Bacteriological and immunological studies towards effective control of "Mycobacterium ulcerans" disease (Buruli ulcer)

Abstract

After tuberculosis and leprosy, Buruli ulcer (BU) which is caused by Mycobacterium ulcerans, is the most common mycobacterial infection in immuno-competent humans. Since the 1980s BU has gained significant public health importance in the tropics especially in West Africa, including Ghana. The establishment of control measures is hampered as a result of the scarcity of understanding of many features of the disease. Priority areas for research defined by WHO include: understanding the mode of transmission, development of simpler methods for early diagnosis, development of effective antibiotic treatment, and the understanding of protective immune responses to support vaccine development. The availability of M. ulcerans isolates from endemic areas is necessary for detailed transmission studies and the analysis of efficacy of antibiotics for the treatment of BU. However, cultivation of M. ulcerans from clinical specimens is burdensome; reported recovery rates are as low as 20%. We evaluated four different decontamination methods and one non-decontamination procedure in combination with four egg-based media for the primary isolation of M. ulcerans from tissue specimens excised from BU lesions. Oxalic acid decontamination and culture on LJ medium supplemented with glycerol was the most efficient procedure and achieved a recovery rate of 75.6%. The success of cultivation depended also on a good sampling procedure. The use of the optimised cultivation method has allowed the production of a large isolate collection. For efficient case management and confirmation of epidemiological data, it is necessary to reconfirm clinical diagnosis by laboratory procedures. We used culture together with PCR and direct AFB staining to establish a system of reconfirming cases clinically diagnosed at the Amasaman Health Centre, Ghana. All three methods showed a comparable sensitivity and the laboratory analysis demonstrated a high accuracy of clinical judgment by an experienced clinician. Current recommendation by the WHO requires that BU patients be treated with a combination of rifampicin and streptomycin for 8 weeks before surgical excision. In many infectious diseases, the development of drug resistance has a serious impact on patient management. It is therefore essential to monitor the drug susceptibility of M. ulcerans. We analysed the susceptibility of 28 isolates to rifampicin, streptomycin isoniazid and ethambutol and identified both streptomycin and rifampicin resistant strains in Ghana. Findings from this study call for reconsideration of the current treatment guidelines. Currently, micro-epidemiological studies aiming to reveal transmission chains cannot be done in BU. This is due to the low degree of genetic polymorphism in M. ulcerans revealed by routinely used genetic fingerprinting procedures. We used VNTR typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1 to detect genetic diversity in isolates from Ghana. Analysis revealed three different genotypes in isolates from Ghana, demonstrating for the first time genetic diversity among M. ulcerans isolates in an African country. Ex vivo ELISpot analysis of IFN-γ secreting cells was carried out by stimulating PBMCs from BU patients with PPD, IPP and IRIV. Data from the study demonstrated for the first time that M. ulcerans infection-associated systemic reduction in IFN-γ responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. We also showed that the immune suppression reversed after surgical treatment and that the suppression is not related to reduction in IL-12 secretions. This indicates that the observed systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria. In a longitudinal study, we compared recovery of immediate effector function of Vγ2Vδ2 T cells in surgically treated BU patients to that of TB patients under chemotherapy. At the time of diagnosis, systemic production of IFN-γ after IPP stimulation was suppressed in both disease states but reverses after treatment. Restoration of Vγ2Vδ2 reactivity was slow such that an optimum response was not yet achieved by two months in both populations. Our result demonstrates that immunosuppression in BU may not be caused by the terpenoid toxin of M. ulcerans (mycolactone) alone.