The kinetics of mycolactone in relation to the microbiological, clinical and immunological responses to antibiotic therapy for mycobacterium ulcerans disease.

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Background: Mycobacterium ulcerans is the causative agent for a chronic necrotising skin infection called Buruli ulcer. Pathology of the disease is closely linked with the elaboration of a unique lipid toxin, mycolactone, which has potent cytotoxic and immunomodulatory properties. In this study, assays were developed to detect and quantify mycolactone concentrations in tissues during curative antibiotic therapy in mice and in humans to understand its dynamics in pathogenesis and to explore its potential as a biomarker for diagnosis and monitoring of patients with Buruli ulcer disease on antibiotic therapy. The currently recommended antibiotic regimen for the management of Buruli ulcer is a combination of daily intramuscular injections of streptomycin and oral rifampicin for 8 weeks (RS8). This regimen was compared with streptomycin/rifampicin for 2 weeks followed by clarithromycin/rifampicin (RS2RC6) for 6 weeks in patients to determine the clinical and bacteriological effectiveness in a pilot study. Methods: Biopsies were obtained from infected human skin tissues and BALB/c mouse hind footpads before, during and after 8-weeks of rifampicin-containing combination antibiotic therapy. Lipids were extracted from tissues using organic solvents, mycolactone concentrations were measured using a cytotoxicity assay and mass spectrometry. Trends in mycolactone concentrations and clinical, bacteriological and immuno-histopathological responses were determined. Concentrations of cytokines in supernates of whole blood assays in humans or murine splenocytes after stimulation with mycobacterial antigens/T-cell mitogens were measured using ELISA. iv Results: Eighty-three patients with confirmed Buruli ulcer were randomized to RS8 or RS2RC6 and monitored for recurrence free-healing. Bacterial load in tissue samples before and after treatment for 6 and 12 weeks was monitored in samples obtained by 4mm punch biopsy by semi-quantitative culture. There was no difference in using RS8 or RS2RC6 with respect to healing rate or the proportion healed in each group after 4, 8, 12, 16, 20, 24 and up to 52 weeks. The success rate was 93% in each group and there was no recurrence after 12-month follow-up. There was no difference in the number of bacteria cultured at the different time points for the two regimens. Mycolactone was detectable in 92% and 77% of human samples (n=80) using cytotoxicity assays and mass spectrometry respectively. Antibiotic therapy was associated with a decline in tissue concentration of mycolactone in both human and murine-infected tissues which was paralleled by resolution of clinical lesions, reductions in bacteriological counts and restoration of local and systemic immune responses. Discussions/Conclusions: This study shows that mycolactone concentrations in tissues is closely associated with the presence of M. ulcerans and provides useful proof-of-concept data that mycolactone detection could potentially be used to monitor response to antibiotic therapy as well as for diagnosis of Buruli ulcer disease. The findings from the pilot study indicate that rifampicin combined with clarithromycin can replace rifampicin and streptomycin for the continuation phase after rifampicin-streptomycin treatment for 2 weeks without any apparent loss of efficacy. The implication is that a controlled trial of fully oral therapy using rifampicin and clarithromycin for 8 weeks (RC8) is justified.
A thesis submitted to the Department of Microbiology, Kwame Nkrumah University of Science and Technology in partial fulfilment of the requirements for the degree of Doctor of Philosophy,