, ces anticorps (anti-NF et anti-tubulines) appartenaient essentiellement à la classe des IgM, alors qu'une proportion équivalente d'IgG et d'IgM de ces anticorps (anti-NF et anti-tubulines) a été observée dans le sérum des sujets témoins. Ils étaient par ailleurs, indétectables dans le LCR des sujets en phase I et dans celui des sujets témoins et pourraient de ce fait contribuer au diagnostic de la phase II. Nous avons montré que ces anticorps anti-NF et anti-tubulines sont induits par des antigènes stables et accessibles du trypanosomes (mimétisme moléculaire). En effet, la sous-unité 160 kDa de NF partage des épitopes avec des antigènes flagellaires de trypanosomes. Utilisant cette protéine de NF (160 kDa) et secondairement des protéines flagellaires de T. B brucei et de T. B. Gambiense, dans des conditions immunisantes, nous avons induit chez des souris swiss, des anticorps anti-NF et anti-flagelles (T. Brucei) qui ont été protecteurs contre l'infection provoquée par des formes de culture de T. B. Brucei AnTat 1-9 et T. B. Brucei AnTat 1-1E chez ces souris

RESUME

La Trypanosomiase Humaine Africaine (THA) appelée également « maladie du sommeil» est une maladie parasitaire provoquée par un protozoaire du genre Trypanosoma dont deux sous-espèces (T. brucei gambiense et T. brucei rhodesiense) sont pathogènes à l’homme. La stratégie de lutte contre cette maladie est essentiellement basée sur le dépistage précoce et le traitement des malades, complété avec le contrôle du vecteur. Cependant, l’utilisation du service de dépistage de la THA par les communautés exposées représente un défi majeur. L’adhésion aux campagnes de dépistage actif avec des équipes mobiles spécialisées était en-dessous de 50% dans certains villages endémiques fin des années nonante. De surcroît, l’utilisation des services de santé fixes en RDC est si faible que ceci compromet le dépistage passif dans les formations sanitaires fixes. Notre hypothèse est que cette faible utilisation des services de santé pourrait elle-même être due à un problème d’acceptabilité du dépistage et traitement de la THA par les communautés vivant dans les zones de transmission de la THA. Tout ceci compromet l’élimination de la THA comme problème de santé publique, un but que s’est fixé la communauté internationale d’ici 2020.

Ce travail a comme objectif d’explorer cette dimension socioculturelle de la maladie qui est souvent négligée dans le contrôle de la THA et générer une meilleure connaissance de ces aspects.

Nous avons réalisé cinq études en total pour adresser la question de la sous-utilisation des services de dépistage et traitement de la THA par les communautés et sa relation avec l’acceptabilité des services. Nous avons d’abord développé une première étude qui évalue les résultats du traitement de la THA en analysant rétrospectivement les données de routine du programme de contrôle de la THA pour l’année 2006 à 2008. Ensuite, nous avons réalisé trois études qualitatives par focus group (groupe focalisé) et entretiens individuels pour documenter la dimension socioculturelle de la lutte contre la THA. D’abord une étude qui a exploré les perceptions sur la THA dans la communauté, suivi par une étude qui explore les perceptions sur le traitement de la THA et une autre qui se concentre sur les pratiques diagnostics des professionnels de santé face à un syndrome neurologique en contexte de ressources limitées.

Une cinquième étude combine une enquête-ménage avec des focus groups et des entretiens individuels pour explorer les perceptions de la communauté sur la santé en général et les services de santé.

Nous avons comparé les obstacles à l’utilisation des services de dépistage et traitement de la THA identifiés dans ce travail avec les messages de sensibilisation sur la THA utilisés au programme de contrôle de la THA en RDC et nous avons développé des recommandations stratégiques.

L’évaluation des indicateurs de performances sur l’issue de traitement montre que le taux de suivi post-thérapeutique est faible dans son ensemble :25 % pour le premier suivi de six mois et moins d’un pourcent des patients revient pour la dernière visite de contrôle au mois 24. Nous avons aussi observé dans cette étude un taux d’échec au mélarsoprol et à la pentamidine respectivement de 30% et de 22 % au Kasaï Oriental qui sont cependant difficilement interprétables, car le dénominateur est incomplet. Comme très peu de patients reviennent au contrôle post-thérapeutique, cette proportion est probablement biaisée vers ceux qui sont en échec de traitement.

L’étude de perception de la THA montre que la maladie est bien connue dans les communautés vivant dans les zones à risque. Par contre, plusieurs obstacles au dépistage et traitement de la THA ont été identifiés. Les plus importants sont :la toxicité des médicaments de la THA, les obstacles financiers, l’inadéquation entre le programme de dépistage des équipes mobiles et les occupations des communautés, les interdits qui accompagnent le traitement de la THA, le manque de confidentialité et la peur de la ponction lombaire.

L’étude sur la perception du traitement de la THA a montré que le mélarsoprol est perçu comme un médicament toxique et est surnommé « médicament des interdits ». Par contre, le régime NECT est perçu comme un nouveau médicament moins toxique qui a rendu les interdits liés au mélarsoprol obsolètes sauf un seul, celui de ne pas avoir de rapport sexuel pendant la période de traitement et de suivi post thérapeutique qui est de 6 mois. Les interdits ont été instaurés de manière empirique par les professionnels de santé et les communautés pour mitiger les effets indésirables du mélarsoprol. Leur violation pourrait entrainer des conséquences graves et mortelles. Ces interdits sont fortement ancrés dans les croyances de la communauté et constituent aujourd’hui un obstacle au dépistage et traitement.

L’étude sur les pratiques diagnostiques des professionnels de santé en matière de syndrome neurologique en contexte de ressources limitées a montré qu’en zone rurale le diagnostic est principalement clinique. Les obstacles perçus au diagnostic de confirmation sont essentiellement d’ordre financier puisque le patient doit tout financer de sa poche. Autres obstacles évoqués sont le manque d’outils de diagnostic et la perception de la communauté qui voit le clinicien comme un devin (petit dieu) ou oracle capable de « deviner » directement la maladie sans passer par un processus diagnostique de laboratoire.

L’étude sur les perceptions de la santé et des services de santé a montré que les capacités de travailler (82%) et les capacités de se mouvoir (66%) sont les signes de bonne santé les plus perçus. 90% des responsables des ménages perçoivent positivement la santé de leur ménage. Les opinions sur le service de santé sont partagées.

Les études présentées dans ce travail ont généré des nouvelles connaissances sur la dimension socioculturelle de la THA. L’analyse des messages de sensibilisation sur la THA utilisés par le programme de contrôle de la THA en RDC en termes de comparaison avec les obstacles au dépistage et traitement de la THA identifiés dans ce travail montre que ces aspects socioculturels bien qu’étant des véritables goulots d’étranglements dans la dynamique de la lutte contre la THA ne sont pas bien ciblés par la communication sur la THA.

Les perspectives des communautés exposées au risque de la THA doivent être adressées par un dialogue continu entre professionnels de santé et communautés adapté aux réalités locales. Ainsi il sera possible d’améliorer de manière opérationnelle les stratégies d’information, éducation et communication, et de façon plus large, le dépistage et traitement de la THA en intégrant la dimension socioculturelle de la THA dans la politique de lutte contre la THA.

SUMMARY

Human African Trypanosomiasis (HAT), also known as “sleeping sickness” is a parasitic disease caused by protozoa of the species Trypanosoma. There are two types that infect humans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. The strategy used to control sleeping sickness consists of early case detection and treatment of patients, together with vector control. Meanwhile, utilization/access to HAT screening by the affected communities remains a major challenge. Adherence to active screening programs with mobile units was below 50% in certain endemic villages end of the 90’s. Moreover, utilization of fixed health facilities in DRC is so low that it compromises passive case finding. Our hypothesis is that this low utilization of health services is caused by a problem of acceptance of case detection and treatment of HAT by the communities living in the HAT transmission zones. This compromises the target of the international community to eliminate HAT as a public health problem by 2020. This thesis wants to explore and tries to generate more knowledge on the socio-cultural aspect that is often neglected in the control of HAT.

We conducted five studies to address the lack of community participation in HAT screening and treatment activities and the relation with acceptance of these services.

The first study evaluated the results of HAT treatment by retrospectively analyzing data of the routine HAT control program for the period 2006-2008.

Afterwards we performed three qualitative studies consisting of focus group discussions and individual interviews to document the socio-cultural dimension of the fight against HAT. The first study explored the community perceptions regarding sleeping sickness. The second study explored the perceptions regarding HAT treatment and a third study focused on diagnostic practices of health professionals in low-resource settings facing a neurological syndrome.

The fifth study consists of a household survey, focus group discussions and individual interviews to explore community perception regarding health in general and health services. We compared the identified barriers to screening and treatment of HAT with awareness messages on sleeping sickness used by the HAT control program in DRC and we developed strategic recommendations. The evaluation of performance indicators for treatment showed that compliance with post-treatment follow-up is very poor: 25% for the first post-treatment follow-up examination at six months and less than 1% of the patients returns for the final examination at 24 months. In this study we also observed a treatment failure rate of respectively 30% and 22% for melarsoprol and pentamidine in Kasai-Oriental. However, these date are difficult to interpret because of an incomplete denominator. As only few patients return for follow-up visits, this proportion is probably biased towards those in treatment failure.

The study on the perception of sleeping sickness shows that the disease is well known amongst the communities living in the endemic areas. However, several screening and treatment barriers were identified. The most important are: drug toxicity, financial barriers, the incompatibility between the itineraries of the mobile screening teams and the local communities’ activities, the prohibitions related to HAT treatment, lack of confidentiality and fear of lumbar punctures. The study on the perceptions regarding HAT treatment show that melarsoprol is perceived as a toxic drug and is nicknamed the ‘taboo drug’. On the other hand the NECT regime is perceived as the new drug that is less toxic and that has abolished all the taboos of melarsoprol with the important exception of sexual intercourse during the treatment period and the post-treatment follow-up period of six months.

The prohibitions have been established empirically by healthcare providers and communities to mitigate the side effects of the melarsoprol regimen. Violating these restrictions is believed to cause severe and sometimes mortal complications. Communities adhere strictly to these prohibitions and this constitutes a barrier for HAT screening and treatment.

The study focusing on diagnostic work-up of neurological syndromes in low-resource settings by health care providers has shown that in rural areas diagnosis is usually clinical. Barriers to confirmation of diagnosis are mainly related to the purchasing power of the patient. Other reported barriers are a lack of diagnostic tools and the communities’ perceptions associated with the care provider. Clinicians are perceived as diviners being able to directly identify the cause of the illness without using laboratory tests. The study regarding the perceptions on health and health services has shown that ability to work (82%) and ability to move (66%) are the most perceived signs of good health. 90% of the household responsibles positively perceive the health of their family. The opinions on the health services are divided.

The studies presented in this thesis have generated new insights on the socio-cultural dimension of HAT. The analysis of the awareness messages on HAT in DRC compared with the reported HAT screening and treatment barriers have shown that

although these sociocultural aspects are real bottlenecks in the dynamic of the fight against HAT, they are not targeted by the communication on HAT.

The prospects for communities at risk of HAT should be addressed through continuous dialogue between health professionals and communities adapted to local realities.

It will thus be possible to operationally improve the information strategies, education and communication, and more broadly, screening and treatment of HAT by integrating the socio-cultural dimension in the fighting policy against sleeping sickness.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Dans la trypanosomose humaine africaine prédominent signes inflammatoires, neurologiques et immunologiques, en particulier anomalies de production de monoxyde d'aazote (NO) et d'auto-anticorps. Les trypanosomes sont détruits par les dérivés S-nitrosylés. Leur synthèse par les macrophages murins activés fait intervenir NO, O2 et H2O2. Chez les malades, des anticorps dirigés contre ces dérivés sont mis en évidence, ainsi que des anticorps contre un épitope "L-tryptophane". Cet épitope est porté par les VSG des trypanosomes du groupe brucei, et absent sur T. Cruzi et T. Musculi. La production de NO est insuffisante pour la destruction des parasites. Ce nouveau mécanisme d'échappement aux défenses de l'hôte est lié à l'induction d'arginase par les trypanosomes. Elle consomme la L-arginine, substrat utisé pour la production de NO. Les facteurs parasitaires induisant l'arginase, purifiés par un anticorps monoclonal, sont des protéines de PM 105 et 70 kDa, dont l'identification est en cours
Neurological and inflammatory signs associated with immunological alterations are a hallmark of human Africal trypanosomiasis. They include alterations in antibody and nitric oxide (NO) productions. Trypanosomes are highly sensitive to S-nitrosylated compounds. Murine macrophages use oxygen and NO-dependent mechanisms to synthesize S-nitrosylated compounds. Antibodies to NO-epitopes and to tryptophan-like epitopes are present in patient sera. Tryptophan-like epitopes are borne by VSG from trypanosomes of the brucei group and absent on T. Cruzi and T. Musvculi. NO production is insufficient to kill parasites. Induction of arginase represents a new escape mechanism in host immune defence elaborated by parasites. L-arginine stock, essential for NO production, is depleted. Parasite factors inducing arginase are purified by monoclonal antibodies. The identification of these 105 and 70 kDa proteins is in progress

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
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Les trypanosomoses sont des maladies parasitaires dues à des protozoaires flagellés du genre Trypanosoma. En Afrique de l'Ouest, elles sont transmises à l'homme et aux animaux par des insectes hématophages, en particulier les glossines. La trypanosomose humaine africaine (THA) à Trypanosoma gambiense sévit sur un mode endémo-épidémique et, non traitée, se termine par une méningo­encéphalite mortelle. L'auteur décrit l’introduction et l'évaluation des techniques immunologiques, en particulier l’immunofluorescence indirecte, au cours des campagnes de dépistage dans les foyers des pays membres de l'OCCGE. Des techniques parasitologiques plus sensibles, en particulier la centrifugation en tubes capillaires et la filtration/centrifugation sur mini-colonnes de DEAE-cellulose, ont été évaluées pour obtenir un diagnostic de certitude. L'épidémiologie de la THA a été étudiée dans les foyers reviviscents de Bouaflé et de Vavoua en Côte d'Ivoire. Le foyer sans glossines d’OUAHIGOUYA au Burkina Faso, situé au nord de la limite de répartition des glossines est décrit. Enfin, une stratégie de dépistage est proposée, pour l’Afrique de l’Ouest, afin d’être incluse dans une stratégie globale comprenant lutte anti-vectorielle, dépistage et traitement des malades. La découverte récente d’un réservoir animal potentiel de trypanosomes pathogènes pour l'homme rend illusoire l'idée d'éradiquer cette maladie. La trypanosomose animale est un obstacle majeur au développement de l'élevage en Afrique Tropicale. Les potentialités des zones de savanes humides sont telles qu'une élimination de cette maladie permettrait de répondre à la demande croissante en produits d'élevage. L'impossibilité d'éradiquer les glossines sur l’ensemble de ces zones et l'apparition de chimiorésistance des trypanosomes aux médicaments trypanocides disponibles, font des bovins trypanorésistants de l'ouest-africain une solution d'avenir pour le développement de l'élevage. Dans le cadre des recherches du CRTA sur la trypanotolérance, l'auteur a isolé un répertoire de Trypanosoma brucei brucei et étudié son expression chez des animaux sensibles et résistants.

La trypanosomose humaine africaine (THA) est une parasitose réémergente qui pose un réel problème de santé publique. Deux stades sont classiquement décrits dans l'évolution de la maladie, le stade lymphatico-sanguin (stade 1) et le stade nerveux (stade 2). Le diagnostic du stade 2 et la discrimination entre les stades, dont dépend le traitement, reste difficile. Le critère habituellement retenu hormis la présence du trypanosome est une élévation de la cytorachie supérieure à 5 éléments/µL de liquide céphalo rachidien (LCR). Ce dernier critère est discutable car non spécifique. Notre travail de thèse a permis de préciser le type de cellules lymphocytaires impliquées dans la THA, à la fois dans le sang et dans le LCR de patients. Nous avons montré que dans le sang des malades, tous stades confondus, la proportion de lymphocytes B (CD19) augmente alors que celle des lymphocytes T diminue confirmant le caractère immunosuppressif de la maladie. Dans le LCR l'augmentation des cellules B CD19 chez les patients en stade 2, pourrait constituer un nouveau critère du stade nerveux. D'autre part nous avons cherché à comprendre quels étaient les mécanismes d'attraction des lymphocytes dans le système nerveux central par la mesure dans le sérum et le LCR de IL-1β, IL-8, MIP-1α, MCP-1 et RANTES. Le taux de MCP-1 dans le LCR semble être caractéristique des patients en stade 2 et pourrait constituer un autre marqueur du stade nerveux. Des travaux antérieurs ont montré que la réponse immunitaire induite au cours de la THA dans le SNC, conduit à la production d'anticorps anti-galactocérébrosides et anti-neurofilaments spécifiques du stade 2. Nous avons développé un test de terrain permettant de rechercher de façon simple ces anticorps dans le LCR des patients pour aider au diagnostic du stade de la maladie. Afin d'améliorer ce test et grâce à des analyses glycolipidiques du parasite, nous avons mis en évidence la présence de glucosylcéramide. La glucosylcéramide synthase pourrait être impliquée dans les mécanismes d'apoptose décrits dans la THA et dans les mécanismes de chimiorésistance. Des essais d'immunisation avec les produits de synthèse de cette enzyme montrent une protection contre le parasite
The human African trypanosomiasis (HAT) is a re emerging disease, which represents a real public health problem. Two stages are classically described in the evolution of the disease, the heamo-lymphatic stage (stage 1) and the nervous stage (stage 2). Both diagnosis of stage 2 and discrimination between the stages, which is crucial to determine the treatment to apply, remain difficult. Except the presence of the trypanosome, the criterion usually taken into account is an increased cell number, upper than 5 cells/µL, in the cerebrospinal fluid (CSF). This last criterion remains controversial because it is considered as not specific enough. Our PhD work specifies the nature of the lymphocytic cells that are involved in the HAT, both in the blood and the CSF of patients. We show that, whatever the HAT stage, the proportion of B cells (CD19) increases in the patients' blood whereas the number of T cells decreases, thus confirming the immunosuppressive character of the disease. In the CSF, the increased number of CD19 cells observed for stage 2 patients could constitute a new criterion of the nervous stage. Furthermore, we tried to understand by which mechanisms lymphocytes were attracted in the central nervous system. To understand this phenomenon, we measured the rates of IL-1β, IL-8, MIP-1α, MCP-1 and RANTES, both in serum and in CSF and showed that MCP-1 rate in CSF seems to be characteristic of the stage 2 and could constitute another marker of the nervous stage. Previous work have shown that, only during stage 2 of HAT in the CNS, the immune system produces anti-galactocerebrosides and anti-neurofilaments antibodies, so we developed a field test to detect these antibodies, such as to help practicers to diagnose the stage of the disease. To improve this test, we analyzed the glycolipids in the parasite membranes and could highlight the presence of both glucosylceramide and its enzyme (glucosylceramide synthase), which could play a role in the apoptosis mechanisms described in HAT. The glucosylceramide synthase is also known for its action in the mechanisms of resistance to drugs. Then, we developed immunization tests with the products of this enzyme and showed a protective effect against the parasite

Une étude longitudinale menée en Angola a montré une corrélation entre le titre des anticorps circulants anti-trypanosome détectés par le test d'agglutination directe pour la trypanosomiase (CATT) et la parasitémie chez les habitants de 503 villages du Nord-Ouest de l'Angola. Une seconde étude transversale a montré une corrélation entre les résultats du CATT et du micro-CATT dans les zones endémiques pour la trypanosomiase humaine africaine (THA) en Angola. Les résultats de ces deux études apportent des éléments importants pour la planification des activités de lutte et pour la mise ne place de la surveillance de l'endémie que ce soit de façon qualitative ou quantitative. Une troisième étude faite de façon rétrospective sur les résultats de l'analyse de 2015 liquides céphalo-rachidiens a révélé qu'un nombre de lymphocytes supérieur à 5 et inférieur ou égal à 20 constitue une phase intermédiaire (Pi) dans la classification de la maladie située entre la phase hémato-lymphatique (P1) et la phase méningo-encéphalitique (P2). A cette phase (Pi) des critères complémentaires sont nécessaires pour faire le choix du médicament à utiliser pour le traitement. L'ensemble des résultats de ces trois études et les informations complémentaires qui ont été obtenus sur le terrain ont permis de localiser les zones atteintes par la THA d'une part et d'identifier un certain nombre de villages de haute (16%) et faible prévalence. De plus, d'autres zones se sont avérées être hautement suspectes. Pour ce pays, qui compte près de 4 millions d'habitants exposés au risque d'infection, où les glossines sont présentes sur près de la moitié du pays et où plus de quinze mille malades ont été dépistés entre 1997 et 1998 alors que la surveillance est largement insuffisante, la trypanosomiase humaine africaine constitue définitivement un problème majeur de santé publique. L'auteur conclut que compte tenu des moyens limités disponibles en Angola pour lutter contre la trypanosomiase, il est essentiel d'optimiser l'utilisation de ces maigres ressources et que les efforts doivent s'orienter vers la surveillance et l'identification des zones prioritaires où il est urgent de mettre en oeuvre une lutte active.

La trypanosomose humaine africaine (tha) est une affection parasitaire due a un protozoaire de l'espece trypanosoma brucei. La complexite de l'evolution clinique de la maladie est frequemment evoquee. Lors de cette etude en cote d'ivoire, l'origine de cette complexite a ete recherchee a la fois chez le parasite par l'etude de la diversite genetique au sein de trypanosoma brucei ssp. , et chez l'hote chez lequel une susceptibilite individuelle a l'infection est suspectee. Deux enquetes ont ete necessaires pour mener a bien ces recherches, la premiere consistant a etudier l'existence d'associations entre la diversite genetique des trypanosomes et la diversite des formes cliniques qu'ils occasionnent, et la deuxieme consistant en un suivi a long terme de sujets refusant le traitement. Les techniques de caracterisation des trypanosomes ont mis en evidence un important monomorphisme genetique au sein des souches circulant en cote d'ivoire alors que l'etude clinique a confirme une grande diversite de tableaux et d'evolutions cliniques. Cependant, les taux d'isolement observes ont ete particulierement faibles, pouvant entrainer un biais dans notre echantillonage de parasites, mais quoiqu'il en soit, l'existence d'une susceptibilite individuelle a l'infection semble devoir etre prise en consideration et devra etre etudiee par des travaux complementaires.

Ce travail étudie l’organisation de la veille-sommeil par une modélisation stochastique et aborde, par des approches chronobiologiques, l’altération des rythmes circadiens et ultradiens lors de la trypanosomose humaine africaine (THA) et expérimentale. La THA est actuellement en réémergence et de nouvelles stratégies de lutte contre cette maladie doivent être mises en oeuvre. Nous espérons, par cette étude, apporter une aide au diagnostic du stade évolutif de la THA (stade I ou II). L’analyse est développée en deux volets : (i) application d’une modélisation stochastique et recherche des périodes inhérentes au sommeil humain pour caractériser le stade de la maladie ; (ii) validation par un modèle expérimental (chez le rat) des résultats obtenus chez l’homme par l’application de ces mêmes méthodes, complétées par une recherche histologique du trypanosome au niveau des tissus nerveux des animaux infestés
A stochastic model is used to study the sleep-wake organization while the disruption of the circadian and ultradian rhythms during Human African Trypanosomiasis (HAT) as well as in a rat experimental model is apprehended by classical chronobiological techniques. Currently we observe a reemergence of HAT and new therapeutic strategies have to be developed. Treatment of HAT depends on the severity (stage I or stage II) of the illness and medication for the second stage has potential harmful side effects. We aim through this work to bring in an additional tool for specifying as early as possible the stage of the illness. This is conducted in two complementary directions : (i) an application of a stochastic modeling and a search for the underlying temporal periods in human sleep process are used to characterize the stage of the illness ; (ii) using the same approaches, the results obtained for humans are validated by a rat experimental model in addition to an histological search for the parasite in brain tissues of the infested animal

La trypanosomose humaine, affection parasitaire rencontree en afrique sub-saharienne, connait actuellement de nouvelles flambees epidemiques dans de nombreux foyers historiques. Les relations qu'entretiennent les differents elements du cycle de transmission, le trypanosome, la glossine vectrice et l'hote, restent mal connues. Les facteurs environnementaux et humains interviennent dans le passage de l'etat d'equilibre, existant en zone endemique, a l'etat d'instabilite, declenchant le phenomene epidemique. Chez l'insecte vecteur, des trypanosomes animaux coexistent avec l'agent pathogene pour l'homme et des competitions s'exercent entre les differentes especes. Cette etude associe des enquetes entomologiques, en zones endemiques de maladie du sommeil au cameroun, a des analyses pcr, permettant l'identification specifique des trypanosomes deceles chez les glossines. La technique d'amplification genomique s'avere particulierement interessante pour detecter les infections mixtes et les pauci-infections. Cependant, la methode montre aussi certaines limites, tous les trypanosomes ne sont pas identifies par les marqueurs moleculaires utilises. La diversite genetique des souches circulant dans des zones eloignees est discutee. Comparativement aux trypanosomes infectant les animaux sauvages ou domestiques, trypanosoma brucei s. L. , l'espece potentiellement pathogene pour l'homme, n'est que rarement identifiee.

Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées mortelles telles que la leishmaniose viscérale (L. donovani et L. infantum) ou la trypanosomiase humaine africaine (T. brucei), pour lesquelles les traitements disponibles sont très limités. Depuis quelques années, on observe un regain d'intérêt pour le développement de nitrohétérocycles aromatiques anti-infectieux tels que le delamanide et le féxinidazole. De récentes études indiquent que l'activité anti-kinétoplastidés de ces dérivés repose sur leur bioactivation sélective par des nitroréductases parasitaires, conduisant à la formation de métabolites réduits électrophiles, fortement cytotoxiques. Suite à des études préliminaires réalisées dans notre équipe en série 8-nitroquinoléin-2(1H)-one, ces travaux de thèse portent sur la synthèse et l'étude in vitro de l'activité antiparasitaire de 80 dérivés notamment fonctionnalisés en positions 3 et 6 du pharmacophore par divers motifs, notamment via la mise au point de réactions d'halogénation sélective et de couplages pallado-catalysés. Ainsi, 5 nouvelles molécules hits (4 anti-kinétoplastidés et 1 sélective de T. brucei) ont été identifiées (0,01 µM ≤ CI50 ≤ 7 µM et 13 < IS < 1500), trois d'entre-elles étant des substrats sélectifs des nitroréductases parasitaires de type I. Afin de préciser les relations structure-activité, une étude des potentiels de réduction a également été menée. Des études physico-chimiques (solubilité, test de perméabilité PAMPA) et pharmacocinétiques in vitro (stabilité microsomale et fixation à l'albumine humaine) sont venues compléter ce travail. Enfin, des évaluations de la mutagénicité et de la génotoxicité de ces hits sur des cellules procaryotes et humaines ont été conduites, dans le but de statuer sur leur potentiel pharmaceutique antiparasitaire humain et vétérinaire
Kinetoplastids are flagellated protozoan parasites responsible for lethal neglected tropical diseases, such as visceral leishmaniasis (L. donovani and L. infantum) or sleeping sickness (T. brucei brucei), for which very few drugs are available. Nowadays, nitroheterocyclic compounds present a renewed interest as anti-infective agents, as illustrated by the development of fexinidazole and delamanid. Some recent studies demonstrated that the antikinetoplastid activity of these derivatives involves their selective bioactivation by parasitic nitroreductases, leading to the formation of electrophilic reduced metabolites, highly cytotoxic. Based on preliminary studies conducted in our team in 8-nitroquinolin-2(1H)-one series, this PhD work is about the synthesis and in vitro antiparasitic study of 80 derivatives mainly functionalized at positions 3 and 6 of the pharmacophore by various substituents, especially via the optimization of selective halogenation and pallado-catalyzed cross coupling reactions. Thereby, 5 new hit compounds (4 antikinetoplastid and 1 selective of T. brucei) were identified (0.01 µM ≤ IC50 ≤ 7 µM and 13 < SI < 1500), three of them being selective substrates of type I parasitic nitroreductases. In order to refine the structure-activity relationship studies, an analysis of reduction potentials was also conducted. In vitro physicochemical (solubility, PAMPA permeability assay) and pharmacokinetic (microsomal stability and human albumin binding) experiments completed this work. Finally, the mutagenicity and genotoxicity evaluations of these new hit compounds toward prokaryotic and human cells were realized, in order to assess their human and veterinary antiparasitic pharmaceutical potential

Dans le but d'evaluer l'apport de la genetique des populations, d'une part a la taxonomie des trypanosomes de l'espece trypanosoma brucei, et d'autre part a une comprehension de l'epidemiologie de la trypanosomiase humaine africaine (t. H. A. ), une etude isoenzymatique par electrophorese en acetate de cellulose a ete entreprise sur 55 stocks isoles de l'homme et de l'animal au congo, au zaire et au cameroun. Sur les 24 loci etudies, 15 se sont averes variables, et ont permis d'individualiser 23 zymodemes, eux-meme divises en deux groupes: le premier correspondrait a la sous-espece classique trypanosoma brucei gambiense, la seconde, a la sous-espece classique trypanosoma brucei brucei. Confirmant la taxonomie connue, ces resultats sont corrobores par l'analyse du polymorphisme des fragments de restriction de l'adn kinetoplastique. L'analyse statistique montre que la reproduction des trypanosomes etudies est principalement clonale dans l'aire consideree. Les zymodemes sont assimilables a des clones naturels ou a des familles de clones etroitement apparentees, stables dans l'espace et dans le temps. Leur repartition geographique confirme le caractere endemo-epidemique de la t. H. A. En afrique centrale: d'une part, la circulation des trypanosomes asymptomatiques semble conditionner la dissemination de la maladie, et d'autre part, les reservoirs humains et animaux contribuent a la persistance a l'etat endemique des principaux foyers historiques au congo et au zaire, qui auraient une origine ancestrale commune

Les glossines (mouches tsétsé) sont les vecteurs des trypanosomes africains, responsables de la Trypanosomose Humaine Africaine (THA) ou maladie du sommeil en Afrique sub-saharienne. De nouvelles stratégies de lutte contre la THA visent à utiliser les symbiontes de la glossine pour augmenter sa réfraction à l'infection par les trypanosomes. La mise en place de telles approches nécessite une bonne connaissance des bases moléculaires et cellulaires des interactions entre les symbiontes, la glossine et le trypanosome. Les objectifs de cette thèse étaient, i) d'évaluer l'évolution des densités des symbiontes (Wigglesworthia glossinidia et Sodalis glossinidius) au cours du cycle de développement du vecteur et ii) de caractériser les gènes de Sodalis, Glossina palpalis gambiensis et Trypanosome brucei gambiense en interaction et qui s'expriment différentiellement au cours de l'infection. Nous avons pu montrer la présence permanente des deux symbiontes quel que soit le stade de développement de la glossine, ce qui permet leur utilisation dans le cadre du contrôle des vecteurs. Par la suite, des infections expérimentales ont été réalisées sur des glossines d'insectarium. Des glossines de l'espèce G. p. gambiensis ont été gorgées sur des souris infectées par T. b. gambiense. L'analyse des métatranscriptomes des glossines infectées versus réfractaires à l'infection nous ont permis de mettre en évidence les gènes de Sodalis, G. p. gambiensis et T. b. gambiense différentiellement exprimés aux étapes clé de l'infection. Les résultats qui découlent de cette thèse mettent la lumière sur la complexité des interactions Sodalis - G. p. gambiensis - T. b. gambiense et soulignent l'implication des bactériophages du symbionte S. glossinidius dans la réfraction des glossines à l'infection. Mots clés : maladie du sommeil, mouche tsétsé, trypanosome, symbiontes, compétence vectorielle, expression de gènes
Tsetse flies are the vectors of African trypanosomes, the causative agents of human African trypanosomiasis (sleeping sickness)in sub-saharan Africa. New sleeping sickness control strategies plan to use tsetse gut symbionts to increase tsetse flies refractoriness to trypanosomes infection. Such approaches require good knowledge on the molecular and cellular basis of interactions between symbionts, tsetse fly and trypanosome. This thesis aimed to i) assess the evolution of Glossina palpalis gambiensis symbionts (Wigglesworthia glossinidia and Sodalis glossinidius) densities throughout the host fly development cycle and ii) to characterize genes of Sodalis, G. p. gambiensis and Trypanosoma brucei gambiense in interaction, which are differentially expressed during the infection. We showed that both symbionts are present in all tsetse fly development stages, allowing their use in the context of vector control. Subsequently, experimental infections were performed on colonies flies. G. p. gambiensis female flies were fed on T. b. gambiense hosting mice. Transcriptome of infected flies and flies that have cleared trypanosome they ingested were analysed. This allow us identifying genes of Sodalis, G. p. gambiensis and T. b. gambiense differentially expressed at the infection key stages. Our results highlight the complexity of interactions between Sodalis, G. p. gambiensis, T. b. gambiense and underline the involvement of bacteriophages hosted by S. glossinidius in tsetse fly refractoriness to trypanosome infection. Key words: sleeping sickness; tsetse fly; trypanosome; symbionts; vector competence; gene expression

La trypanosomose est la maladie parasitaire la plus dévastatrice en Afrique, et affecte à la fois les hommes et le bétail. Vu l’inefficacité des stratégies de contrôle actuelles, une stratégie alternative dite “anti-maladie” a été proposée dans le cadre de la trypanosomose animale. Elle vise à neutraliser les effets de la maladie plutôt qu’à éliminer le parasite. Une telle stratégie nécessite une meilleure compréhension du développement de la pathologie ainsi qu’une caractérisation détaillée des facteurs de virulence impliqués. Dans ce contexte, nous nous sommes intéressés à l’étude de l’interaction hôte/pathogène entre les trypanosomes Africains et l’endothélium de l’hôte mammifère. En comparant quatre espèces différentes de trypanosomes Africains, nous avons montré que leurs capacités d’activation des cellules endothéliales étaient distinctes. Nous avons clairement démontré que T. congolense, T. vivax et T. b. gambiense activent les cellules endothéliales via la voie de NF-ƘB, alors que T. b. brucei est incapable d’activer cette voie. Cette activation a induit une résponse pro-inflammatoire in vitro et in vivo, ce qui souligne l’importance de ce mécanisme dans le développement de la maladie. Pour la première fois, nous avons identifié une activité sialidase chez le parasite de l’homme T. brucei gambiense, et nous avons démontré que les trans-sialidases trypanosomales sont les médiateurs de cette activation endothéliale et de la réponse inflammatoire consécutive, et ceci à la fois chez les trypanosomes africains d’homme et d’animaux. De plus, nous avons montré que l’activation endothéliale implique l’activité lectin-like des trans-sialidases et non pas l’activité catalytique, ainsi que des récepteurs sialylés sur la surface endothéliale. En conclusion, ce travail a apporté des avancées considérables dans la compréhension de la relation hôte/pathogène et a permis de désigner les sialidases comme un facteur de virulence central dans le dialogue intermoléculaire durant les trypanosomoses, en faisant une cible de choix pour le vaccin « anti-maladie »
Trypanosomiasis remains by far the most devastating parasitic disease in Africa affecting both humans and livestock. The current control strategies being not efficient, an alternative “anti-disease” strategy aiming to neutralize the pathological effects of the parasite rather than to eliminate it, was proposed. Therefore, it is essential to understand the development of pathogenesis and characterize the involved pathogenic factors. In this context, we wanted to elucidate the host-pathogen interaction between the African trypanosomes and the mammalian host endothelium. By comparing four different trypanosomes species, we showed that they displayed distinct capacities for activation of endothelial cells. We clearly demonstrated that T. congolense, T. vivax and T. b. gambiense activate the endothelial cells via the NF-ƘB pathway, but not T. b. brucei. This activation caused a pro-inflammatory response in vitro and in vivo, showing the importance of this mechanism in the development of pathogenesis. For the first time, we identified sialidase activity in the human parasite T. brucei gambiense, and demonstrated that the trypanosomal trans-sialidases are the mediators of this endothelial activation and its consequent inflammatory response, for both human and animal trypanosomes. Additionnally, we showed that endothelial cell activation is mediated by the lectin-like domain of the trans-sialidase rather than the catalytic site, and involves sialylated receptors of the endothelial cell surface. In conclusion, our study brings considerable insights into the host-pathogen relationship and designates sialidases as a central virulence factor in the molecular crosstalk during trypanosomiasis, which makes it a perfect target for the anti-disease strategy

Ce travail porte sur les differentes politiques sanitaires definies lors de la colonisation francaise en afrique occidentale francaise, particulierement dans les pays voltaiques (burkina faso actuel). Le choix des trois affections, lepre, trypanosomiase humaine africaine et onchocercose permet de suivre les circonstances qui obligerent l'administration a modifier le mode de colonisation et a mettre l'accent sur la sauvegarde du "capital humain". Aussi, on decouvre les rapports difficiles entre les detenteurs du savoir et ceux du pouvoir. La colonisation politique aurait-elle reussi sans l'apport des sciences bio-medicales? c'est la question a laquelle cette recherche tente de repondre
This study concentrates on the different sanitary policies defined during french colonisation in french occidental africa, mainly in upper volta (nowadays burkina faso). Choosing those three diseases, leprosy, african human trypanosomiasis and onchocerciassis permit to follow the circumstances which obliged administration to modify colonial system and put all energy into the protection of "reserve of health". Woud colonisation succeed without helping by bio-medical sciences? it is question that this research tries to solve

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

La maladie du sommeil ou Trypanosomose Humaine Africaine (THA) est une parasitose vectorielle due à un protozoaire flagellé sanguicole du genre Trypanosoma et d'espèce brucei. Deux sous-espèces de ce parasite sont pathogènes pour l'Homme : T. b. gambiense et T. b. rhodesiense ; transmis par les mouches Tsé-Tsé présentes en Afrique subsaharienne. Cette maladie évolue classiquement en deux stades : le stade hémolyphatique qui est marqué par la présence du parasite dans le sang et la lymphe et le stade nerveux caractérisé par la présence du trypanosome dans le Système Nerveux Centrale. En l’absence de traitement cette maladie est mortelle. Actuellement les traitements accessibles à la population sont stades-dépendants. Pour contrôler un jour cette pathologie, la recherche et l’amélioration des outils de diagnostic de la maladie et le diagnostic de stade sont essentielles. C’est dans ce but que nous avons exploité une biobanque d’échantillons composée de patients infectés par T. b. gambiense et d’individus non-infectés pour : 1) Évaluer l’efficacité de biomarqueurs de stade déjà existants -Néoptérine et CXCL-13- et nous avons évalué leur potentiel sur les échantillons recueillis lors du suivi des patients post-traitements. 2) Rechercher de nouveaux biomarqueurs protéiques par spectrométrie de masse LCMS/MS. Notre étude a permis d’identifier, grâce à l’établissement d’un nouveau catalogue protéomique un grand nombre de biomarqueurs potentiels dans le liquide céphalo-rachidien, l’urine et la salive de patients. Certaines de ces protéines pourraient améliorer la prise en charge et le suivi des patients à l’avenir
Sleeping sickness, or Human African Trypanosomiasis (HAT), is a parasitic disease caused by a flagellar protozoan of the genus Trypanosoma and brucei species. Two subspecies of this parasite are pathogenic for humans: T. b. gambiense and T. b. rhodesiense; transmitted by Tsé-Tse flies present in sub-Saharan Africa. This disease classically evolves in two stages: the hemolymphatic stage which is define by the presence of the parasite in the blood and lymph and the nervous stage characterized by the presence of trypanosome in the central nervous system. Without treatment, this disease is lethal. Currently the available treatments for patients are stage-dependent. In order to control this pathology one day, research and improvement of tools for the diagnosis of the disease and the staging is fundamental. In this context, we have exploited a samples biobank composed of T. b. gambiense-infected patients and uninfected controles to: 1) evaluate the efficacy of existing stage biomarkers -Neopterin and CXCL-13- and we assessed their potential on the samples collected during post-treatment followup of patients. 2) determine new protein biomarkers using LC-MS/MS mass spectrometry. Our study identified a large number of potential biomarkers in cerebrospinal fluid, urine and saliva through the establishment of a new proteomic catalogue. Taking into account some of these proteins may improve patient management and follow-up in the future

Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées, pouvant être potentiellement mortelles chez l’Homme, telles que la leishmaniose viscérale (L. donovani et L. infantum), la trypanosomiase humaine africaine (T. brucei) ou la maladie de Chagas (T. cruzi), et pour lesquelles les traitements actuellement disponibles présentent des limitations. Ces dernières années, un regain d’intérêt est porté aux molécules anti-infectieuses nitroaromatiques, à l’image du fexinidazole. L’activité anti-kinétoplastidés de ces composés résulte de leur bioactivation sélective par des nitroréductases parasitaires, conduisant à la formation d’espèces réactives toxiques pour le parasite. Deux hits antileishmaniens et deux hits anti-Trypanosoma, substrats des nitroréductases parasitaires de type 1, ont été précédemment décrits en série 3-nitroimidazo[1,2-a]pyridine. Ces travaux de thèse portent sur la synthèse et l’étude des relations structure-activité de cent-trois dérivés originaux, dont soixante-et-un dérivés sont fonctionnalisés en positions 2, 6 et 8 du pharmacophore 3-nitroimidazo[1,2-a]pyridine, et trente-cinq dérivés sont fonctionnalisés en positions 5 ou 7, via des réactions de SN2, SNAr, ou de couplages pallado-catalysés. Deux dérivés en série 3-nitroimidazo[1,2-b]pyridazine et cinq dérivés 5-nitroimidazoles sont obtenus par miodification structurale. Ainsi, deux nouveaux hits ont été identifiés en série 3-nitroimidazo[1,2-a]pyridine, un premier antileishmanien, et un second anti-Trypanosoma, possédant tous deux de meilleurs paramètres pharmacocinétiques et physicochimiques que les hits précédents
Kinetoplastids are flagellated protozoa responsible for life-threatening neglected tropical diseases in humans, such as visceral leishmaniasis (L. donovani and L. infantum), human African trypanosomiasis (T. brucei), or Chagas disease (T. cruzi), for which currently available treatments have limitations. The antikinetoplastid activity of these compounds results from their selective bioactivation by parasitic nitroreductases, leading to the formation of reactive species toxic to the parasite. Two antileishmanial and two anti-Trypanosoma hits, substrates of type 1 parasitic nitroreductases, have been previously described in 3-nitroimidazo[1,2-a]pyridine series. This thesis work focuses on the synthesis and structure-activity relationships of one hundred and three original derivatives, of which sixty-one derivatives are functionalized at positions 2, 6, and 8 of the 3-nitroimidazo[1,2-a]pyridine pharmacophore, and thirty-five derivatives are functionalized at positions 5 or 7, via SN2, SNAr, or pallado-catalyzed coupling reactions. Two 3-nitroimidazo[1,2-b]pyridazine series derivatives are also obtained from a scaffold-hopping strategy, and five 5-nitroimidazole derivatives are obtained by structural simplification. Thus, two new hits were identified in 3-nitroimidazo[1,2-a]pyridine series, a first one antileishmanial, and a second one anti-Trypanosoma, both showing better pharmacokinetic (microsomal stability, human albumin binding) and physicochemical (water solubility, PAMPA permeability tests) properties than the previous hits

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.

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.

The trypanolytic factor of human serum :a trojan horse.

African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised.

Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I).

We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process.

ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon.

ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the auxotrophic parasites contributes to both growth rate and resistance against host oxidative burst. In human serum, the trypanosome receptor is unable to discriminate between Hp and the closely related TLF-bound Hpr, explaining TLF efficient endocytosis.

As such, the TLF acts as a Trojan horse, killing the parasite from inside the cell after having deceived its vigilance through the high similarity between heme-delivering haptoglobin and toxin-associated Hpr.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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.

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.

This research describes the exploration of a new scaffold with the potential to be developed in to a new drug for the treatment of Human African Trypanosomiasis (HAT), a neglected disease endemic in sub-Saharan Africa. Derivatives of an isoquinoline scaffold were synthesised and evaluated for their in vitro activity against T.b.rhodesiense, the causative agent of HAT. Five derivatives were identified with inhibition of T.b.rhodesiense in the sub-micromolar range with good selectivity over mammalian cells.

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.

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.

For centuries, African natives have been facing various infectious tropical illnesses, among which African trypanosomiases are some of the most frequent relevant parasitic diseases. African trypanosomiases, commonly called sleeping sickness in humans (HAT; Human African Trypanosomiasis) and Nagana in domestic livestock, affect a huge number of people living in poverty in 36 sub-Saharan countries, resulting in a key socioeconomic impact. After a century of outbreaks, due to political instability and lack of funding, around 70 million people and 50 million cattle are still at risk of exposure in Africa. Trypanosomiasis is transmitted by the bite of insects from the Glossina spp (Glossinidae) and is fatal in humans, if untreated. While taking a blood meal, infected Glossina flies can spread extracellular protozoans from the species Trypanosoma brucei. There are three morphologically indistinguishable subspecies of T. brucei. The subspecies T. b. gambiense is responsible for a chronic form of the human disease, while T. b. rhodesiense causes an acute form, which more rapidly leads to death. Both subspecies are infective to humans, whereas T. b. brucei is only infective to animals. During the early stage of the disease or hemolymphatic phase, the parasite is restricted to the blood and lymph and after months or years it invades the central nervous system resulting in various neurological symptoms including sleeping disturbance. As for other neglected tropical diseases, the chemotherapeutical arsenal against HAT is based on limited, expensive and often toxic medicines that are administered parentally in a context of poverty and lack of qualified personnell in healthcare centers. The few drugs that are available are pentamidine and suramin for the early stage disease and eflornithine (also in combination with nifurtimox) and melarsoprol for the late stage when the parasite infects the brain. Overall, the situation described above highlights the critical nature of this phenomena and the urgent need to explore new sources of potentially effective and safe compounds for therapy. In this scenario the naturally-occurring products may play a crucial role as source of bioactive drug candidates. With this vision in mind, in Chapter 2 I performed a complete phytochemical analysis on both polar and volatile compounds of T. diversifolia collected from a geographically isolated population living in Dschang, Cameroon and I assessed their biological activities (antitrypanosomal and amtimicrobial activities). The main secondary metabolites occurring in the T. diversifolia methanolic extract were isolated by column chromatography and structurally elucidated by MS and NMR techniques. Tagitinins C emerged as the most active compound against T. brucei (TC221) with an IC50 value  of  0.0042  μg/mL.  This  activity  was  4.5  times  better  than  that of the reference drug suramin. Then I analysed the chemical composition and the antimicrobial effects of the essential oil (EO) hydrodistilled from inflorescences of T. diversifolia. Results showed that T. diversifolia EO was mostly active against Staphylococcus aureus and selectively inhibited in vitro the NAD biosynthetic enzyme NadD from S. aureus (IC50 of ∼60 g/mL). Besides its extensive utilizations in the traditional medicine, the plant is believed to have a great potential in agriculture. For this reason, I decided to evaluate the T. diversifolia polar extracts against the two-spotted spider mite Tetranychus urticae (Tetranychidae), which is one of the most economically important arthropod pests worldwide. The ethyl acetate extract resulted as the most active oviposition inhibitor, with an ED50 value of 44.3 µg.cm-3 and an ED90 of 121.5 µg.cm-3. In Chapter 3, I investigated a lipophilic extract of Onosma visianii roots containing 12% of shikonin derivatives. The phytochemical investigation of the lipophilic extract resulted in the isolation of 12 naphtoquinone derivatives which were evaluated against Trypanosoma brucei. Isobutylshikonin and isovalerylshikonin emerged as the most active naphtoquinone derivatives, showing an IC50 of 3.3 and 2.7 g/mL, respectively. Furthermore, isovalerylshikonin provided an inhibition of Glossina palpalis acetylcholinesterase (gpAChE) (IC50 =  7.1  μg/mL),  stronger  than   isobutyrylshikonin (IC50 =  91.3  μg/mL),  with  a  significant  tse-tse fly versus human selectivity (SI = 7.2). In Chapter 4, I oriented my attention to the Apiaceae family, which is a class of aromatic plants rich of EOs. Four out of nine Apiaceae EOs resulted active against T. brucei showing an IC50 in the range 2.7-10.7 g/mL. Terpinolene, one the major isolated component of these oils, was particularly active with an IC50 value of 0.035 g/mL (0.26 µM) and a selectivity index (SI) of 180. As part of the extended family of naturally-occurring products, sesquiterpenes hold promising inhibitory effects against the bloodstream forms of T. brucei. For this reason, in Chapter 5, I decided to explore the potential of Smyrnium olusatrum EOs obtained and its main oxygenated sesquiterpenes,  namely  germacrone,  isofuranodiene,  and  β-acetoxyfuranoeudesm-4(15)-ene, as potential inhibitors of T. brucei. The EOs obtained efficiently inhibited the growth of parasite with IC50 ranging from 1.9 to 4.0 g/mL. Among the isolated main EOs components, isofuranodiene exhibited a significant and selective inhibitory activity against T. brucei (IC50 = 0.6 g/mL, SI = 30). In Chapter 6, I finally selected six medicinal and aromatic plants traditionally used in Cameroon to treat several disorders, including infections and parasitic diseases. Then I evaluated the activity of their EOs against T. brucei TC221 and their selectivity against Balb/3T3 cells, used as counter-screen for cytotoxicity. The most relevant outcomes showed that the EOs from A. indica, A. daniellii and E. giganteus were the most active ones, with IC50 values of 15.21, 7.65 and 10.50 g/mL, respectively. Overall, the results of my PhD thesis provided new insights into the potential of naturallyoccurring compounds as valuable sources for the development of innovative trypanocidal drugs or botanical insecticides.

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.

This research aims to utilise ligand-based target prediction to (i) understand the mechanism of action of African natural products (ANPs), (ii) help identify patterns of phylogenetic use in African traditional medicine and (iii) elucidate the mechanism of action of phenotypically active small molecules and natural products with anti-trypanosomal activity. In Chapter 2 the objective was to utilise ligand-based target prediction to understand the mechanism of action of natural products (NPs) from African medicinal plants used against cancer. The Random Forest classifier used in this work compares the similarity of the input compounds from the natural product dataset with compound-target combinations in the training set. The more similar they are in structure, the more likely they are to modulate the same target. Natural products from plants used against cancer in Africa were predicted to modulate targets and pathways directly associated with the disease, thus understanding their mechanism of action e.g. "flap endonuclease 1" and "Mcl-1". The "Keap1-Nrf2 Pathway" and "apoptosis modulation by HSP70", two pathways previously linked to cancer (which are not currently targeted by marketed drugs, but have been of increasing interest in recent years) were predicted to be modulated by ANPs. In Chapter 3, we aimed to identify phylogenetic patterns in medicinal plant use and the role this plays in predicting medicinal activity. We combined chemical, predicted target and phylogenetic information of the natural products to identify patterns of use for plant families containing plant species used against cancer in African, Malay and Indian (Ayurveda) traditional medicine. Plant families that are close phylogenetically were found to produce similar natural products that act on similar targets regardless of their origin. Additionally, phylogenetic patterns were identified for African traditional plant families with medicinal species used against cancer, malaria and human African trypanosomiasis (HAT). We identified plant families that have more medicinal species than would statistically be expected by chance and rationalised this by linking their activity to their unique phyto-chemistry e.g. the napthyl-isoquinoline alkaloids, uniquely produced by Acistrocladaceae and Dioncophyllaceae, are responsible for anti-malarial and anti-trypanosome activity. In Chapter 4, information from target prediction and experimentally validated targets was combined with orthologue data to predict targets of phenotypically active small molecules and natural products screened against Trypanosoma brucei. The predicted targets were prioritised based on their essentiality for the survival of the T. brucei parasite. We predicted orthologues of targets that are essential for the survival of the trypanosome e.g. glycogen synthase kinase 3 (GSK3) and rhodesain. We also identified the biological processes predicted to be perturbed by the compounds e.g. "glycolysis", "cell cycle", "regulation of symbiosis, encompassing mutualism through parasitism" and "modulation of development of symbiont involved in interaction with host". In conclusion, in silico target prediction can be used to predict protein targets of natural products to understand their molecular mechanism of action. Phylogenetic information and phytochemical information of medicinal plants can be integrated to identify plant families with more medicinal species than would be expected by chance.