Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease

Abstract

Background: Mycobacterium ulcerans (M. ulcerans) causes a devastating necrotising infection of skin tissue leading to progressive ulceration. M. ulcerans is the only human pathogen that secretes mycolactone, a polyketide molecule with potent cytotoxic and immunomodulatory properties. These unique features make mycolactone an attractive biomarker for M. ulcerans disease. We sought to measure the concentration of mycolactone within lesions of patients with Buruli ulcer before, during and after antibiotic treatment to evaluate its association with the clinical and bacteriological response to therapy.

Methods: Biopsies of M. ulcerans infected skin lesions were obtained from patients before, during and after antibiotic therapy. Lipids were extracted from the biopsies and concentration of mycolactone was assayed by mass spectrometry and a cytotoxicity assay and correlated with clinical and bacteriological response to therapy.

Results: Baseline concentration of mycolactone measured by mass spectrometry predicted time to complete healing of small nodules and ulcers. Even though intra-lesional concentrations of mycolactone declined with antibiotic treatment, the toxin was still present after antibiotic treatment for 6 weeks and also 4 weeks after the end of treatment for 8 weeks in a subgroup of patients with slowly healing lesions. Additionally viable bacilli were detected in a proportion of these slowly healing lesions during and after treatment.

Conclusions: Our findings indicate that baseline intra-lesional mycolactone concentration and its kinetics with antibiotic therapy are important prognostic determinants of clinical and bacteriological response to antibiotic treatment for Mycobacterium ulcerans disease. Mycolactone may be a useful biomarker with potential utility in optimising antibiotic therapy.

Figure 1

Mycolactone concentration in punch biopsies of untreated M. ulcerans disease lesions before antibiotic treatment. (A) Mycolactone concentration in ASL measured by cytotoxicity assay using synthetic mycolactone A/B generated calibration curves. Horizontal lines represent medians and each dot represents one lesion. (B) Mycolactone concentration in punch biopsies of untreated M. ulcerans disease lesions before antibiotic treatment. Mycolactone A/B concentration in ASL measured by multiple reaction monitoring (MRM) methodology with liquid chromatographic separation coupled to a tandem mass spectrometer. Horizontal lines represent medians and each dot represents one lesion.

Figure 2

Tissue mycolactone concentration in serial biopsies during and after antibiotic treatment. Antibiotic treatment was started at week 0 and completed at week 8. Biopsies were taken at weeks 0 in all patients and at week 6 and/or 12 when it was clinically indicated. (A) Mycolactone concentration in ASL measured by cytotoxicity assay using synthetic mycolactone A/B generated calibration curves. (B) Tissue mycolactone concentration in serial biopsies during and after antibiotic treatment. Mycolactone A/B concentration in ASL measured by multiple reaction monitoring (MRM) methodology with liquid chromatographic separation coupled to a tandem mass spectrometer.

Figure 3

Correlation between baseline mycolactone A/B concentration measured using mass spectrometry and time to complete healing. (A) All categories and forms of buruli lesion. (B) All category I lesions. (C) Nodular lesions. (D) Category I ulcers (less than 5 cm in widest diameter).