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

IntroductionBuruli 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.MethodsFine 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.ResultsOut 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.ConclusionsCurrent 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.

Challenges associated with the treatment of Buruli ulcer

“…A recent publication by Sarpon‐Duah et al. indicated high bacterial load at baseline contributes to persistent infection leading to slow healing . In TB, subpopulations consisting of dormant or semi‐dormant, antibiotic tolerant persisters survive longest during chemotherapy and are difficult to kill even with new antibacterial drug .…”

IFN-γ and IL-5 whole blood response directed against mycolactone polyketide synthase domains in patients withMycobacterium ulceransinfection

“…For patients with Buruli ulcer, basic demographic data including age, sex, lesion form, lesion site and category were recorded on standard WHO BU01 forms ( WHO, 2018 ). Swabs from ulcers and fine needle aspirates from pre-ulcerative lesions were obtained for M. ulcerans IS2404 PCR and also combined with the M. ulcerans 16S rRNA as previously described ( Phillips et al, 2009 ; Sarpong-Duah et al, 2017 ; Sarfo et al, 2010 ). Patients were treated with combination oral rifampicin 10 mg/kg and intramuscular streptomycin 15 mg/kg daily for 8 weeks or oral clarithromycin.…”

“…Fine needle aspirates and swab samples were transported from study site to the KCCR stabilized in 500 μl RNA protect (Qiagen, Manchester, UK) for the M. ulcerans combined 16S rRNA reverse transcriptase/IS2404 qPCR viability assay as described elsewhere ( Sarpong-Duah et al, 2017 ). Briefly, whole transcriptome RNA and whole genome DNA were extracted from the same clinical sample.…”

“…Briefly, whole transcriptome RNA and whole genome DNA were extracted from the same clinical sample. The RNA and DNA isolation was carried out using the AllPrep DNA/RNA Micro kit (Qiagen, Manchester, UK) as previously described ( Beissner et al, 2012 ; Sarpong-Duah et al, 2017 ). A total of 12 μl RNA extracts were immediately reverse transcribed ( Sarpong-Duah et al, 2017 ).…”

Buruli Ulcer in Animals and Experimental Infection Models

“…For CFU determination the infected tissue is usually ground up, sometimes de-contaminated to avoid overgrowth with faster growing microorganisms and further processed [22,52]. Very recently an optimized procedure for extraction and quantification of bacterial RNA from infected mouse tissue was published, which provides a surrogate marker for viability of the bacteria [66,67]. Parallel to measuring foot pad thickness, a grading system can be applied to estimate the severity of the disease (Fig.…”