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

Beißner, Marcus; Symank, Dominik; Phillips, Richard Odame; Amoako, Yaw Ampem; Awua-Boateng, Nana-Yaa; Jansson, Moritz; Huber, Kristina L.; Herbinger, Karl‐Heinz; Battke, Florian; Löscher, Thomas; Adjei, Ohene; Bretzel, Gisela

doi:10.1371/journal.pntd.0001756

Cited by 41 publications

(63 citation statements)

References 11 publications

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“…Standard IS 2404 PCR was performed according to the protocol described by Stinear et al [15], [17]. IS 2404 qPCR was performed as recently described using a BioRad CFX96 real-time PCR detection system [27], [43]. All PCR assays included negative extraction controls, positive, negative (no template) and inhibition controls.…”

Section: Methodsmentioning

confidence: 99%

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

et al. 2013

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BackgroundIn a previous study PCR analysis of clinical samples from suspected cases of Buruli ulcer disease (BUD) from Togo and external quality assurance (EQA) for local microscopy were conducted at an external reference laboratory in Germany. The relatively poor performance of local microscopy as well as effort and time associated with shipment of PCR samples necessitated the implementation of stringent EQA measures and availability of local laboratory capacity. This study describes the approach to implementation of a national BUD reference laboratory in Togo.MethodologyLarge scale outreach activities accompanied by regular training programs for health care professionals were conducted in the regions “Maritime” and “Central,” standard operating procedures defined all processes in participating laboratories (regional, national and external reference laboratories) as well as the interaction between laboratories and partners in the field. Microscopy was conducted at regional level and slides were subjected to EQA at national and external reference laboratories. For PCR analysis, sample pairs were collected and subjected to a dry-reagent-based IS2404-PCR (DRB-PCR) at national level and standard IS2404 PCR followed by IS2404 qPCR analysis of negative samples at the external reference laboratory.Principal FindingsThe inter-laboratory concordance rates for microscopy ranged from 89% to 94%; overall, microscopy confirmed 50% of all suspected BUD cases. The inter-laboratory concordance rate for PCR was 96% with an overall PCR case confirmation rate of 78%. Compared to a previous study, the rate of BUD patients with non-ulcerative lesions increased from 37% to 50%, the mean duration of disease before clinical diagnosis decreased significantly from 182.6 to 82.1 days among patients with ulcerative lesions, and the percentage of category III lesions decreased from 30.3% to 19.2%.ConclusionsHigh inter-laboratory concordance rates as well as case confirmation rates of 50% (microscopy), 71% (PCR at national level), and 78% (including qPCR confirmation at external reference laboratory) suggest high standards of BUD diagnostics. The increase of non-ulcerative lesions, as well as the decrease in diagnostic delay and category III lesions, prove the effect of comprehensive EQA and training measures involving also procedures outside the laboratory.

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Section: Methodsmentioning

confidence: 99%

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

et al. 2013

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“…PCR standards were generated by exact quantification of whole-genome DNA from two M. ulcerans cultures from Ghana by IS2404 quantitative real-time PCR (12,13). The limits of detection for the two assays were determined by testing 10-fold serial dilutions of PCR standards.…”

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confidence: 99%

“…In combination with a recently described 16S rRNA-based viability assay (12), molecular drug resistance testing would also allow a reliable differentiation of individuals harboring viable drug-resistant organisms opposed to mycobacterial DNA residues detectable in secondary lesions (4). Furthermore, novel real-time PCR highresolution melt analysis assays without allele-specific primers or probes may constitute a promising tool for screening clinical isolates in future studies (20).…”

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confidence: 99%

Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

et al. 2014

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“…ulcerans in human tissue [15]. In this study, we have investigated the time taken for the 16S rRNA assay to become negative during antibiotic treatment for 8 weeks.…”

Section: Discussionmentioning

confidence: 99%

“…IS 2404 qPCR were performed by well established methods as previously described [17][18] [15]. IS 2404 qPCR was also performed on all samples.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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.

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

Cited by 41 publications

References 11 publications

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

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

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