Development of new molecular genetic tools to study "Mycobacterium ulcerans" infection (Buruli ulcer)

Abstract

Buruli ulcer is an infectious disease caused by an environmental pathogen, Mycobacterium ulcerans, which is the third major mycobacterial pathogen of man, after M. tuberculosis and M. leprae. Since 1980, dramatic increases in the incidence of Buruli ulcer have been reported from West African countries, sometimes associated with man-made environmental changes. After the first international conference on Buruli ulcer in 1998 (Yamoussoukro meeting), attention has been drawn to the severity of this neglected disease and to its many poorly understood features. Since then new initiatives have been undertaken to promote control and research efforts. Within the framework of WHO identified research priorities, the present PhD project focused on the development of new molecular genetic tools to investigate M. ulcerans epidemiology and pathology. Apart from the association of Buruli ulcer with swampy environments, little is known about risk factors, environmental reservoirs and pathways of transmission. One factor that impairs research on these issues is the lack of suitable fine typing methods to track different M. ulcerans subclones and their spreading within a community. The comprehension of the population structure itself and of the mechanisms leading to genetic variability also suffers from this lack of tools. For this reason, we developed a new plasmid-based microarray approach, which was used to perform a comparative genomic analysis of 30 M. ulcerans strains, from different geographical origins. Fifteen large sequence polymorphisms were identified affecting genes of all major functional categories. Results obtained with this prototype microarray demonstrated that insertional/deletional events, often associated with insertion sequences are the most important mechanisms of genetic diversification in M. ulcerans. Analysis of strain diversity with a larger microarray should represent a suitable tool for micro-epidemiological studies. Within the framework of a Buruli ulcer survey in Cameroon, an optimized diagnostic PCR was developed. The method, operating on genetic material extracted directly from swab samples, demonstrated the usefulness of such highly sensitive technique for epidemiological studies. Neglected Buruli ulcer foci have been rediscovered and an association between Buruli ulcer cases and slow flowing water basins have been reconfirmed. A quantitative PCR specific for M. ulcerans DNA, the IS2404 real-time PCR, was developed with the aim to gain insights into the pathology of the disease. The very high sensitivity and specificity of the method allowed the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions, and its comparison with histopathological changes. Although the heaviest mycobacterial burden was detected in the central foci of the lesions, we could measure significant amounts of mycobacterial DNA and microcolonies in samples from peripheral regions and occasionally in healthy appearing excised tissue margins. Additional peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing. Even when granulomas provided evidence for the development of cell-mediated immunity, development of satellite lesions by contiguous spreading was not completely prevented. The technique offers also the potential to predict recurrences: in one case we could demonstrate that a relatively small number of mycobacteria that have spread into healthy appearing tissue can lead to the development of a recrudescence. These data altogether support the concept that wider surgical excision improves the chance of healing of Buruli ulcer. The application of our approach for assessing the mycobacterial burden in excision margins, combined with long term follow-up of patients, should help to improve current guidelines for surgical treatment of Buruli ulcer. It is becoming more and more evident that mycobacterial spreading can occur even at distant sites from the original primary ulcer, producing so called “metastatic lesions”. The contribution of re-activation versus re-infection is not clear, neither is the mode of spreading into the body known. In the case of a HIV+ patient, we could report the insurgence of multifocal aggressive lesions leading to osteomyelitis. The time span interposing between the primary Buruli manifestations and the recurrence at the new sites, together with the physical distance of the patient from the endemic area, is such to argue about eventual persistence of M. ulcerans in an immunocompromised individual.