Development of better methods for the laboratory diagnosis of Mycobacterium ulcerans disease (Buruli ulcer) and search for environmental reservoirs of Mycobacterium ulcerans.

摘要

Mycobacterium ulcerans causes cutaneous and bone lesions known as Buruli ulcer (BU). BU is largely a problem of the poor in remote rural areas near wetlands in warm regions. After tuberculosis and leprosy, it is the third most common mycobacterial disease in humans.;BU diagnosis is most often made in health centers lacking suitable laboratory facilities on clinical grounds. A specimen is then transported to a reference laboratory for microbiological confirmation. We developed a semisolid transport medium which allowed obtaining positive cultures even from clinical specimens kept for more than two months. While microbiological tests on ulcerative lesions can be performed on swabs, for nonulcerative forms tissue fragments are needed, requiring local anesthesia. Fine-needle aspiration (FNA) would seem to offer an alternative technique since it is minimally invasive. We compared the sensitivities of microbiological tests on FNA and tissue specimens from nonulcerative BU lesions, and they were not significantly different. To improve the diagnosis of BU, we therefore recommend (i) to further explore FNA as a promising alternative to invasive punch biopsies in nonulcerative lesions; and (ii) to process clinical specimens as much as possible locally since any delay reduces the positivity rate of laboratory tests especially of primary culture. The environmental reservoir and mode(s) of transmission of M. ulcerans are largely unknown. M. ulcerans DNA has been found in aquatic organisms obtained from stagnant water in BU endemic areas. M. ulcerans may persist in the environment associated with other organisms such as free-living amoebae that protect the bacilli. We explored the role of fish and free-living amoebae as potential reservoirs. Fish found positive for M. ulcerans DNA all appear to feed on insects or plankton and are believed to concentrate M. ulcerans from this usual food source. A greater proportion of water bodies in areas of high BU endemicity contained amoebae than in areas of low endemicity. We demonstrated intracellular bacilli soon after experimental infection of Acanthamoeba polyphaga by mycolactone-producing or non-producing M. ulcerans strains and no cytotoxic effects. Moreover, M. ulcerans persisted and survived within A. polyphaga. The present work has shed some light on the involvement of fish, free-living amoebae, and biofilms, and indirectly of plankton and insects.