Dissertations / Theses on the topic 'Buruli, Ulcère de'

Les mycobactérioses cutanées restent prévalentes en Afrique de l’Ouest, malgré l’existence de traitements du fait des problèmes liés au diagnostic précoce. Le but de cette thèse est de contribuer à l’amélioration de leur diagnostic microbiologique. Les résultats issus des travaux de cette thèse permettent de comprendre l’épidémiologie des mycobactérioses cutanées au Burkina Faso, Afrique de l’Ouest. Ce travail est une première qui s’intéresse de façon simultanée au diagnostic microbiologique de l’ulcère de Buruli, de la lèpre et de la tuberculose cutanée au Burkina Faso par des approches innovantes. Une revue de la littérature réalisée sur la microbiologie des ulcères cutanées et sous cutanées en Afrique de l’Ouest a permis de dégager les objectifs suivants : i) étudier la prévalence moléculaire de Mycobacterium ulcerans dans les échantillons cliniques de plaies chroniques chez les patients vivant dans les milieux ruraux du Sud –Ouest et de l’Ouest du Burkina Faso ;ii)de mettre en place une technique microscopique d’hybridation de sondes moléculaires fluorescentes pour une détection précise de Mycobacterium leprae dans les échantillons cliniques de peau ; iii) d’utiliser cette technique pour rechercher dans des échantillons non invasifs(selles et secrétions nasales ) de Mycobacterium ulcerans afin de proposer des méthodes non invasifs de diagnostic de la lèpre au Burkina Faso, Afrique de l’Ouest ;iii) de mettre en place une technique de coloration microscopique permettant de distinguer les mycobactéries vivantes ,dormantes ou mortes dans des échantillons cliniques pour le suivi des traitements et fournir aux chercheurs un outil de sélection des échantillons éligibles pour les tentatives de culture de Mycobacterium leprae. Les échantillons cliniques pour la recherche de Mycobacterium ulcerans a été faite à Bobo-Dioulasso, à Djikologo (Diébougou) et à Bomborokuy (Nouna) soit 64 écouvillons de plaies cutanés chroniques. Quant aux échantillons de peau (biopsies et incisions cutanées), ils ont été réalisés dans le service de dermatologie du Centre Hospitalier Universitaire Souro Sanou de Bobo –Dioulasso. Les tests moléculaires réalisés pour la recherche de Mycobacterium ulcerans ciblaient les marqueurs suivants : IS2404, IS2606 et le KR-B. Une sonde oligonucléotide spécifique designer sur le rpoB de Mycobacterium leprae et marquée au fluorochrome Alexa555 a été utilisée par la microscopie à Fluorescence –lèpre. La Fluorescéine diacétate, le Nile red et le DAPI ont été utilisés pour la caractérisation de Mycobacterium leprae selon les trois stades : vivants, dynamiques et morts. Pour le Burkina Faso, il y a une première évidence moléculaire de la circulation de Mycobacterium ulcerans dans les plaies chroniques de patients ruraux. Un outil microscopique plus spécifique a permis de poser un diagnostic précis qui a permis aux dermatologues du CHUSS de soigner les malades. Pour les cliniciens, le DDD-lèpre reste un outil de suivi des patients sous médicaments anti lépreux. Les chercheurs trouveront dans le DDD un outil pour relancer les recherches sur la culture de la lèpre basée sur des échantillons ils auront un aperçu de la proportion des mycobactéries vivantes. Ces résultats confirment d’une part une extension géographique de l’ulcère de Buruli en Afrique de l’Ouest et d’autre part interpellent à un renforcement des efforts dans la lutte contre la lèpre au Burkina Faso, Afrique de l’Ouest avec à la fin une perspective de renforcement des approches de culture de la lèpre pour les scientifiques.Mots-clés : Mycobactérioses cutanées / Afrique de l’Ouest / Burkina Faso/ Mycobacterium ulcerans/ Mycobacterium leprae / FISH / DDD
Cutaneous mycobacterioses remains prevalent in West Africa, despite the existence of treatments due to problems linked to early diagnosis. The aim of this thesis is to contribute to the improvement of their microbiological diagnosis. The results of the work of this thesis allow us to understand the epidemiology of cutaneous mycobacterioses in Burkina Faso, West Africa. This work is a first that focuses on the simultaneous microbiological diagnosis of Buruli ulcer, leprosy and cutaneous tuberculosis in Burkina Faso using innovative approaches. A review of the literature carried out on the microbiology of skin and subcutaneous ulcers in West Africa made it possible to identify the following objectives: i) to study the molecular prevalence of Mycobacterium ulcerans in clinical samples of chronic wounds in patients living in the rural areas of South West and West of Burkina Faso; ii) set up a microscopic technique for hybridization of fluorescent molecular probes for precise detection of Mycobacterium leprae in clinical skin samples; iii) used this technique to search in non-invasive samples (stool and nasal secretions) of Mycobacterium ulcerans in order to propose non-invasive methods of diagnosing leprosy in Burkina Faso, West Africa; iii) to put in place a microscopic staining technique making it possible to distinguish living, dormant or dead mycobacteria in clinical samples for monitoring treatments and to provide researchers with a tool for selecting samples eligible for attempts to culture Mycobacterium leprae. The clinical samples for the research of Mycobacterium ulcerans were made in Bobo-Dioulasso, in Djikologo (Diébougou) and in Bomborokuy (Nouna) that is 64 swabs of chronic skin wounds. As for skin samples (biopsies and skin incisions), they were carried out in the dermatology department of the “Centre Hospitalier Universitaire Souro Sanou de Bobo – Dioulasso”. Molecular tests carried out for Mycobacterium ulcerans targeting the following markers: IS2404, IS2606 and KR-B. A specific oligonucleotide probe designer on the role of Mycobacterium leprae and labeled with fluorochrome Alexa555 was used by Fluorescence microscopy - leprosy. Fluorescein diacetate, red Nile and DAPI were used for the characterization of Mycobacterium leprae according to the three stages: living, dynamic and dead. For Burkina Faso, there is a first molecular proof of the circulation of Mycobacterium ulcerans in chronic wounds of rural patients. A more specific microscopic tool made it possible to make a precise diagnosis which enabled the dermatologists of CHUSS to treat the sick. For clinicians, DDD-leprosy remains a tool for monitoring patients on anti-leprosy drugs. The researchers will find in DDD a tool to relaunch research on the culture of leprosy based on samples they have an overview of the proportion of living mycobacteria. These results confirm on the one hand a geographical extension of Buruli ulcer in West Africa and on the other hand call for a strengthening of efforts in the fight against leprosy in Burkina Faso, West Africa with the end a prospect of strengthening approaches to leprosy culture for scientists.Keywords: Cutaneous mycobacterioses / West Africa / Burkina Faso / Mycobacterium ulcerans / Mycobacterium leprae / FISH / DDD

Notre travail porte sur tous les cas UB enregistrés entre 1997 et 2003 au Centre Sanitaire Nutritionnel Gbemoten (CSNG) de Zagnanado dans la région du Zou au Bénin. Une base de données a été créée et a permis d’analyser divers aspects de l’UB. Ces analyses ont donné lieu à diverses publications dont les résultats essentiels sont repris ci-dessous.

Une première publication porte sur 1700 cas consécutifs admis au CSNG entre 1997 et 2001. Ces données nous ont permis d’illustrer l’évolution du nombre des cas d’UB au cours des années, par département et par sous-préfecture. Le nombre de patients UB qui se présentent au centre est fonction de différents facteurs comme par exemple l’ouverture d’autres centres de traitement, l’organisation de campagnes d’information sur l’UB mais aussi de changements dans l’environnement. Au niveau des formes de la maladie, nous avons montré que les formes non ulcérées sont aussi fréquentes que les formes ulcérées. La forme de la maladie est liée au délai à consulter le CSNG. Les atteintes osseuses sont fréquentes et sont présentes chez plus de 13% des cas. Au fil des années, le délai à consulter le CSNG ainsi que la durée de l’hospitalisation ont été fortement réduits, passant de 4 mois à 1 mois dans le premier cas et de 9 mois à 1 mois en ce qui concerne l’hospitalisation. Les personnes ressources à cibler lors des campagnes de sensibilisation sur la maladie ont été identifiées. Il s’agit des anciens malades, qui dans 68% des cas, réfèrent les nouveaux malades au CSNG.

Une deuxième publication, portant sur la même période, nous a permis de mettre en évidence pour la première fois des taux d’UB élevés chez les sujets de 60 ans et plus, probablement du fait d’une diminution de leur immunité. Chez les 60 ans et plus, les hommes sont plus à risque de développer un UB que les femmes. Par contre aucune différence dans le risque d’UB en fonction du sexe n’est trouvée chez les moins de 60 ans. Les lésions d’UB prédominent au niveau des membres inférieurs pour tous les groupes d’âge. Du fait de leur petite stature, les enfants ont une répartition des lésions qui touchent tout le corps. Chez les adultes, les femmes présentent plus de lésions que les hommes au niveau de la tête, du cou et du tronc. Les sujets de moins de 15 ans développent souvent des lésions multifocales, associées à des atteintes osseuses. Ces atteintes osseuses constituent les formes graves de la maladie.

Notre troisième publication porte sur le suivi des malades au niveau des villages, entre mars 2000 et février 2001, afin de déterminer les taux de récurrences de l’UB. Le taux de récurrence de la maladie est faible (6.1%) pour un temps de suivi des malades allant jusqu’à 7 ans. La majorité des malades soignés au CSNG étaient en bonne santé. Nous n’avons néanmoins pu retrouver qu’un nombre limité de malades.

Dans notre quatrième publication, nous avons pu mettre en évidence le développement d’un UB sur le site d’une morsure humaine. La surface de la peau du malade a pu être contaminée par M. ulcerans et la morsure (= traumatisme) a occasionné l’entrée de M. ulcerans dans le derme. Une autre explication plausible est que la morsure ait réactivé un foyer latent de M. ulcerans au niveau du site de la morsure.

Notre base de données nous a également permis de contribuer à d’autres études, notamment celles sur l’influence du BCG et de la schistosomiase sur l’UB. Dans les deux cas, nous trouvons une association significative avec les formes graves de l’UB, l’absence de vaccination BCG et la schistosomiase favorisant le développement de formes osseuses. Aucun lien n’a pu être établi entre la présence ou l’absence de l’infection à Schistosoma haematobium et l’UB.

Dans la dernière partie de notre travail, nous nous sommes attachés à l’étude de quelques facteurs de risque de l’UB. Nous avons montré que l’UB est essentiellement associé à l’âge, le lieu de résidence et le type d’eau utilisé. Pour les sujets de 5 ans et plus, le risque d’UB est plus élevé chez les sujets vaccinés à la naissance avec le BCG. L’analyse par strate d’âge des sujets âgés de 5 ans et plus, nous a permis de voir que dans le groupe des 50 ans et plus l’influence du type d’eau utilisé est moindre que dans les autres groupes d’âge, laissant envisager qu’un autre facteur entre en jeu :une réactivation de la maladie à la faveur d’une baisse d’immunité.

Notre étude démontre que la prise en charge de l’UB doit être pluridisciplinaire et doit pouvoir englober diverses interventions telles que :

- les campagnes d’information à la population,

- la formation du personnel sanitaire ainsi qu’une implication de l’état,

- une bonne prise en charge des malades au niveau des centres de traitement de l’UB en collaboration avec les laboratoires régionaux et internationaux,

- la confirmation microbiologique des cas,

- la mise en place d’un suivi des malades en s’appuyant sur les structures déjà mises en place pour d’autres maladies,

- la mise à disposition des populations de sources d’eau potable (puits, pompes) qui par la même occasion permettront de limiter/diminuer d’autres affections liées à la consommation d’eau non potable,

- la poursuite des activités de recherche sur un éventuel vaccin ou une médication appropriée, ainsi que sur le réservoir de M. ulcerans afin de pouvoir mettre en place des stratégies de prévention de la maladie.

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

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

Une des 17 maladies tropicales négligées déclarées par l’OMS, l’ulcère de Buruli constitue aujourd’hui un réel problème de santé publique en Afrique centrale et de l’Ouest où il sévit dans près de 30 pays. Cette maladie touchant principalement les populations des zones enclavées reste un mythe car l’on ne connaît pas aujourd’hui le mode de transmission exacte de la bactérie responsable, Mycobacterium ulcerans, à l’humain. Une meilleure connaissance de l’écologie de ce bacille constitue pourtant un levier important tant pour apporter des informations concernant sa transmission que pour lutter contre ce fléau. Fortement soupçonnés de jouer un rôle d’hôtes ou de vecteur de M. ulcerans, certains taxons de punaises aquatiques semblent être prédisposés de par leur écologie ou leur comportement à héberger le bacille voire à le transmettre. Les travaux que nous présentons ici visent dans un premier temps à identifier sur des bases anatomiques complétées par des approches moléculaires les différents taxons de punaises aquatiques présents au Cameroun. Au-delà de proposer une clé d’identification d’espèces et une classification des punaises aquatiques du Cameroun reposant sur des séquences nucléotidiques, nous discutons du rôle important joué par l’écologie de certaines espèces de punaises dans la transmission de M. ulcerans. Les différentes approches comparatives et biostatistiques développées dans ce travail de thèse étayent l’hypothèse que certains taxons de punaises aquatiques, et notamment issus des familles Belostomatidae et Naucoridae, acquièrent la mycobactérie à partir de l’environnement en partageant des habitats (fréquentation de la végétation aquatique émergée) où le bacille prolifère, et en se nourrissant de proies, comme des mollusques ou de petits poissons, eux-mêmes contaminés par la bactérie. La capacité de vol chez les punaises aquatiques n’apparaît pas faciliter l’acquisition de M. ulcerans mais semble impliquée dans la dissémination de ce dernier en contaminant de nouveaux environnements. Au terme de cette thèse, nous discutons de l’écologie des communautés de punaises aquatiques et de leur macroécologie au Cameroun, et examinons l’importance de l’échantillonnage comme élément important de tout travail de recherche en écologie et évolution des maladies infectieuses
One of 17 neglected tropical diseases declared by WHO, Buruli ulcer is now a real public health problem in Central and West Africa, where it occurs in nearly 30 countries. This disease mainly affecting people in remote areas remains a myth because we don't know today the exact mode of Mycobacterium ulcerans transmission, the causal agent to humans. A better understanding of the ecology of this bacillus is an important lever to make the information about its transmission as the fight against this scourge. Strongly suspected to playing a role of host or vector of M. ulcerans, some aquatic bugs’ taxa seem to be predisposed by their ecology or behavior to harbor the bacillus or to convey it. The works presented here are intended firstly to identify on anatomical bases complemented by molecular approaches different taxa of aquatic bugs present in Cameroon. Beyond proposing an identification key of taxa and classification of aquatic bugs in Cameroon based on the nucleotide sequences, we discuss the important role of the ecology of some taxa of aquatic bugs in the transmission of M. ulcerans. Different comparative and biostatistics approaches developed in this thesis support the hypothesis that some taxa of water bugs, especially from the Belostomatidae and Naucoridae families acquire the mycobacteria from the environment by sharing habitats (attendance of emerged aquatic vegetation) where the bacilli proliferate, and feeding on prey such as molluscs and small fish themselves contaminated with the bacteria. The flight capacity of aquatic bugs do not appear to facilitate the acquisition of M. ulcerans but seems to be involved in their dissemination by contaminating new the environments. At the end of this thesis, we discuss the ecology of aquatic bugs’ communities and their macroecology in Cameroon, and examine the importance of sampling as an important component of any research work in ecology and evolution of infectious diseases

L'ulcère de Buruli (UB), infection à Mycobacterium ulcerans, troisième mycobactériose mondiale, connait une émergence rapide depuis 1980, essentiellement dans les pays d'Afrique subsaharienne. Jusqu’ici, les connaissances épidémiologiques sur l’UB étaient fondées sur des séries de cas cliniques non confirmés par laboratoire. Nous avons constitué la plus grande cohorte de cas confirmés à ce jour rassemblant plus de 1200 patients traités au CDTUB de Pobè au Bénin entre 2005 et 2011, afin de décrire l'épidémiologie clinique de la maladie et d'explorer l’architecture génétique de la susceptibilité à cette maladie. Les patients atteints d’UB sont des enfants (âge médian au diagnostic de 12 ans), présentant une lésion unique (96%), large (plus de 15 cm, 36%), ulcérative (66%) du membre inférieur (60%). Nous rapportons une présentation clinique atypique de l’UB, dans laquelle les patients présentent exclusivement une ostéomyélite à M. ulcerans. Le sex-ratio varie avec l’âge : les garçons sont majoritaires parmi les enfants (57% de patients masculins chez les moins de 15 ans), et les femmes parmi les adultes (33% de patients masculins). La présentation clinique dépend de l’âge et du sexe. 9% des patients masculins ont présenté une ostéomyélite contre 4% des patients féminins. Un an après la fin du traitement, 22% des patients présentent des séquelles fonctionnelles fixées. Une présentation clinique comportant une lésion oedémateuse, osseuse, de grande taille ou plusieurs lésions est significativement associée avec le développement de séquelles fonctionnelles (OR 7.64, IC95% [5.29-11.31]). Les patients coinfectés par le VIH ont un risque significativement plus élevé de développer un UB sévère (OR 2.77, IC95% [1.32-6.33]). Nous avons exploré l’architecture génétique de la susceptibilité à l’UB dans une perspective mendélienne et une perspective complexe. Le cas le plus sévère de la maladie observé dans ce centre appartient à une famille consanguine dans laquelle la ségrégation du phénotype suggère un défaut génétique mendélien récessif. Une analyse de liaison génétique par cartographie d'homozygotie suggère l’implication du locus des béta-défensines sur le chromosome 8 dans la pathogénèse de l'UB, et mène à l’identification d’une délétion homozygote ségrégeant parfaitement avec la maladie. Dans une perspective complexe, une étude d’association pangénomique a été réalisée après génotypage d’une cohorte de 400 cas et 400 témoins exposés sur plus de 2 millions de SNPs par la puce Illumina Omni2.5 et a permis l’identification de nombreux signaux d’intérêt. L’étude de réplication est en cours. La compréhension de la physiopathologie de l'infection à M. ulcerans est cruciale pour générer de nouvelles pistes thérapeutiques et vaccinales. La dissection du contrôle génétique de l'infection par l'hôte est en ce sens indispensable
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most frequent mycobacteriosis worldwide. It has been rapidly emerging in sub-Saharan African countries since 1980. Until now, knowledge of BU epidemiology relied on series of non laboratory-confirmed clinical cases. From 2005-2011, we recruited the current largest cohort of laboratory-confirmed cases (more than 1,200 patients) at the Pobe CDTUB, Benin, to describe the clinical epidemiology of the disease and to explore the genetic architecture of human susceptibility to BU. Typically, patients with BU were children (median age at diagnosis 12 years) presenting with a unique (96%) large (≥15 cm, 36%) ulcerative (66%) lesion of the lower limb (60%). Atypical clinical presentation of BU included osteomyelitis with no identifiable present or past BU skin lesions. The sex ratio of BU widely varied with age, with male patients accounting for 57% of patients aged 15 years and younger, but only 33% of those older than 15 years. Clinical presentation of BU was significantly dependent on age and sex. 9% male patients had BU osteomyelitis, whereas only 4% of female patients did. 1 year after treatment, 22% of patients with follow-up information presented with permanent functional sequelae. Presentation with oedema, osteomyelitis, or large (≥15 cm in diameter), or multifocal lesions was significantly associated with occurrence of permanent functional sequelae (OR 7•64, 95% CI 5•29–11•31) and operationally defines severe BU. When coinfected with HIV, patients had a significantly higher risk to develop severe BU (OR 2.77, IC95% [1.32-6.33]). We explored the genetic architecture of susceptibility to BU in both mendelian and complex genetic frameworks. The most severe case of the disease to have been treated at the Pobe CDTUB belonged to a consanguineous family in which the segregation of the phenotype was indicative of a recessive mendelian genetic defect. Genetic linkage analysis by homozygosity mapping suggested the implication of the beta-defensin locus on chromosome 8 in BU pathogenesis and lead to the identification of a homozygous deletion, which co-segregated perfectly with the disease in the family. In a complex genetics approach, we undertook a genome-wide association study, which involved the genotyping of more than 2 million SNPs (Illumina Omni2.5) in a cohort of 400 cases and 400 exposed controls. We identified many signals of interest. The replication study is ongoing. Understanding BU physiopathology is crucial to the development of efficient vaccines and drugs. Dissection of the genetic control of the infection by M. ulcerans by its human host therefore constitutes an indispensable step

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

L’ulcère de Buruli ou infection à Mycobacterium ulcerans, est la troisième mycobactériose après la tuberculose et la lèpre. M. Ulcerans produit une toxine, la mycolactone, aux propriétés cytotoxiques, responsables des destructions du tissu cutané. L’arsenal préventif et thérapeutique est limité. Le mode de transmission du bacille n’est pas totalement élucidé. Des modèles expérimentaux montrent que les punaises aquatiques pourraient jouer le rôle d’hôte et de vecteur de la bactérie. Dans ce contexte, le premier axe de notre travail a consisté à étudier le rôle des punaises aquatiques comme hôte et vecteur du bacille lors d’une étude environnementale réalisée au Cameroun. Nos résultats ont clairement établi que ces insectes pouvaient être des hôtes du bacille et que la recherche de M. Ulcerans dans leur salive pouvait être utilisée pour apprécier le risque d’infection à M. Ulcerans. Le deuxième axe de notre étude a consisté à caractériser un nouveau modèle murin original qui mime les différentes phases de l’infection observées chez l’homme et tout particulièrement le phénomène de cicatrisation spontanée. La charge bactérienne au niveau du tissu cicatriciel reste élevée, suggérant que la synthèse de la toxine est inhibée. Lors de la cicatrisation, la réponse inflammatoire locorégionale est importante, alors que la mise en place de la réponse adaptative ne semble pas jouer un rôle majeur. Cette première phase ouvre de nouvelles perspectives pour le développement de nouvelles stratégies préventives et thérapeutiques. L’absence d’un outil nous permettant de détecter la mycolactone est un obstacle pour la compréhension de la physiopathologie de l’infection à M. Ulcerans. Nous nous sommes intéressés à réaliser les premières étapes de sélection en vue d’obtenir un aptamère nucléotidique dirigé contre la mycolactone
Buruli ulcer or M. Ulcerans infection is the third most common mycobacterial disease in the world. M. Ulcerans produces a toxin, mycolactone, which induces extensive destruction of the skin and soft tissues with the formation of large ulcers. Therapeutic and preventive arsenals are still limited. The mode of transmission of M. Ulcerans is unclear and the role of water bugs was evocated for 10 years without strong evidence in natura. Firstly, we conducted a large study in Cameroon with the aim to assess the role of water bugs as hosts and vectors of M. Ulcerans. Our results could be put to advantage for surveillance and prevention purposes. More precisely, our work suggests that the detection of M. Ulcerans in water bug saliva could be used as an environmental indicator of the risk of M. Ulcerans infection. Secondly, we have studied the infection control in an original mouse model that mimic all clinical disease stages in particular the spontaneous healing. Indeed, FVB/N mice are sensitive as all other mouse strains with respect to M. Ulcerans infection, but present a spontaneous healing response after the ulcerative stage. A fascinating observation was the dynamic of the bacterial load in healing tissues suggesting that mycolactone production is regulated. During the healing process, the loco-regional inflammatory response is important, while adaptive response seems to play a minor role. Altogether, these results represent new perspectives for the development of preventive and therapeutic strategies. Currently, the lack of a powerful tool to detect mycolactone remains an obstacle for bacilli-host interaction understanding. In this context, we realized the critical steps to obtain nucleotidic aptamer against mycolactone

L'ulcère de Buruli est une maladie nécrosante de la peau et des tissus mous accompagnée d'ulcères de grandes tailles, causée par une mycobactérie. L’Organisation Mondiale de la Santé a lancé en 1998 une initiative mondiale contre cette maladie tropicale négligée, présente notamment en Afrique et en Australie. L'infection est causée par Mycobacterium ulcerans, mycobactérie faisant partie de la même famille que les mycobactéries responsables de la tuberculose et de la lèpre. Mycobacterium ulcerans sécrète une toxine nommée mycolactone, responsable de cette infection. Cette toxine a des propriétés cytotoxiques et immunosuppressives. Le projet de recherche développé dans l'équipe concerne la synthèse et l'évaluation biologique des mycolactones A et B et de leurs analogues. A l'heure actuelle, la première étude relation structure-activité conduisant à une meilleure compréhension du mode d'action des mycolactones a été établie. La stratégie de synthèse choisie implique plusieurs réactions de couplage catalysées par des métaux (fer, cuivre, palladium) pour la création de liaisons carbone-carbone. La stéréochimie des sept alcools secondaires a quant à elle été contrôlée par des réactions d'allyl-et crotylboration asymétrique ainsi que par des réactions de dihydroxylation catalytique et asymétrique. Au cours de ce travail de thèse, 12 analogues ont ainsi été obtenus en appliquant cette voie de synthèse
Buruli ulcer, a severe necrotizing skin disease caused by Mycobacterium ulcerans, is one of the most neglected tropical diseases. The World Health Organization has started in 1998 a Global Initiative against this disease. The pathogen belongs to the same family as the mycobacteria responsible for tuberculosis and leprosy and 6000 new cases are registered each year. Infection leads to extensive destruction of the skin and soft tissues with the formation of large ulcers usually on the legs or the arms and can caver up 15% of the skin surface. These effects are due to the presence of the bacterial toxins mycolactones, secreted by M ulcerans. Mycolactones are the first polyketides isolated from a human pathogen. Deciphering their functional interactions is of fundamental importance for the understanding and ultimately the control of this devastating mycobacterial infection. A Diverted Total Synthesis approach of mycolactones analogues has been developed and provides the first insights into their structure-activity relationship based on cytopathic assays on L929 fibroblasts

L’ulcère de Buruli est une maladie nécrotique de la peau présente dans plus de trente pays dans le monde, et affectant principalement le continent africain et l’Océanie. L’infection est due à Mycobacterium ulcerans (M. ulcerans), un micro-organisme qui sécrète une exotoxine appelée mycolactone, représentant le premier polycétide isolé d’un pathogène humain. La maladie est caractérisée par la formation progressive de lésions nécrotiques combinée à une absence de réponse immunitaire et de sensation de douleur ; la mycolactone est connue pour être directement impliquée dans ce mécanisme biologique. A ce jour, aucun traitement totalement performant et spécifique contre l’ulcère de Buruli n’a été développé, ce qui révèle le manque crucial de connaissances sur les mécanismes chimique et biologique. Dans ce contexte, le projet développé s’intéresse à l’élucidation du mécanisme d’action des mycolactones en utilisant la synthèse totale comme outil principal. Pour cela, notre équipe a mis au point une voie de synthèse modulaire permettant la préparation de la toxine naturelle et de ses différents analogues en vue de les tester biologiquement et d’affiner ainsi notre compréhension mécanistique de cette infection
Buruli ulcer is a necrotizing skin disease present in more than thirty countries in the world, located mainly in West and Central Africa but also in Australia and in Japan. This infection is caused by Mycobacterium ulcerans (M. ulcerans) that secretes a macrolide toxin called mycolactone, which is the first polyketide isolated from a human pathogen. The disease is characterized by the formation of painless progressive necrotic lesions combined with a lack of acute inflammatory response, and mycolactone is known to be directly involved in the biological mechanism. To date no specific and completely efficient treatment of Buruli ulcer has been developed which correlates with the dramatic lack of understanding of the associated chemical and biological mechanisms. In this context, this research project aims at a better understanding of mycolactone A/B molecular interactions by using total synthesis as main tool. To this end, our research team has developed an efficient synthetic pathway allowing the preparation of the natural toxin and its differents analogues for purposes of their biological evaluation and fine-tuning our mechanical understanding of this infection

Dans l'infection cutanée à Mycobacterium ulcerans (ulcère de Buruli), maladie de pays tropicaux humides, le mécanisme exact de la transmission du bacille n'est pas très clair. Une enquête épidémiologique et immunologique a été menée dans une région endémique du Cameroun. Une étude cas-témoin a permis de discerner trois facteurs de protection: le port de vêtements longs pour les travaux des champs, l'utilisation de la moustiquaire, et le traitement des lésions cutanées. Considérant que les punaises d'eau peuvent être des réservoirs et des vecteurs de M. Ulcerans, nous avons montré expérimentalement que leur salive inoculée à des souris pouvait avoir un rôle protecteur contre le développement de lésions cutanées. Nous avons observé de ce fait dans deux populations, béninoise et camerounaise, une signature immunologique distincte par un taux d'anticorps IgG plus élevé chez les sujets indemnes avec la reconnaissance de plusieurs constituants salivaires adhérant à la paroi de M. Ulcerans
The exact mechanism of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, an emerging tropical disease, remains elusive. An epidemiological and immunological investigation was carried out in an endemic area of Cameroon. A case-control study enabled to identify three protective factors: covering limbs during farming activities, use od bed nets and wound treatment. Given that water bugs can be reservoirs and vectors of M. Ulcerans, we have reported that exposure of laboratory mice to aquatic insect saliva could protect against M. Ulcerans lesion development. Furthermore we have observed in two cohorts of individuals in Benin and Cameroon distinct human immune signatures with a level of antibodies IgG higher for the safe subjects than Buruli patients and the specific recognition of several saliva molecules bound to M. Ulcerans

Comprendre les rétroactions entre les maladies infectieuses, la structure des écosystèmes et le développement économique est nécessaire pour alléger le fardeau des maladies tropicales négligées. Ce groupe d'infections parasitaires, virales, et bactériennes est étroitement associé à des conditions géographiques, environnementales, sanitaires et économiques particulières aux régions tropicales. A travers l'étude de l'ulcère de Buruli, une maladie émergente et négligée associé à une morbidité et handicap très importants dans des régions tropicales, ce travail de thèse s'intéresse aux interactions complexes entre ces différents composants des systèmes épidémiologiques. Combinant un travail de terrain important pour la collecte des données environnementales avec une approche de recherche multidisciplinaire, cette thèse vise à améliorer notre compréhension des différents aspects de l'écologie et de l'épidémiologie de cette maladie infectieuse. Notamment, la dynamique de son agent pathogène, M. ulcerans, est caractérisée pour un large éventail d'écosystèmes et communautés aquatiques au Cameroun, permettant d'identifier les facteurs environnementaux permettant sa propagation. En outre, nous évaluons la transmission de l'agent pathogène de l'environnement à l'homme et l'impact de la maladie sur le développement économique des populations endémiques. Ainsi, ce travail montre comment les dynamiques écologiques, épidémiologiques, environnementales et économiques interagissent de concert, mettant en évidence de façon criante le besoin d'une telle approche interdisciplinaire dans l'étude des maladies tropicales négligées
Understanding the feedbacks between infectious diseases, ecosystem structure and economic development is necessary to alleviate the burden of Neglected Tropical Diseases. This group of parasitic, viral, and bacterial infections is closely associated with particular geographical and environmental conditions mainly present in the tropics, thriving under conditions of poverty, inefficient sanitation and malnutrition. This PhD thesis works through the case study of Buruli ulcer, an emerging and neglected infectious disease associated with a great morbidity and disability burden in tropical regions. Relying on an extensive environmental field survey and a multidisciplinary research approach, this PhD attempts to gain a better understanding of different aspects of the ecology and epidemiology of Buruli ulcer. Notably, the dynamics of its pathogen, M. ulcerans are characterized for a wide range of freshwater ecosystems and aquatic communities in Cameroon, and the environmental drivers of M. ulcerans presence are investigated. Furthermore, we assess the transmission of the pathogen from the environment to humans and the impact of the disease on the economic development of endemic populations. Thus, this work shows how the interplay between ecological, epidemiological and economic dynamics interact together and calls for an urgent need to apply such inter-disciplinary approach to decrease the burden of neglected tropical diseases

Mycobacterium ulcerans (MU), agent causatif de l'ulcère de Buruli (UB), une maladie infectieuse humaine émergente, est associé aux milieux aquatiques tropicaux, et en particulier ceux modifiés par les activités humaines. L’écologie de cette mycobactérie est encore peu informée, et des interrogations demeurent sur son cycle de transmission au sein des écosystèmes et à l’humain. La tendance observée aujourd’hui en recherche est de montrer l’existence d’une multitude de taxa porteurs du bacille au sein des écosystèmes aquatiques mais aussi terrestres, laissant donc imaginer qu’un ou quelques facteurs en commun puissent expliquer sa présence et son développement. Dans ce contexte, nous avons développé des approches expérimentales au laboratoire pour analyser les effets de paramètres environnementaux, sélectionnés comme être importants dans la définition de la niche écologique de MU, sur le maintien et le développement des populations bactériennes. En tenant compte de gammes de valeurs rencontrées dans des régions endémiques et non endémiques, où sévit, nous avons testé dans un premier temps l'effet de deux polysaccharides très largement présents dans les écosystèmes naturels (la chitine et l’amidon) ainsi que cinq composants chimiques (le fer, le calcium, le zinc, le phosphate et le sulfate), représentant des nutriments indispensables pour les bactéries, sur la croissance de MU. Notre travail montre que la chitine augmente de manière très significative la croissance de MU. A l’inverse, la présence d’amidon ne favorise pas son développement. Le calcium est le seul élément chimique à contribuer à l’augmentation de quantité de cellules de MU avec le temps, mais ce paramètre reste toutefois très marginal. L’absence d’effet joué par le fer, le zinc, le sulfate et le phosphate sur la croissance in vitro de MU tend à indiquer que les valeurs utilisées dans nos expériences correspondent à des valeurs limites pour expliquer la distribution géographique de MU dans les écosystèmes aquatiques tropicaux. Dans un deuxième temps, eu égard au peu d‘informations existant concernant le rôle joué par le pH sur la présence et le développement de MU, nous avons reproduit des environnements pour étudier la croissance de MU en fonction de différentes valeurs de pH rencontrées dans des régions du Cameroun et de Guyane française où la mycobactérie peut être présente ou absente. Nos résultats montrent que le pH présente un effet significatif sur la dynamique de croissance de MU avec un effet plus marqué pour des valeurs de pH proches de 6,0. De plus, il existe une forte interaction entre pH et chitine puisque pour des mêmes valeurs de pH, la croissance bactérienne est 10 fois plus importante en présence de milieu avec chitine. Ces résultats suggèrent aussi que des pH trop acides, inférieurs à 5,0, sont défavorables à la croissance de la mycobactérie.Finalement, nous nous sommes intéressés à l’expression génique de différents isolats de MU générés à partir de différents environnements expérimentaux. Pour ce faire, et en nous servant d’une nouvelle approche de séquençage des ARN, nous avons étudié l’expression de MU dans différents environnements ; Nous nous sommes notamment intéressés à l’expression des gènes impliqués dans les voies métaboliques de production de la mycolactone, le peptide responsable des ulcérations chez l’humain. Des contextes environnementaux spécifiques pourraient conduire à une sur-expression de toxine peptidique traduisant ainsi une écologie et une épidémiologie (micro-)contexte-dépendant ayant des incidences pathologiques et cliniques très particulières.Dans leur ensemble, nos résultats participent à une recherche permettant de mieux comprendre les paramètres clés de la niche écologique de M. ulcerans et au-delà contribue à l’identification des écosystèmes aquatiques favorables, ou non, au maintien et au développement de cette mycobactérie responsable de l’ulcère de Buruli
Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer (BU), an emerging human infectious disease, is associated with tropical aquatic environments, particularly those modified by human activities. The ecology of this mycobacterium is still poorly understood, and questions remain about its transmission cycle within ecosystems and from nature to humans. Nowadays the research orientation is to show the existence of a multitude of host taxa carrying the bacillus in both aquatic and riparian ecosystems. Thus, it is likely to think that one or a few common factors might contribute and explain the presence and development of this bacillus across distinct localities and regions.In this context, we have developed experimental approaches in the laboratory to analyze the effects of several environmental parameters, selected as being important in the definition of the MU ecological niche and in its growth and persistence in natural ecosystems. Considering ranges of values encountered in endemic and non-endemic regions where BU occurs, we first tested the effect on MU growth of two polysaccharides widely present in nature (chitin and starch) and five chemical components (iron, calcium, zinc, phosphate and sulfate) representing essential nutrients for bacteria. Our work shows that chitin increases significantly the growth of MU. Conversely, the presence of starch does not favor its development with time. Calcium is the only chemical element contributing to increase MU cell number over time, but this effect remains very marginal. The lack of effect exerted by iron, zinc, sulfate and phosphate on the in vitro growth of MU suggests that values used in our experiments correspond to the limit values to explain the geographical distribution of MU in tropical aquatic ecosystems.Secondly, given the few existing information about the role of pH on the presence and development of MU in natural settings, we have reproduced in the lab some environments to study the growth of MU depending on different pH values encountered in regions of Cameroon and French Guiana where the mycobacterium can be present or absent. Our results show that pH has a significant effect on MU growth with a greater effect at pH values close to 6.0. In addition, there is a strong interaction between pH and chitin as to the same pH bacterial growth is 10 times greater in the presence of medium with chitin. These results also suggest that pH too acidic, lower than 5.0 are unfavorable for MU growth.Finally, we looked at gene expression of different MU cultures from different experimental frameworks. Here, and by making use of a new RNA sequencing approach, we studied the genetic expression of MU in differents environments. We are especially interested in the expression of genes implicated in the metabolic pathways of mycolactone production, the peptide toxin responsible of ulcerations in human. Specific environmental contexts could lead to an over-expression of these genes by MU populations, thus pinpointing the fact that MU ecology and epidemiology could be (micro-) context-dependent having some pathological and clinical implications. Taken together, our results participate in a research allowing to better understand the key parameters of the ecological niche of MU, and beyond helping to identify the aquatic ecosystems favorable or not to the maintenance and development of this mycobacterium responsible for Buruli ulcer

L’ulcère de Buruli est la troisième mycobactériose la plus prévalente dans le monde après la tuberculose et la lèpre. L’ulcère de Buruli sévit dans au moins 33 pays dont l’Afrique de l’Ouest qui présente la prévalence la plus élevée. Parmi ces pays dans lesquels l’ulcère de Buruli est rapporté, la Côte d’Ivoire dont nous sommes originaires présente la plus forte incidence de 36% déclarée à l’Organisation Mondiale de la Santé. L’ulcère de Buruli est causé par Mycobacterium ulcerans, une mycobactérie environnementale dont le réservoir et les sources de transmission à l’homme, ne sont pas élucidés. Notre revue de la littérature a porté sur les sources environnementales de cette mycobactérie en Afrique de l’Ouest. Sur cette base, nous avons réalisé une vaste campagne de prélèvements d’échantillons environnementaux en Côte d’Ivoire et détecté par PCR en temps réel M. ulcerans dans l’eau stagnante, le sol et les selles d’un mammifère herbivore Thryonomys swinderianus. Ensuite, nous avons prouvé expérimentalement que M. ulcerans pouvait survivre dans le sol pendant au moins quatre mois et que le sol était source d’infection expérimentale par M. ulcerans dans un modèle animal. Ces résultats suggèrent que, en Côte d’Ivoire, l’eau stagnante, le sol et cet animal pourraient jouer un rôle dans le cycle de vie de la bactérie. Dans le second travail, nous avons valorisé des plantes tropicales aquatiques par l’utilisation de leurs extraits dans le milieu de culture de M. ulcerans, mycobactérie à croissance lente pour accélérer sa croissance. Dans le troisième travail, nous avons montré l’efficacité in vitro et dans un modèle animal, du bleu de méthylène contre M. ulcerans
Buruli ulcer is the third mycobacteriosis in the world after tuberculosis, leprosy. Buruli ulcer is widespread in at least 33 countries including West Africa which has the highest prevalence. Among the countries in which Buruli ulcer is reported, Ivory Coast which we come from has the highest incidence of 36% reported in the World Health Organization. Buruli ulcer is caused by Mycobacterium ulcerans, an environmental mycobacterium whose reservoir and source of transmission to humans, are not understood. Our review of the literature focused on environmental sources of this mycobacterium in West Africa, which converge stagnant aquatic environment. On this basis, we conducted an extensive campaign of environmental sampling in Ivory Coast and detected by real-time PCR M. ulcerans in stagnant water, soil and feces of an herbivorous mammal Thryonomys swinderianus. Then we experimentally proved that M. ulcerans could survive in the soil for at least four months. These results suggest that, in Ivory Coast, stagnant water, soil and animal could play a role in the life cycle of the bacterium. In the second work, we have upgraded aquatic tropical plants by the use of extracts in the culture medium of M. ulcerans, slow growing mycobacteria to accelerate its growth. In the third work, we proposed the methylene blue, less expensive, easy to access as an alternative treatment for Buruli ulcer. It is appropriate to resume the experiment on methylene blue by other teams of researchers and after reproducing our experimental data, suggest the topical use of purified, non-toxic methylene blue in human clinical

L'ulcère de Buruli est une maladie émergente tropicale négligée. Il provoque une défiguration et une incapacité permanente pour les victimes. L'agent causal est Mycobacterium ulcerans. Cependant, le réservoir environnemental et le mode de transmission de cette bactérie ne sont pas connus. Les tentatives visant à gérer la maladie ont été freinées par le manque de connaissances concernant le mode de transmission ainsi que le réservoir environnemental de M. ulcerans. Certains écosystèmes et habitats ont été associés au risque de contracter cette mycobactérie, notamment les milieux aquatiques d'eau douce stagnants et perturbés par les activités humaines des régions tropicales. S'il n'existe pas de vecteur bien identifié, des insectes aquatiques Hémiptères sont fortement suspectés d'intervenir dans la vectorisation de cet agent infectieux à l'humain. Une compréhension complète de la distribution et du mode de transmission de la bactérie aiderait ainsi à la gestion de la maladie. Dans cette thèse, nous utilisons les outils issus de la modélisation de la niche écologique pour décrire la distribution de M. ulcerans. Suite à la construction d'un modèle au Cameroun, en Afrique Centrale, et testé avec une seconde base de données en Guyane Française (Amérique du Sud), nous trouvons que l'agent pathogène montre des variations saisonnières notables dans la répartition de nos sites d'étude, au Cameroun. Pendant la saison humide, M. ulcerans est plus fréquente dans les grands bassins versants et en absence de marécages, tandis que durant la saison sèche, la bactérie est plus présente dans les petits bassins versants où peuvent être présents dans les zones marécageuses. Notre étude a permis de générer des cartes de répartition de l'agent pathogène dans la région d'étude, qui pourront être utilisées dans des études ultérieures contribuant à mieux gérer le risque infectieux pour cette maladie. De plus, nous avons développé une modélisation des niches écologiques des insectes aquatiques soupçonnés d'être des vecteurs de l'agent pathogène. Basé sur un protocole d'échantillonnage d'insectes aquatiques qui couvre l'ensemble du Cameroun, nous avons construit un modèle suivant la méthode de l'entropie maximale. Ceci nous a permis d'interpoler notre modèle sur toute l'Afrique de l'Ouest. Nous avons ensuite testé une corrélation entre la répartition prévue des insectes potentiellement vecteurs, et la prévalence de l'ulcère de Buruli. Nous mettons en évidence une corrélation positive significative entre la répartition des insectes et la répartition de la maladie, et trouvons que cette corrélation varie significativement dans l'espace et le temps. Ceci est cohérent avec la possibilité d'une transmission multi-hôte pour cet agent pathogène.Enfin, en collaboration avec d'autres auteurs, nous avons pu explorer différents facteurs influençant la distribution de M. ulcerans, tels que les réseaux et la structure de la communauté biotique, ou encore l'impact de l'occupation du sol sur la distribution de l'ulcère de Buruli dans notre région d'étude d'Akonolinga au Cameroun. Et enfin nous avons testé le changement de distribution de la maladie à une plus grande échelle, entre le Bénin et le Nigeria. Cette thèse contribue à une meilleure compréhension de la distribution de Mycobacterium ulcerans et de l'ulcère de Buruli, fournissant des éléments de preuve d'une transmission multi-hôtes de la mycobactérie, ainsi que les premières cartes de répartition de l'agent pathogène pour la région d'Akonolinga au Cameroun
The Buruli ulcer is an emerging environmentally acquired infectious neglected tropical disease. It causes permanent disability and disfigurement in victims. The causative agent is Mycobacterium ulcerans; however the environmental reservoir and mode of transmission of this bacterium are not known. Attempts to manage the disease have been hampered by lack of knowledge of the mode of transmission and the environmental reservoir of M. ulcerans. Certain environments have been associated with the disease, notably disturbed aquatic environments composed of small bodies of stagnant water. There is no known vector, though aquatic insects have been implicated as possible vectors. A full understanding of the distribution and mode of transmission of the bacterium would help in management of the disease.In this thesis, we use the tools developed in ecological niche modelling to describe the distribution of M. ulcerans. Following the construction of a model in Cameroon, Central Africa, and tested against a second database in French Guiana (South America), the pathogen is found to have notable seasonal changes in its distribution in our study sites in Cameroon. In the wet season, M. ulcerans is more common in large watersheds, while in the dry season the bacterium is more common in small watersheds. This enabled the generation of hazard maps of the pathogen distribution in the study region, which will be used in future studies and management of the disease. Following this we undertook ecological niche modelling to describe the distribution of the aquatic insects suspected to be vectors of M. ulcerans. Based on a sampling protocol that spanned the country of Cameroon, we undertake maximum entropy modelling, which enabled us to interpolate our model across all of West Africa. With these maps we explore the correlation between the predicted distribution of the insects to the prevalence of the Buruli ulcer. We find a significant positive correlation between the distribution of the insects and the distribution of the disease, and find that this correlation undergoes significant changes in space and time, consistent with the model of multi-vectorial transmission of the disease.Finally, in collaboration with other authors, we have assisted in exploring how the distribution of M. ulcerans changes according to community structure networks, how the distribution of the Buruli ulcer disease changes in our study region of Akonolinga, Cameroon, and how the distribution of the disease changes at a larger scale, between Benin and Nigeria. This thesis contributes to our understanding of the distribution and drivers of Mycobacterium ulcerans and the Buruli ulcer, providing evidence of multi-vectorial transmission of the disease, and the first hazard maps of the pathogen for Akonolinga, Cameroon

L'ulcère de Buruli est une maladie infectieuse tropicale présente dans des foyers endémiques. Cette infection essentiellement cutanée est causée par Mycobacterium ulcerans. M. ulcerans est un pathogène opportuniste dont le réservoir est environnemental. Notre revue de la littérature a répertorié les sources environnementales potentielles de cette mycobactérie. Seulement cinq souches de M. ulcerans ont été isolées à partir de prélèvements de l’environnement. Il existe une corrélation inverse entre réchauffement climatique et incidence de l’ulcère de Buruli, peut-être liée à la sensibilité intrinsèque de M. ulcerans aux variations de température, ou bien à des modifications de son écosystème. Dans la perspective d’améliorer les protocoles d’isolement de M. ulcerans à partir de l’environnement, nous avons entrepris une analyse phénotypique à haut débit des substrats carbonés métabolisés par M. ulcerans et le profil obtenu nous a orientés après une recherche bibliographique des principales sources environnementales de ces substrats, vers des interactions plus spécifiques de M. ulcerans avec les bactéries, algues, mollusques et champignons. Les résultats de ce premier travail ont servi de base pour la mise au point de milieux de culture innovants qui nous ont permis d’isoler pour la première fois, une microcolonie de M. ulcerans à partir de fèces d’agouti. Nous avons mis au point une méthode de lecture automatisée des échantillons colorés par la méthode de Ziehl-Neelsen. Notre travail de thèse a produit des protocoles qui ont pour objectif d’être mis en œuvre dans les pays d’endémie, pour préciser les sources et modes de transmission de M. ulcerans aux populations
Buruli ulcer is a tropical infectious disease present in endemic foci/This mainly cutaneous infection is caused by Mycobacterium ulcerans. M. ulcerans is an opportunistic pathogen from the environment. Our literature review has listed the potential environmental sources of this mycobacterium.. However, only five strains of M. ulcerans have been isolated from environmental samples. There is an inverse correlation between global warming and incidence of Buruli ulcer, possibly related to the intrinsic sensitivity of M. ulcerans to temperature, or to changes in its ecosystem. In order to improve the isolation protocols of M. ulcerans from the environment, we conducted a high-throughput phenotypic analysis of the carbon substrates metabolized by M. ulcerans and the profile obtained oriented us afterwards a bibliographic search of the main environmental sources of these substrates, towards more specific interactions of M. ulcerans with other bacteria, algae, molluscs and fungi. The results of this first work served as a basis for the development of innovative culture media which, allowed us to isolate for the first time a microcolony of M. ulcerans from feces of agouti. We also developed a method for automated reading of samples stained by Ziehl-Neelsen staining. Our thesis work has produced protocols that are intended to be implemented in African endemic countries, in order to clarify the sources and modes of transmission of M. ulcerans to populations

La récente pandémie de Covid19 nous rappelle, si cela était encore nécessaire, que la propagation des maladies infectieuses ignore les frontières géographiques. Les changements combinés de biodiversité locale et l’utilisation des terres, l’augmentation de la connectivité internationale par le transport et le commerce ainsi que la menace imminente du changement climatique a accru le risque d’émergence et de réémergence des maladies infectieuses (EMI). Jusqu’à présent la réponse des politiques de santé publique a été la surveillance passive sans toutefois s’avérer réellement efficace dans la prévention et le contrôle des épidémies. Le choix qui a été fait ici est celui d’une nouvelle approche anticipative, par identification des zones à haut risques d’EMI en se basant sur la détection des facteurs environnementaux les plus favorisant. Parmi ces facteurs on trouve la conversion des terres, la diminution drastique de la biodiversité ou encore le changement climatique. Ainsi la méthode biogéographique a permis d’étudier et d’analyser les EMI à travers différents groupes de taxons de pathogènes comme les bactéries, les virus, les protozoaires et les champignons. L’étude a été portée globalement, ainsi que localement, en Guyane Française, un territoire français d’outre-mer situé en Amérique du Sud. Dans les deux cas, à travers les différents groupes de pathogènes, les risques d’inondation, les récentes conversions de parcelles de forêts en terres agro-minières et l’augmentation du minimum de température due au changement climatique se sont avérés être des facteurs significatifs dans l’émergence globale et locale des maladies infectieuses étudiées. Les principaux résultats de cette thèse sont les suivantes :1. Une approche biogéographique de modélisation de la distribution des EMI en utilisant les bases de données existantes sur les cas cliniques, l’imagerie satellite et un modèle statistique non conventionnel est efficace pour détecter précocement les régions à risque, permettre d’améliorer la prévention, et contrôler leur diffusion.2. Il est possible d’anticiper les EMI en identifiant et en gérant précocement les facteurs favorisant ayant un lien direct avec l’anthropisation de l’environnement
The COVID-19 pandemic highlights that the spread of infectious diseases goes beyond geographical boundaries. Simultaneous changes in local biodiversity and land use, the increasing international connectivity through human transport and trade and the imminent threat of climate change have increased the risk of the emergence and reemergence of infectious diseases. The current public health response to emerging infectious diseases (EID) by passive surveillance has proven largely ineffective in preventing and controlling disease outbreaks. The way toward is to “get ahead of the curve” by identifying potential hotspots of disease emergence and detecting the environmental triggers such as land transformation, biodiversity loss and climate change. I used a biogeographic approach to study and analyze disease emergence across different taxonomic pathogen groups such as bacterial, viral, protozoal and fungal, globally and in French Guiana, a French Overseas territory located in South America. I found that regions at risk of floods, recent conversion of forest to agricultural lands and increasing minimum temperature (i.e. temperature at night) caused by cli mate change were drivers for disease emergence locally and globally across the different pathogen groups. The main findings of the PhD thesis are the following:1. Biogeographic approach to mapping the distribution of EIDs with using existing human cases data, remote sensing imagery and unconventional statistical models is effective to “get ahead of the curve” in the detection of regions at risk and the management of EIDs.2. EIDs are not unprecedented but predictable by identifying and managing the triggers of disease emergence, which have a direct link with the anthropization of the environment

Ce projet de recherche se focalise sur les infections par mycobacterium ulcerans (maladie de l’ulcère de Buruli), une maladie de la peau dévastatrice caractérisée par la formation de lésions nécrotiques progressives et l’absence d’une réponse inflammatoire. Bien que négligée, cette infection est la troisième maladie mycobactérienne la plus répandue après la tuberculose et la lèpre et des cas sont rapportés dans plus de 30 pays à travers le monde. Mycobacterium ulcerans sécrète une toxine polycétidique complexe, appelée mycolactone A/B, qui est directement responsable des effets pathogènes de la maladie. Depuis sa découverte, les propriétés biologiques inhabituelles de la mycolactone A/B ont suscité de nombreux efforts de recherche dans différents domaines. Dans ce contexte, ce projet s’intéresse à l’élucidation du mécanisme d’action des mycolactones en utilisant la synthèse totale comme outil principal. Dans cette optique, notre équipe a mis en place une voie de synthèse permettant un accès facile et robuste à différents mimes de mycolactone. L’utilisation de cette méthode a conduit à la préparation de 13 mimes de la toxine au cours de cette thèse. D’autre part notre équipe s’intéresse également à la préparation de mimes possédant un ou plusieurs atomes de fluor. Ces derniers présentent un intérêt particulier pour améliorer la compréhension des interactions ayant lieu entre la toxine et sa cible cellulaire. Les travaux réalisés autours de la synthèse de mycolactones fluorés ont conduit à la mise au point d’une méthode générale et simple pour introduire un groupe trifluorométhyle sur un alcyne terminal, permettant ainsi des modulations inédites de la structure de la toxine
This research project focuses on mycobacterium ulcerans infection (Buruli ulcer disease), a severe skin disease characterized by the formation of progressive necrotic lesions and the lack of an acute inflammatory response. Although neglected, this infection is the third most common mycobacteriosis after Mycobacterium tuberculosis and Mycobacterium leprae, and cases are reported in more than 30 countries worldwide. Mycobacterium ulcerans secretes a complex polyketidic macrolide, called mycolactone A/B, which is directly involved in the biological effects of the disease. Since its discovery, the unusual biology triggered by this toxin has spurred research efforts. In this context, this research project aims at a better understanding of mycolactone A/B molecular interactions by using total synthesis as main tool. To this end, our research team has developed an efficient synthetic pathway allowing the preparation of different mimetics of the toxin. This synthesis has been used to prepare thirteen new mycolactone mimetics during this thesis. Moreover our team has also been interested in the synthesis of fluorinated mycolactone analogs. Such fluorinated mycolactones are of great interest to improve the interactions that occur between the toxin and its biological binding site. Work in this field led to the development of a simple and general method to introduce a trifluoromethyl group onto a terminal alkyne, allowing novel modulation of the structure of the toxin

La mycolactone est un lipide diffusible produit par Mycobacterium ulcerans, la bactérie responsable d'une maladie tropicale de la peau appelée ulcère de Buruli. La production de mycolactone dans la peau infectée par M. ulcerans provoque une nécrose tissulaire locale, tout en induisant des anomalies immunosuppressives au niveau systémique. Lorsque j'ai commencé mon doctorat, les mécanismes moléculaires à l'origine de ces effets étaient inconnus. Au cours de ma thèse, j'ai contribué à démontrer que la mycolactone est un nouvel inhibiteur du translocon Sec61,le canal régulant la biogenèse de la plupart des protéines sécrétées et membranaires dans les cellules eucaryotes. J’ai démontré qu’une mutation ponctuelle dans la sous-unité alpha de Sec61 protège les cellules des effets cytotoxiques et immunosuppresseurs de la mycolactone. J'ai montré que le blocage du translocon Sec61 par la mycolactone empêche efficacement la synthèse des principaux récepteurs immunitaires et des molécules de signalisation du système immunitaire,blockant la communication entre les cellules immunitaires et inhibant l'immunité antimycobactérienne. Par une série d'études protéomiques à grande échelle, j'ai démontré que la mycolactone est un inhibiteur à large action de Sec61 et j’ai identifié les substrats de Sec61 les plus impactés dans différents types cellulaires. Ces analyses m'ont également permis de décrire la réponse au stress induite par le blocage de la translocation des protéines, qui inclut des éléments de la réponse au stress protéostatique (UPR) et de la réponse intégrée au stress (ISR), provoquant finalement l'apoptose cellulaire. Plusieurs études ont impliqué le translocon Sec61 dans des processus qui requièrent le transport rétrograde de protéines à travers les membranes : la Dégradation Associée au Réticulum Endoplasmique (ERAD), processus essentiel du contrôle de la qualité des protéines et la cross-présentation, une voie essentielle à l'activation de l'immunité adaptative aux pathogènes intracellulaires et au cancer. J'ai montré que le blocage de Sec61 par la mycolactone n'affecte pas l'export de protéines vers le cytosol dans ces deux voies, suggérant que Sec61 ne peut pas fonctionner comme un rétrotranslocon. Mes travaux ont permis d’élucider le moléculaire responsable des divers effets de la mycolactone observés chez les patients atteints d’ulcère de Buruli et de révéler l’importance majeure de la translocation des protéines dans la régulation des réponses immunitaires et de l’homéostasie des protéines
Mycolactone is a diffusible lipid produced by the human pathogen Mycobacteriumulcerans, the causative agent of a tropical skin disease called Buruli ulcer. Bacterial production of mycolactone in infected skin causes local tissue necrosis, while inducing immunosuppressive defects at the systemic level. When I started my PhD, the molecular mechanism(s) underpinning these effects were unknown. Over the course of my thesis, I contributed to demonstrate that mycolactoneis a novel inhibitor of the Sec61 translocon, a channel regulating the biogenesis of most secretedand membrane proteins in eukaryotic cells. Indeed, a single point mutation in the alpha subunit ofSec61 protected cells from the cytotoxic and immunosuppressive effects of mycolactone. I showed that mycolactone-mediated blockade of the Sec61 translocon efficiently prevents the synthesis ofkey immune receptors and signaling molecules, impeding the communication between immunecells that is required for the development of anti-mycobacterial immunity. Through a series of larges caleproteomic studies, I demonstrated that mycolactone is a broad-acting inhibitor of Sec61 and identified the Sec61 clients that are primarily down regulated by mycolactone in physiologicallyrelevant cell types. These analyses also allowed me to describe a unique stress response,encompassing elements of the unfolded protein response and integrated stress response, that isinduced upon protein translocation blockade and ultimately causes cell apoptosis. The Sec61 translocon has been proposed to play a role in other cell functions that require the retrograde transport of proteins across membranes, namely Endoplasmic Reticulum-Associated Degradation(ERAD), an essential process in protein quality control, and antigen export to the cytosol during cross-presentation, a pathway essential to the activation of adaptive immunity to intracellular pathogens and cancer. Using mycolactone, I showed that Sec61 blockade does not affect protein export to the cytosol in either of these pathways, arguing against Sec61 operating as are trotranslocon. Altogether, my work provided a molecular mechanism for the diverse effects of mycolactone in Buruli Ulcer patients, and thus for M. ulcerans virulence. Mycolactone representing the most potent Sec61 blocker identified to date, my studies also revealed the key importance of Sec61-mediated protein translocation in the regulation of immune responses and protein homeostasis

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.

Background Reducing social distance between hospital staff and patients and establishing clear lines of communication is a major challenge when providing in-patient care for people afflicted by Buruli ulcer (BU) and chronic ulcers. Research on hospitals as therapeutic communities is virtually non-existent in Africa and is currently being called for by medical anthropologists working in the field of health service and policy planning. This paper describes a pioneering attempt to establish a therapeutic community for patients suffering from BU and other chronic ulcers requiring long term hospital care in Benin. Methods A six-month pilot project was undertaken with the objectives of establishing a therapeutic community and evaluating its impact on practitioner and patient relations. The project was designed and implemented by a team of social scientists working in concert with the current and previous director of a hospital serving patients suffering from advanced stage BU and other chronic ulcers. Qualitative research initially investigated patients' understanding of their illness and its treatment, identified questions patients had about their hospitalization, and ascertained their level of social support. Newly designed question-answer health education sessions were developed. Following these hospital wide education sessions, open forums were held each week to provide an opportunity for patients and hospital staff to express concerns and render sources of discontent transparent. Patient group representatives then met with hospital staff to problem solve issues in a non-confrontational manner. Psychosocial support for individual patients was provided in a second intervention which took the form of drop-in counseling sessions with social scientists trained to serve as therapy facilitators and culture brokers. Results Interviews with patients revealed that most patients had very little information about the identity of their illness and the duration of their treatment. This knowledge gap surprised clinic staff members, who assumed someone had provided this information. Individual counseling and weekly education sessions corrected this information gap and reduced patient concerns about their treatment and the status of their healing process. This led to positive changes in staff-patient interactions. There was widespread consensus among both patients and staff that the quality of communication had increased significantly. Open forums providing an opportunity for patients and staff to air grievances were likewise popular and patient representative meetings resulted in productive problem solving supported by the hospital administration. Some systemic problems, however, remained persistent challenges. Patients with ulcers unrelated to BU questioned why BU patients were receiving preferential treatment, given special medicines, and charged less for their care. The idea of subsidized treatment for one disease and not another was hard to justify, especially given that BU is not contagious. Conclusion This pilot project illustrates the basic principles necessary for transforming long term residential hospitals into therapeutic communities. Although the focus of this case study was patients suffering from chronic ulcers, the model presented is relevant for other types of patients with cultural adaptation.

Understanding disease ecology is vital in preventing future outbreaks of established infections and to predict the emergence of new pathogens. In recent decades there have been a number of high profile infectious diseases which have swept across countries and in some cases the world. Many of these begin as generalist emerging infections; such microbes are difficult to study in the wild due to their inherently ambiguous life histories and complex associations with numerous hosts and the environment. In this PhD a number of techniques are used to pinpoint and further understand the life history of one such pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer, in the hope that this data can be used to predict and prevent future outbreaks and can be applied to other emerging infections. The results of this study include the first identification of the pathogen in the environment for a whole new continent, South America. Further to this it has led to the discovery of the likely ecological niche of the bacilli by linking its presence to specific functional groups of organisms. In turn the occurrences of these groups have been related to anthropogenic conditions such as deforestation and human mediated land use. Finally complex links between climatic fluctuations and outbreaks of the disease in Southern America and Cameroon, central Africa help complete our understanding of this mysterious disease.

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).

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.

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.

Mycobacterium Ulcerans (also known as Buruli Ulcer) is a rare skin disease which is prevalent in rural communities in the developing world mostly in Africa. Even though the mortality rate is low morbidity and consequent disabilities which affect the quality of life of sufferers is very high. If the disease is reported early treatment is available but many people receive help late in the disease process. Many reasons have been given in the literature why people receive treatment late. The aim of this study was to explore the experiences of people living with Buruli ulcer in a rural district in central Ghana in order to understand their diagnosis and support needs. The study was qualitative in design and used Glaser‟s version of grounded theory. It consisted of semi-structured and unstructured interviews, participant observation, conversation with opinion leaders, traditional healers, people living with Buruli ulcer (who were not part of the study) and a focus group interview with six health care professionals. The Dermatology Life Quality Index (DLQI) tool was also used to determine the quality of life of people living with Buruli ulcer in the study area. Three principal categories of Delayed treatment, Quality of life as a mirage and Seeking to be myself and a Basic Social Psychological Problem (BSPP) of Reliving the trauma of my ulcer were derived. These led to an encompassing core category of My needs matter. The theory explains the needs of people living with Buruli ulcer and that this followed a three stage process whereby Delayed diagnosis was the cause of the BSPP, Quality of life as a mirage the consequences and Seeking to be myself as the strategies required to deal with the BSPP of Reliving the trauma of my ulcer. How to deal with the consequences of this disease and the approaches to dealing with the needs of this vulnerable group have also been explored. The outcome of this thesis and its contribution to knowledge is a theory which explains the needs of people in the early stages of and living with Buruli ulcer and why their needs matter.

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).

Tese de Doutoramento em Ciências da Saúde
Buruli ulcer (BU) is an emerging neglected tropical skin disease caused by Mycobacterium ulcerans and remains the third most common mycobacteriosis worldwide, exceeded only by tuberculosis and leprosy. Infection with M. ulcerans starts off as a nonulcerative cutaneous lesion and can eventually break down into an ulcer if left untreated. Development of severe disabling deformities is extremely frequent in BU patients, having a significant socioeconomical impact and undermining the potential development of the affected populations. BU disease is closely related with the low temperature requirement of M. ulcerans (30- 33ºC), which favors the development of cutaneous lesions; with its slow growth rate that translates into slowly progressing lesions; and, most importantly, with the secretion of a lipidic exotoxin known as mycolactone, which is responsible for the induction of cell death and tissue necrosis. All these particular features impact on the mycobacterial nature of this intriguing pathogen. Mycolactone has been attributed cytotoxic and immunosuppressive properties and is suggested to inhibit both local and systemic protective immune responses. Indeed, the histological hallmarks for advanced BU lesions are extensive necrosis and minimal or absent inflammatory infiltrates, in the presence of abundant extracellular bacilli. In contrast, infections with the genetically related pathogenic mycobacteria M. tuberculosis and M. marinum have been consistently associated with the recruitment of acute and chronic inflammatory infiltrates to the site of infection. Despite the histological pattern associated to BU, several studies have associated cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) responses as being protective against M. ulcerans infections, suggesting that the predominantly described histopathology of BU lesions may not reflect the actual host-parasite interaction at the active site of infection. Therefore, to more extensively reassess the dynamics of the inflammatory response we performed a comprehensive histological analysis of footpads of mice infected with M. ulcerans, throughout the whole course of the infectious process. We found that experimental infections with M. ulcerans induced persistent, virulence-dependent inflammatory responses. A mycolactonenegative avirulent strain induced an initial acute neutrophilic inflammatory process that was gradually replaced by a predominantly chronic mononuclear infiltrate with granuloma-like organization. On the other hand, infection with mycolactone-positive strains was associated with a persistent influx of a mixed inflammatory infiltrate composed of neutrophils and macrophages with the frequent presence of intraphagocytic bacilli. The recruited cells were later depleted in the center of the infectious focus, by apoptosis and lytic secondary necrosis, which led to the generation of characteristic necrotic acellular areas with abundant extracellular bacilli. At the periphery of this necrotic area cellular infiltrates with intraphagocytic bacilli were consistently observed. At later stages of infection, these necrotic areas devoid of inflammatory cells expanded by progressive invasion of healthy tissues reproducing at a lower scale the scenario typical of human biopsies. Our results led to the proposal that M. ulcerans would be, like the other pathogenic mycobacteria, a facultative intracellular pathogen with phases of intramacrophage and extracellular multiplication, a hypothesis confirmed by subsequent studies in our laboratory. This interpretation allows the reconciliation of the cytological, histopathological, microbiological and immunological features of M. ulcerans infection with the above highlighted particular traits of this peculiar mycobacterium. We have provided evidence for the development of a local cellular immune response at the site of M. ulcerans infection. However, several reports have suggested that mycolactone inhibits local as well as systemic protective immune responses. In this study, we found that M. ulcerans infection triggered the early development of a T cell response in the draining lymph node (DLN) characterized by an increase in the number of mycobacteria-specific IFN-γ-producing cells. However, mice remained susceptible to infection with a highly virulent strain, and disease progression was accompanied by a depletion of CD4+ cells, not only in the active infection foci, but also in the DLN, contributing to the loss and thus ineffective immune response. The dissemination of M. ulcerans to the DLN and its ultimate destruction further impaired a sustained protective immune response. Despite this, advanced infection with M. ulcerans did not render mice more susceptible to a systemic co-infection with Listeria monocytogenes, suggesting that the local and regional immunosuppression associated to M. ulcerans infection is not accompanied by systemic immunosuppression. Given our previous observations, and bearing in mind the reported association of CMI, DTH and IFN-γ with host resistance to BU, we next focused on the role of this cytokine in M. ulcerans infections. In vivo and in vitro experimental infections demonstrated that IFN-γ is required for the control of both an avirulent and an intermediate virulent strain of M. ulcerans. However, for a highly virulent strain, no differences were found in bacterial control, regardless the presence of IFN-γ. When cultured macrophages were infected with different multiplicities of infection (MOI), we found that the lack of protection was due to a virulence-dependent/MOI dependent impairment of the macrophage antimicrobial mechanisms triggered by IFN-γ, such as phagosome maturation, phagolysosome fusion, and generation of reactive nitrogen intermediates. This inhibition of macrophage antimicrobial activity was due to the production of mycolactone, since the addition of increasing concentrations of purified toxin to cultured macrophages infected with avirulent mycolactone-negative M. ulcerans had a dose-dependent inhibitory effect on phagosome acidification/maturation and nitrite production. The fact that adaptive immunity is initially triggered against M. ulcerans infection suggests that the development of improved vaccines that boost this type of response could lead to an early recall immune response before the build-up of mycolactone compromised T cell differentiation/maintenance. Therefore, we then characterized the immunological mechanisms underlying the transient protection conferred by vaccination with M. bovis BCG or with a mycolactone-negative strain of M. ulcerans, in the footpad murine model of experimental BU. We observed that BCG vaccination was effective in delaying the onset of disease and bacterial proliferation, regardless the experimental dose of M. ulcerans footpad infection. At the site of infection, previous BCG vaccination triggered a strong chronic inflammatory response with increased levels of IFN-γ expression. In the DLN, a faster, heightened and longer T cell and IFN-γ responses were also mounted. However, in vaccinated mice, the eventual increase in bacterial loads and the development of extensive areas of tissue necrosis in footpads resulted in a delayed progression to ulceration and DLN necrosis. Immunization with the mycolactone-negative M. ulcerans strain also significantly delayed the progression of infection by virulent M. ulcerans, but pathology in the DLN and in the footpad also eventually prevailed with emergence of ulceration. The findings presented here help clarify several aspects of host response to M. ulcerans infection and have important implications for the development of new prophylactic and therapeutic strategies against BU.
A úlcera do Buruli (BU) é uma doença tropical negligenciada emergente, causada pela infecção por Mycobacterium ulcerans. A BU é a terceira micobacteriose mais comum a nível mundial, a seguir à tuberculose e à lepra. A BU é caracterizada por lesões cutâneas não ulcerativas que podem evoluir para úlceras necróticas, caso não sejam tratadas numa fase precoce. O desenvolvimento de deformações incapacitantes é muito frequente, tendo a BU um grande impacto socioeconómico nas populações afectadas. O processo fisiopatológico da BU está associado à baixa temperatura de crescimento de M. ulcerans (30-33ºC), que favorece o desenvolvimento de lesões cutâneas; à lenta taxa de crescimento da micobactéria, resultando em lesões progressivas e lentas; e, principalmente, à acção de uma potente exotoxina lipídica, a micolactona, associada às lesões ulcerativas. Têm sido atribuídas propriedades citotóxicas e immunosupressoras à micolactona, tendo a toxina sido associada à inibição das respostas imunológicas, quer localmente, quer a nível sistémico. De facto, a histopatologia da BU tem sido classicamente caracterizada por extensas zonas de necrose, com um reduzido, ou mesmo ausente, infiltrado inflamatório, assim como pela presença numerosos bacilos extracelulares. Esta descrição contrasta com o que é sabido para outras micobactérias patogénicas, como o Mycobacterium tuberculosis ou o Mycobacterium marinum, cujas infecções induzem um recrutamento de um infiltrado inflamatório agudo, seguido de um crónico, para o local de infecção. No entanto, vários estudos sugerem que a imunidade mediada por células e as reacções de hipersensibilidade do tipo retardado estão associadas à protecção contra o M. ulcerans, o que nos levou a considerar que a histopatologia da BU pode não reflectir a totalidade das interacções que ocorrem entre o agente infeccioso e o seu hospedeiro. Assim, efectuou-se uma análise histológica detalhada da almofada plantar de ratinhos infectados com M. ulcerans, ao longo de todo o processo infeccioso. Demonstrou-se que a infecção experimental induziu uma resposta inflamatória cujas características dependem da virulência da estirpe. Uma estirpe micolactona-negativa e avirulenta induziu inicialmente um infiltrado inflamatório neutrofílico agudo, gradualmente substituído por um infiltrado predominantemente mononuclear, crónico, com organizações granulomatosas. Por outro lado, a infecção com estirpes micolactona-positivas estava associada a um influxo inflamatório misto, persistente, composto por neutrófilos e macrófagos, incluindo a presença de bacilos intrafagocíticos. No entanto, posteriormente, ocorria uma destruição das células inflamatórias recrutadas, mediada por apoptose, possivelmente seguida de necrose secundária e lise, o que levou à formação das áreas necróticas, acelulares, típicas, com numerosos bacilos extracelulares libertados após a lise dos fagócitos infectados. No entanto, na periferia das áreas necróticas centrais, bacilos eram persistentemente observados no interior de fagócitos. Em fases mais tardias, as áreas acelulares expandiam, através da invasão progressiva dos tecidos não afectados. Estes resultados levaram-nos a propor que M. ulcerans seria, tal como outras micobactérias patogénicas, um agente patogénico intracelular facultativo, com fases de replicação intramacrofágica e extracelular, uma hipótese mais tarde confirmada no nosso laboratório. Vários estudos têm vindo a sugerir que a micolactona inibe as respostas imunológicas locais, assim como as sistémicas. No entanto, conforme descrito acima, os nossos resultados mostram que numa fase inicial da infecção por M. ulcerans ocorre uma resposta imune celular. Assim, fomos de seguida caracterizar qual o impacto de uma infecção com M. ulcerans no desenvolvimento e manutenção de uma resposta imunológica adaptativa. Neste estudo, mostrámos que a infecção por M. ulcerans leva ao desenvolvimento inicial de uma resposta T no nódulo linfático que drena o local de infecção (DLN), caracterizada pelo aumento de células produtras de IFN-γ. Apesar desta resposta, os ratinhos não controlaram a infecção na almofada plantar, uma vez que a progressão da infecção levou ao aumento da carga bacteriana e a uma diminuição do número de células CD4+ no foco de infecção, contribuindo para uma resposta imunológica ineficaz. Adicionalmente, a apoptose celular associada à destruição do DLN, com a consequente diminuição de células T, também contribui para uma deficiência na manutenção de uma resposta imunológica protectora. Relativamente à eventual ocorrência de imunosupressão sistémica, demonstrou-se que uma infecção avançada com M. ulcerans no tecido subcutâneo não torna os ratinhos mais susceptíveis a uma posterior co-infecção sistémica por Listeria monocytogenes, o que sugere que apesar de ocorrer imunosupressão local e regional, tal não se verifica a nível sistémico. Tendo em consideração os nossos dados, e atendendo às associações anteriores na literatura entre IFN-γ e resistência à BU, estudou-se o papel desta citocina em infecções experimentais por M. ulcerans. Mostrou-se in vivo e in vitro que o IFN-γ era necessário para o controlo das estirpes avirulenta e de virulência intermédia de M. ulcerans. Contudo, para a estirpe de virulência elevada, não foram observadas quaisquer diferenças no controlo bacteriano por parte do hospedeiro, independentemente da presença de IFN-γ. Demonstrou-se que esta ausência de protecção se deve à inibição dos mecanismos antimicrobianos do macrófago activado por IFN-γ, como sejam a maturação do fagosoma, a formação do fagolisosoma e a geração de espécies reactivas de azoto. Verificou-se, ainda, que esta inibição era devida à produção de micolactona, uma vez que a adição de concentrações crescentes da toxina a culturas de macrófagos activados com IFN-γ, e infectados com a estirpe micolactona-negativa, resultou num efeito inibitório dose-dependente na maturação/acidificação do fagosoma e na produção de nitritos. O facto do hospedeiro ter a capacidade de desenvolver, inicialmente, uma resposta imunológica adaptativa contra o M. ulcerans, sugere que uma vacina com as características apropriadas pudesse levar a uma resposta imunológica secundária precoce, antes da eventual acumulação de micolactona comprometer a diferenciação e manutenção de células T protectoras. Neste estudo, caracterizámos os mecanismos imunológicos por detrás da protecção transiente conferida pela vacinação por M. bovis BCG ou por uma estirpe micolactona-negativa de M. ulcerans. Observou-se que a vacinação por BCG atrasou o início da patologia e da proliferação bacteriana associada, independentemente da dose de inicial de M. ulcerans. No local de infecção, em ratinhos vacinados com BCG, observou-se uma resposta inflamatória crónica intensa, com níveis elevados de IFN-γ. No DLN, observou-se uma resposta mediada por células T e por IFN-γ que era mais rápida, mais intensa e mais prolongada do que em ratinhos não imunizados. Contudo, mesmo nos ratinhos vacinados, o aumento da carga bacteriana e o desenvolvimento de extensas áreas de necrose no local de infecção resultou na progressão para ulceração e na destruição do próprio DLN. A imunização com uma estirpe micolactona-negativa de M. ulcerans também atrasou a progressão da infecção com a estirpe virulenta de M. ulcerans, mas o aparecimento de patologia no DLN e na almofada plantar prevaleceu, com a consequente emergência de ulceração. Em suma, os resultados apresentados nesta dissertação contribuem para clarificar vários aspectos da resposta imunológica do hospedeiro contra o M. ulcerans, com implicações para o desenho de novas estratégias profiláticas e terapêuticas.
Fundação para a Ciência e a Tecnologia (FCT)
Grant SFRH/BD/15911/2005.

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.

Yes
Background Buruli ulcer (BU) is an infectious disease caused by Mycobacterium ulcerans and considered the third most prevalent mycobacterial disease in humans. Secondary bacterial infections in open BU lesions are the main cause of pain, delayed healing and systemic illness, resulting in prolonged hospital stay. Thus, understanding the diversity of bacteria, termed the microbiome, in these open lesions is important for proper treatment. However, adequately studying the human microbiome in a clinical setting can prove difficult when investigating a neglected tropical skin disease due to its rarity and the setting. Methodology/Principal findings Using 16S rRNA sequencing, we determined the microbial composition of 5 BU lesions, 3 non-BU lesions and 3 healthy skin samples. Although no significant differences in diversity were found between BU and non-BU lesions, the former were characterized by an increase of Bacteroidetes compared to the non-BU wounds and the BU lesions also contained significantly more obligate anaerobes. With this molecular-based study, we were also able to detect bacteria that were missed by culture-based methods in previous BU studies. Conclusions/Significance Our study suggests that BU may lead to changes in the skin bacterial community within the lesions. However, in order to determine if such changes hold true across all BU cases and are either a cause or consequence of a specific wound environment, further microbiome studies are necessary. Such skin microbiome analysis requires large sample sizes and lesions from the same body site in many patients, both of which can be difficult for a rare disease. Our study proposes a pipeline for such studies and highlights several drawbacks that must be considered if microbiome analysis is to be utilized for neglected tropical diseases.

No
Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans, have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir.
National Health and Medical Research Council of Australia (NHMRC), an NHMRC Senior Research Fellowship to T.P.S. (grant GNT1105525); and an NHMRC Practitioner Fellowship to B.P.H. (GNT1105905). A.H.B. was supported by an Australian Postgraduate Award Ph.D. scholarship.

Tese de Doutoramento - Doutoramento em Medicina
Buruli ulcer (BU) is an infectious disease found in tropical regions of Africa, America, Asia, and Australia. Most of the cases are reported in West Africa and BU is considered a neglected tropical disease by the World Health Organization (WHO). This necrotising skin infection is caused by Mycobacterium ulcerans that secretes the exotoxin mycolactone as its main virulence factor. There is emerging evidence for a major role of genetic factors contributing to either BU development or disease progression. Indeed, not all individuals, even after sustained exposure to M. ulcerans in endemic wetlands, develop BU and when patients present these lesions, a wide spectrum of clinical manifestations can be observed, including non-ulcerative and ulcerative lesions. In addition, spontaneous healing of BU lesions has been previously reported, as well as clustering of cases within specific families. In that sense, it is clear that, to further evaluate the influence of host genetic variability in BU susceptibility and disease severity, new tools are needed. Therefore, we have built a bank of biological samples from a cohort of fully characterized BU patients from Benin, West Africa, as well as from a cohort of unrelated controls matched for age, gender and water contact habits. With this biological bank, the impact of several immune-related polymorphisms can be evaluated, not only for BU susceptibility or resistance, but also for differences in disease phenotypes. Since a precise BU phenotype definition is critical to proceed to any genetic study, we first planned a clinical-based study, retrospectively examining records of patients in Benin, who had laboratory confirmed M. ulcerans infection. We started by evaluating the relationship between the type of lesion presentation and the time delay to seek medical attention, defined as the time between the first remembered signs of disease and attendance for medical care. Of the 476 BU patients studied, 32% had a non-ulcerated form of skin lesion (nodule, oedema or plaque), while 67% presented ulcerated lesions. Only 1% of the patients had disease with bone involvement. In the non-ulcerated forms, the average time delay between symptoms and medical care was 32.5 days, while for ulcerated forms a statistically significant different value was observed (60 days), confirming disease progression from non-ulcerated to ulcerated skin lesions. In addition to the classical forms of BU presentation, the WHO has recognized three levels of increasing severity of lesions. In our cohort, we registered that 66% of the patients presented WHO categories 1 and 2, while 34% presented severe WHO category 3 lesions. Interestingly, time delay in seeking medical assistance did not contribute to the progression of less severe to more severe lesions, since the median time delay for WHO category 1 and 2 lesions (60 days) and category 3 severe lesions (60 days) was the same. The same behaviour was observed restricting analysis to multi-focal lesions and larger lesions, with a diameter superior to 15cm. These observations indicate that, rather than lesions becoming progressively more severe over time, severe lesions present a stable form and therefore should represent a separate phenotype of disease. These results also suggest that host genetic variability can have a strong contribution to the different disease presentations. Taking into consideration the knowledge obtained with this retrospective analysis, we took advantage of the population-based biological bank, composed of DNA samples from 208 BU patients and 300 unrelated healthy individuals. In the first immunogenetic study, we focused on Tumour Necrosis Factor-alpha (TNF-α), a relevant immune-mediator involved in the innate branch of the immune response, known to play a key role in controlling M. ulcerans infection in animal models. Based upon the functional role of the TNFA [-308 Glow → Ahigh (rs1800629)] single nucleotide polymorphism (SNP) on protein production levels, we evaluated the genetic frequency of each allelic variant in cases and controls. We observed that -308 A allele was associated with increased risk susceptibility to BU, considering a recessive model of transmission. This data further reinforces the relevance of TNF-α in BU development, and is in line with data from a previous meta-analysis showing an increased susceptibility to Mycobacterium tuberculosis in individuals carrying this SNP with impact on promoter activity of TNFA. In our second immunogenetic-based study, we evaluated genetic variants in Nucleotidebinding Oligomerization Domain-containing protein 2 ( NOD2), Parkinson disease protein 2 ( PARK2) and Autophagy-related protein 16-1 ( ATG16L1) genes and their impact in susceptibility to BU, given the relevance of autophagy in the host response to mycobacteria. Our data show that the rs1333955 SNP in PARK2 is significantly associated with increased susceptibility to BU. In addition, both the rs9302752 and rs2066842 SNPs in NOD2 genes significantly increased the predisposition of patients to develop category 3 lesions, respectively, whereas the rs2241880 SNP in ATG16L1 was found to significantly protect patients from presenting the ulcer phenotype. In this case, our findings indicate that specific genetic variants in autophagyrelated genes influence susceptibility to BU and its progression to severe phenotypes. Although further studies are needed, our data suggest that disturbances on microtubules and dynein constitution (integrative part of the cytoskeleton of the autophagosome), a process that is known to be affected by mycolactone, might potentially impair the autophagy process and impact the risk and progression of M. ulcerans infection. Overall, our results reinforce the relevance of host genetic variability in the susceptibility/resistance to BU, as well as in the evolution of lesions to distinct phenotypes. Next steps are crucial in functionally validating the relevance of the associated variants on the mechanisms of host susceptibility or resistance to BU, through functional studies with particular emphasis on the immunological function.
A úlcera do Buruli (UB) é uma doença infeciosa das regiões tropicais de África, América, Ásia e Austrália. A maioria dos casos ocorre na África Ocidental e esta doença é classificada pela Organização Mundial de Saúde (OMS) como uma doença tropical negligenciada. Esta infeção cutânea necrotizante é causada por Mycobacterium ulcerans, que tem como principal fator de virulência a exotoxina micolactona. Evidências recentes apontam para um papel da variabilidade genética do hospedeiro como contributo relevante para a suscetibilidade na aquisição da UB, bem como para a sua progressão. De fato, nem todos os indivíduos igualmente expostos a M. ulcerans nas zonas húmidas endémicas desenvolvem infeção e os doentes que desenvolvem a doença podem apresentar um largo espectro de manifestações clínicas, incluindo lesões não ulcerativas e ulcerativas. Adicionalmente, pode ocorrer resolução espontânea da infeção e está descrito uma concentração de casos de UB em determinadas famílias. Neste sentido, fica evidente que, para avaliar a influência da variabilidade genética do hospedeiro na suscetibilidade e/ou agressividade da UB, são necessárias novas abordagens. Assim, construímos um banco de amostras biológicas de doentes do Benim, África Ocidental, com caracterização clínico-epidemiológica completa, em associação a uma coorte de controlos endémicos não relacionados, ajustada para idade e género e mantendo contacto e atividades semelhantes com os meios aquáticos. Com esta ferramenta, pode-se avaliar o impacto de diversos polimorfismos genéticos, na sua maioria relacionados com a imunidade inata, não só na suscetibilidade ou resistência à UB, mas também nos distintos fenótipos das manifestações clínicas. Sendo fundamental uma definição clara de fenótipos associados à UB, primeiro planeámos um estudo clínico retrospetivo com pacientes do Benim com diagnóstico de infeção confirmado laboratorialmente. Começámos por avaliar a relação entre a forma clínica de UB e o tempo decorrido entre os primeiros sinais recordados pelos pacientes e a observação clínica (e terapêutica) inicial. Dos 476 pacientes selecionados, 32% apresentavam formas não ulceradas (nódulo, edema ou placa), 67% apresentavam úlcera e apenas 1% apresentava osteomielite. O grupo de doentes com formas não ulceradas teve uma mediana de 32.5 dias de atraso entre os primeiros sintomas e a procura de cuidados médicos, enquanto que o grupo que apresentava úlceras registou um período de atraso significativamente distinto, com uma mediana de 60 dias. Estes dados confirmam que a doença, tipicamente, progride de formas não ulcerativas para úlceras cutâneas. Para além das lesões clássicas de UB, a OMS reconheceu 3 níveis de severidade crescente das lesões de UB. No presente estudo, foram registadas lesões de categoria 1 e 2 em 66% dos doentes, enquanto que 34% dos doentes apresentavam lesões severas de categoria 3. Numa análise subsequente, mostrou-se que o atraso no tempo de procura de cuidados médicos não foi fator contributivo para apresentação de formas mais severas da doença, uma vez que a mediana do atraso para as lesões menos severas de categoria 1 e 2 (60 dias) e para as lesões severas de categoria 3 (60 dias) era a mesma. O mesmo comportamento foi observado quando a análise foi restrita a lesões múltiplas ou lesões de maior diâmetro, acima de 15cm. Mostrámos, assim, que o tempo de progressão natural da doença não determina formas clínicas mais agressivas de UB, sustentando a hipótese de que a variabilidade genética do hospedeiro pode contribuir para manifestações clinicas distintas (fenótipos) da doença. Tendo em conta o conhecimento adquirido com a análise retrospetiva, recorremos ao banco biológico constituído por amostras de DNA de 208 pacientes de UB e 300 controlos saudáveis. No primeiro estudo imunogenético desta tese, investigámos o papel do Fator de Necrose Tumoral-alfa (TNF-α), dado tratar-se de um imunomediador relevante, previamente implicado na resposta inata e com um papel protetor na infeção por M. ulcerans em modelos animais. Com base na alteração funcional resultante do polimorfismo TNFA [-308 Glow → Ahigh (rs1800629)], com impacto nos níveis de TNF-α, avaliámos a frequência alélica da variante nos casos e nos controlos. Observámos que o alelo A estava associado a aumento do risco de suscetibilidade para UB, de acordo com um modelo recessivo de transmissão alélica. Este achado reforça o papel do TNF-α no desenvolvimento da UB e está de acordo com o verificado recentemente numa meta-análise que associa o mesmo alelo ao aumento de suscetibilidade de infeção pelo Mycobacterium tuberculosis. Num segundo estudo imunogenético, avaliámos variantes genéticas nos genes Nucleotide-binding Oligomerization Domain-containing protein 2 ( NOD2), Parkinson disease protein 2 ( PARK2) e Autophagy-related protein 16-1 ( ATG16L1), uma vez que as proteínas que codificam fazem parte, de uma forma complementar, do processo de autofagia. De facto, a maquinaria da autofagia tem vindo a ser implicada, de forma crescente, no controlo e eliminação intracelular de micobactérias, levando-se a hipótese de que variantes genéticas/funcionais tenham impacto na suscetibilidade à UB. Utilizando as mesmas coortes, demonstrámos que o polimorfismo rs1333955 no PARK2 está significativamente associado a um aumento da suscetibilidade a UB. Associámos ainda os polimorfismos rs9302752 e rs2066482 do gene NOD2 ao aumento de predisposição para desenvolvimento de fenótipos mais agressivos de UB (categoria 3 da OMS), respetivamente. Mostrámos também que o polimorfismo rs2241880 do gene ATG16L1 protege os pacientes de desenvolverem o fenótipo de úlcera. Desta forma, demonstrámos que variantes específicas em genes relacionados com o processo de autofagia influenciam, quer a suscetibilidade para o desenvolvimento de UB, quer a progressão para fenótipos distintos da doença. Apesar de serem necessários mais estudos funcionais, os nossos resultados sugerem que interferências na constituição dos microtúbulos e da dineína (parte integrante do citoesqueleto dos auto-fagossomas), processo que é sabido ser afetado pela micolactona, podem ter impacto no risco de infeção e na progressão do processo infecioso por M. ulcerans. Globalmente, os nossos resultados reforçam a importância do papel da variabilidade genética do hospedeiro na suscetibilidade/resistência à UB, assim como na determinação da sua evolução para os diferentes fenótipos característicos desta doença negligenciada. Os próximos passos para validar a relevância destes achados, no que respeita aos mecanismos da suscetibilidade/resistência do hospedeiro à UB, passarão por estudos funcionais, com ênfase na função imunológica.
The work presented in this thesis was performed in the Microbiology and Infection Research Domain of the Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal (ICVS/3B`s – PT Government Associate Laboratory, Braga/Guimarães, Portugal) and in Centre de Dépistage et de Traitement de l’Ulcère de Buruli (CDTUB) in Allada, Benin. The financial support was given by the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement N° 241500 (BuruliVac); by Infect-ERA (BU_SPONT_HEAL); by Fundação Calouste Gulbenkian under the grant Proc.N° 94776 LJ; from the Fundação para a Ciência e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte); from the Quadro de Referência Estratégico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estratégico – LA 26 – 2013–2014 (PEst- C/SAU/LA0026/2013).