Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay

Buruli ulcer disease (BUD) caused by Mycobacterium ulcerans involves the skin and soft tissue. If left untreated, extensive destruction of tissue followed by scarring and contractures may lead to severe functional limitations. Following the introduction of standardized antimycobacterial chemotherapy with rifampicin and streptomycin, recurrence rates of less than 2% were reported. However, treatment failures occur and a variety of secondary lesions necessitating customized clinical management strategies have been reported. True recurrences by definition occur more than three months after completion of antibiotic treatment, are characterised by the presence of viable bacilli, and require a second course of antibiotics. ‘‘Non-healers’’ may harbour viable, possibly drug-resistant M. ulcerans strains and may benefit from surgical intervention. Early-onset immune-mediated paradoxical reactions emerging during or shortly after treatment do not contain viable bacilli and may heal under conventional wound care and/or minor surgery; lateonset secondary lesions presumably attributable to secondary infection foci may clear spontaneously through enhanced immune responses primed by initial treatment. None of the current diagnostic techniques is applicable to rapidly address the pivotal question of the presence of viable bacilli in non-healers and patients with secondary BUD lesions, and optimal time points for collection of follow-up samples have not yet been investigated. Therefore, to date treatment monitoring is mainly based on clinical observation [1– 5]. Reverse transcriptase assays targeting 16S rRNA and mRNA were successfully applied for the rapid detection of viable mycobacteria in clinical samples from patients with tuberculosis and leprosy [6,7]. To employ this technique for classification of BUD lesions and monitoring of treatment success we developed a M. ulcerans–specific RNA-based viability assay combining a 16S rRNA reverse transcriptase real-time PCR (RT-qPCR) to determine bacterial viability with an IS2404 quantitative real-time PCR (qPCR) for increased specificity and simultaneous quantification of bacilli.
An article by PLOS Neglected Tropical Diseases
PLoS Negl Trop Dis 6(8): e1756. doi:10.1371/journal.pntd.0001756