Clearance of viable Mycobacterium ulcerans from Buruli ulcer lesions during antibiotic treatment as determined by combined 16S rRNA reverse transcriptase /IS 2404 qPCR assay

Abstract

Introduction: Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing.

Methods: Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms.

Results: Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4.

Conclusions: Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Kaplan-Meier analysis of M. ulcerans 16S rRNA in Buruli patients on antibiotic treatment.

Blue line: Median time (weeks) for detection of M. ulcerans 16S rRNA. Red line: Proportion of patients with positive M. ulcerans 16S rRNA at week 4.

Fig 2. Comparison of baseline M. ulcerans IS2404 in Buruli ulcer patients with a positive or negative 16S rRNA result at week 4.

Fig 3. Survival curve for time to healing in Buruli patients with a negative or positive M. ulcerans 16S rRNA at week 4.

Purple lines: Median time to healing.

Fig 4. Rate of wound healing at week 4 in Buruli patients with a negative or positive M. ulcerans 16S rRNA.

Rate of healing was highest in patients where M. ulcerans 16S rRNA was negative at baseline or 4 weeks after starting antibiotic treatment. The rate of healing at week 4 (ROH) was computed in millimeters per week by subtracting the mean diameter of the lesion at week 4 from that at week 0 and dividing this result by 4.