Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay
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Date
2012-08-28
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Publisher
PLOS Neglected Tropical Diseases
Abstract
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.
Description
An article by PLOS Neglected Tropical Diseases
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Citation
PLoS Negl Trop Dis 6(8): e1756. doi:10.1371/journal.pntd.0001756