The kinetics of mycolactone in relation to the microbiological, clinical and immunological responses to antibiotic therapy for mycobacterium ulcerans disease.
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Date
2014
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Abstract
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.
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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.
Description
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,