Yes / Background. Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology. We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings. The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions. To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development. / This work was supported by the UBS Optimus Foundation (Zurich, Switzerland) and the Department of Economy, Science and Innovation of the Flemish Government (Belgium). KV was supported by a VLADOC PhD scholarship of VLIRUOS (Belgium).

Mycobacterium ulcerans is an acid-fast bacillus that is the causative agent of Buruli ulcer, a necrotizing skin disease. The transmission route for M. ulcerans is unknown, but many insects have been posited as part of the web, including Belostomatids, Naucorids, and Culicids. Aedes albopictus was selected for use in a set of experiments where the first-generation larvae were inoculated with M. ulcerans, and mosquitoes were reared throughout the third generation to interrogate presence and quantity of the bacteria. Using qPCR, second and third generations displayed positivity (22% and 5.6% respectively). 16S V4 sequencing was used to obtain microbiota for all life stages as well as environmental samples, and many relationships between generations, life stages, and treatments displayed statistical significance in alpha diversity, beta diversity, and relative abundance of microbiomes. This study opens multiple avenues of further investigation into the transmission web of Buruli ulcer.

Buruli ulcer disease (BUD) remains a ‘mysterious disease’ due to the unknown mode of M. ulcerans transmission and pathogenesis. To understand these, it is important to determine the reservoir of the organism in its natural environments, and stress response and interactions of M. ulcerans in its natural niche and during infection of a host. The major virulence factor of M. ulcerans is mycolactone, a lipid cytotoxin that is encoded on a giant plasmid pMUM001. Genetic analysis suggests that plasmid pMUM001 was acquired by M. ulcerans during evolution from its progenitor, M. marinum. Coincidental evolution of virulence hypothesis suggests that many microbes evolve to acquire traits to outcompete or overcome biotic and abiotic forces during their normal life cycle in the outside-host environment, which can confer virulence during infection of a human host. Hence in this study, we exposed M. ulcerans to selective abiotic forces such as UV, and dynamic oxygen and temperature conditions to determine their effect on M. ulcerans growth, and mycolactone and global gene expression. We also studied the role of mycolactone in determining polymicrobial interaction of M. ulcerans in its natural aquatic habitat by exposing mycolactone coated and uncoated slides in M. ulcerans endemic and non-endemic aquatic locations and determining differences in microbial community composition between them. Further, we studied quorum quenching ability of mycolactone against an opportunistic pathogen, S. aureus. The results obtained showed that exposure of M. ulcerans to abiotic stresses such as higher temperature and lower than optimal oxygen conditions modulate its global and mycolactone gene expression. Further, we also showed that mycolactone can impact overall microbial community structure in a polymicrobial environment in its natural, aquatic habitat. Mycolactone also effected virulence and quorum sensing in an opportunistic pathogen, S. aureus, without inhibiting its growth. These findings are important as they provide insight toward potential reservoirs or environmental niches which may harbor M. ulcerans and inform new potential mechanisms of pathogenesis. Further, our novel research of synergistic or antagonistic interactions within the complex polymicrobial communities colonizing skin and aquatic habitats is a powerful approach in determining M. ulcerans colonization efficiency, resiliency, and transmission mechanisms.

Mycobacterium ulcerans is sweeping across sub-Saharan Africa, but little is known about the mode of transmission and its natural reservoirs. Since the only effective treatment is excision of the infection and surrounding tissue, early diagnosis and treatment is the only way to reduce the havoc associated with Buruli ulcer. Using data from a national case search survey conducted in Ghana during 2000 and suspected risk factors this study tests the hypothesized factors and probes the challenges of developing a spatial epidemiological regression model to explain Buruli ulcer prevalence in the southwestern region of Ghana representing 42 districts. Results suggest that prevalence is directly related to the degree of land cover classified as soil, elevation differential, and percent rural population of the area.

L'ulcère de Buruli, maladie négligée, pouvant entraîner déformations et incapacités permanentes, causée par Mycobacterium ulcerans, une mycobactérie associée aux écosystèmes aquatiques. Les sources et réservoirs de M. ulcerans ne sont pas précisément connus limitant la prophylaxie. Ma thèse contribue à démasquer les sources de contamination par M. ulcerans. Ma revue de la littérature répertoriant les sources potentielles, met en exergue les pièces manquantes pour la compréhension de l’épidémiologie de l’ulcère de Buruli. Mon étude de coculture M. ulcerans-Acanthamoeba griffini indique que cette amibe n’est pas un réservoir de M. ulcerans. Ensuite, mon étude du métabolisme des substrats carbonés indique que les bactéries, champignons, algues et mollusques peuvent combiner des sources carbonées au profit de M. ulcerans dans l’environnement. Capitalisant sur ce résultat, mon étude de l’effet des mycolactones secrétées par M. ulcerans sur les champignons, montre une attraction sur Mucor circinelloides. Ces observations démasquent une nouvelle activité de la mycolactone d’attraction d’organismes fungiques. Le mode de transmission de M. ulcerans à l’homme reste également un mystère. Dans ce sens, j’ai détecté des séquences d'ADN spécifiques de M. ulcerans sur la peau saine d’individus asymptomatiques en zones d’endémie. Ces données pourraient aider à promouvoir une prophylaxie fondée sur le port des habits de protection au contact des environnements à risques. En perspective à mon travail de thèse, une collaboration est mise en place avec le PNLUB et l’Institut Pasteur de Côte d’Ivoire pour étudier les réservoirs de M. ulcerans en Côte d'Ivoire. / Buruli ulcer is a dermis, epidermis and sometimes bone infection leading to deformities and permanent disabilities. It is caused by Mycobacterium ulcerans, a mycobacterium associated to the aquatic ecosystems but its sources and reservoirs are not yet defined. Therefore, no prophylaxis is established. This thesis contribute to unmask the sources of contamination of M. ulcerans. My review has identified potential sources of M. ulcerans in the environment and highlighted the missing pieces for understanding the epidemiology of Buruli ulcer. My study on the role of amoeba in the survival of M. ulcerans in the environment, install M. ulcerans as susceptible to amoeba rendering amoeba an unlikely host of M. ulcerans. Hereafter, i studied carbon substrates metabolized by M. ulcerans strains. Literature survey indicated that the environmental sources of carbon substrates metabolized by M. ulcerans were bacteria, fungi, algae and mollusks. I therefore studied the interactions of M. ulcerans with fungi by testing the effect of mycolactones on fungi. Mycolactones showed an attraction effect on Mucor circinelloides. This observation suggest a novel role for mycolactones as chemoatractants to fungi. The mode of transmission of M. ulcerans to humans remains unknown. I showed that M. ulcerans DNA can be detected on the healthy skin of asymptomatic persons, suggesting an asymptomatic carriage. These data could help promote prophylaxis based on wearing protective clothing in contact with risky environments. In perspective to my thesis work, we set up a collaboration with the Buruli Ulcer Program (PNLUB) and Institut Pasteur Côte d'Ivoire to study the reservoirs of M. ulcerans.

In the fields of development and public health, the decisions of the rural poor are often treated as simple, unanimous, and driven by cultural preconceptions and beliefs. This is particularly the case for sub-Saharan Africa, where a dehistoricizing tendency presupposes an ontological link between an African culture and its tendency to interpret the world through the lens of belief. Generally, household activities are not seen as the kinds of modes of objectifying social practice that are the outcome of complex historical struggles over representation, and pre-disposing cultural factors are presumed to be the key determinants of household behavior. The three papers that constitute this alternative-format, article-based dissertation interrogate these assumptions. Although they address diverse subjects (the rise of West African Pentecostalism; the logic of treatment-seeking behavior in Benin; credit and savings strategies in rural Mali), they share a methodological concern with close analysis of the complexity of household decision-making in the moment, study over time, and attention to local concerns in the context of larger social transformations. In both medical and economic contexts, this approach demonstrates not only that behavior is primarily determined by enabling factors, but that the cultural factors that do condition behavior can be understood as creative, rational, and instructive of larger concerns, rather than merely as an impediment to development goals.

Buruli ulcer (BU) is an environmental bacterium caused by Mycobacterium ulcerans. Modes of transmission and hosts of the disease remain unknown. The purposes of this study are to explore the environmental factors that are possibly explain the spatial distribution of BU, to predict BU cases by using the environmental factors, and to investigate the impact of land use and land cover change on the BU distribution. The study area covers the southwest portion of Ghana, 74 districts in 6 regions. The results show that the highest endemic areas occur in the center and expand to the southern portion of the study area. Statistically, the incidence rates of BU are positively correlated to the percentage of forest cover and inversely correlated to the percentages of grassland, soil, and urban areas in the study area. That is, forest is the most important environmental risk factor in this study. Model from zero-inflated Poisson regression is used in this paper to explain the impact of each land use and land cover type on the spatial distribution of BU. The results confirm that the changes of land use and land cover affect the spatial distribution of BU in the study area.

Buruli ulcer (BU), an infectious disease caused by Mycobacterium ulcerans, is the third most common mycobacterial disease after leprosy and tuberculosis and a WHO-defined neglected tropical disease. Despite years of research, the mode of transmission of BU remains unknown. This master’s thesis provides an integrated spatial analysis of disease dynamics in Ghana, West Africa, an area of comparatively high BU incidence. Within a case/matched control study design, environmental factors associated with BU infection and spatial behaviors are investigated to uncover possible links between individual daily activity spaces and terrains of risk across disturbed landscapes. This research relies upon archival and field-collected data and analyses conducted with geographical information systems (GIS).

L'ulcère de Buruli (UB), ou infection à Mycobacterium ulcerans, est une maladie tropicale négligée qui se manifeste par des lésions cutanées, responsable d'une morbidité importante et de lourdes incapacités chez les personnes atteintes. Cette maladie touche surtout des populations rurales en Afrique de l'Ouest et Afrique Centrale, où elle sévit dans des foyers localisés. Le mode de transmission de M. ulcerans à l'humain est inconnu et les connaissances épidémiologiques limitées, ce qui restreint les possibilités de contrôle de la maladie. Les objectifs de cette thèse étaient d'identifier les circonstances associées au risque d'UB, et d'améliorer la compréhension de la chaîne de transmission de M. ulcerans, à partir de l'étude de deux foyers d'UB situés au Cameroun. Nos travaux ont permis d'identifier des environnements, saisons et activités à risque d'UB à l'échelle individuelle, et à celle du territoire. Un travail d'entomologie de terrain a permis de compléter ces analyses en vérifiant l'existence d'une circulation de M. ulcerans dans l'environnement domestique : plusieurs groupes d'arthropodes porteurs de M. ulcerans ont été identifiés dans l'environnement domestique, parmi lesquels plusieurs possibles candidats vecteurs. Ces travaux apportent des précisions sur l'épidémiologie de d'UB au Cameroun, qui sont transposables à d'autres foyers endémiques africains présentant des environnements similaires. Ces résultats permettront de cibler les populations, zones et périodes à risque, afin de rendre plus efficace les activités de santé publique de contrôle de l'UB. Ces résultats proposent enfin de nouvelles approches pour la recherche du mode de transmission de M. ulcerans / Buruli ulcer (BU) is a tropical disease caused by Mycobacterium ulcerans. It appears as cutaneous lesions causing severe morbidity and permanent incapacities in affected populations. BU occurs mainly in West and Central Africa, where it is found endemic in very localized regions. The transmission mode of M. ulcerans to humans remains mysterious, which, combined with the lack of basic epidemiological knowledge on the disease, hampers considerably the design of effective control strategies. The goals of this thesis were to identify the different risks associated with Buruli ulcer, and to contribute to the improvement of knowledge on the circulation and transmission of M. ulcerans. We studied two Buruli ulcer foci in Cameroon, and analyzed the individual, behavioral, spatial and temporal risk factors for BU. We described populations groups, environments and seasons at higher risk. We also engaged in entomological fieldwork to verify if M. ulcerans was circulating in the domestic environment, an hypothesis never tested in African endemic regions. We identified several taxa carrying M. ulcerans, and possible vector candidates among them.This work proposes an update of the knowledge on Buruli ulcer epidemiology in Cameroon, and several results can be transposed to other endemic regions, especially in Central or West Africa, where endemic areas present similar environments. These results could provide useful elements to design more efficient public health strategies against Buruli ulcer and to target them accurately to populations, places and persons at highest risk. These results finally suggest new leads for future research on the mode of transmission of M. ulcerans.

Yes / Buruli ulcer is a neglected tropical disease of skin and subcutaneous tissue caused by infection with the pathogen Mycobacterium ulcerans. Many critical issues for disease control, such as understanding the mode of transmission and identifying source reservoirs of M. ulcerans, are still largely unknown. Here, we used genomics to reconstruct in detail the evolutionary trajectory and dynamics of M. ulcerans populations at a central African scale and at smaller geographical village scales. Whole-genome sequencing (WGS) data were analyzed from 179 M. ulcerans strains isolated from all Buruli ulcer foci in the Democratic Republic of the Congo, The Republic of Congo, and Angola that have ever yielded positive M. ulcerans cultures. We used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our phylogeographic analysis revealed one almost exclusively predominant sublineage of M. ulcerans that arose in Central Africa and proliferated in its different regions of endemicity during the Age of Discovery. We observed how the best sampled endemic hot spot, the Songololo territory, became an area of endemicity while the region was being colonized by Belgium (1880s). We furthermore identified temporal parallels between the observed past population fluxes of M. ulcerans from the Songololo territory and the timing of health policy changes toward control of the Buruli ulcer epidemic in that region. These findings suggest that an intervention based on detecting and treating human cases in an area of endemicity might be sufficient to break disease transmission chains, irrespective of other reservoirs of the bacterium.