Citation: Beissner M, Symank D, Phillips RO, Amoako YA, Awua-Boateng N-Y, et al. (2012) Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay. PLoS Negl Trop Dis 6(8): e1756. doi:10.1371/journal.pntd.0001756
Editor: Pamela L. C. Small, University of Tennessee, United States of America
Published: August 28, 2012
Copyright: © Beissner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement N° 241500. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
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; late-onset 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 –. 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 , . 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.
Development and Validation
The study was approved by the Committee of Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (CHRPE/28/09). Written informed consent was obtained from all study participants, or their legal representatives.
Bacterial Strains, DNA Extracts, and Clinical Samples
Technical validation of the assay was performed with 29 M. ulcerans strains originating from Cameroon  and Ghana (Table 1), as well as DNA extracts from 18 closely related human pathogenic mycobacterial species and five bacterial species frequently colonizing human skin (Table 2).
Clinical validation was conducted on pre-treatment swab samples in PANTA (BD, Heidelberg, Germany) from 24 suspected BUD cases from Agogo Presbyterian Hospital (n = 14) and Tepa Government Hospital (n = 10), Ghana (Protocol S1). In addition, post-treatment swab samples from seven IS2404 PCR confirmed BUD patients with incomplete wound healing were collected at week nine (Figures 1 and 2).
Figure 1 describes enrolment criteria for clinically suspected BUD patients presenting at Agogo Presbyterian Hospital (n = 14) and Tepa Governmental Hospital (n = 10), Ghana, respectively. None of the eligible study participants was excluded.
Figure 2 describes enrolment criteria for IS2404 PCR confirmed BUD patients with incomplete wound healing (collection of swab samples feasible) who presented at Agogo Presbyterian Hospital, Ghana (n = 7), following completion of 56 doses of rifampicin and streptomycin administered within eight weeks. None of the eligible study participants was excluded.
All clinical samples were subjected to routine diagnostics (microscopy and IS2404 dry-reagent-based [DRB] PCR) at the Kumasi Centre for Collaborative Research (KCCR) .
Primers and Probes
Primers and a hydrolysis probe (TibMolBiol, Berlin, Germany) for specific amplification of M. ulcerans 16S rRNA were designed using DNAsis Max (MiraiBio, San Francisco, USA) by alignment of 16S rRNA gene sequences (GenBank, National Center for Biotechnology Information [NCBI]) from closely related mycobacteria and other bacteria potentially contaminating the human skin (Table 2).
For simultaneous quantification by IS2404 qPCR, the primers described by Fyfe et al.  were used in combination with a hydrolysis probe (Table 3) that was re-designed by DNAsis Max for thermodynamic reasons.
Combined RNA/DNA Extraction, Reverse Transcription, and Real-Time qPCR
Culture suspensions and swab samples were stabilized by RNA protect (Qiagen, Hilden, Germany) and subjected to AllPrep DNA/RNA extraction kit (Qiagen) (Protocols S1 and S2).
M. ulcerans whole transcriptome RNA from cultures and swab samples was transcribed to cDNA by QuantiTect Reverse Transcription Kit (Qiagen) including genomic DNA (gDNA) wipeout (Protocol S2). DNA and cDNA were subjected to IS2404 qPCR and 16S rRNA RT-qPCR, respectively, with corresponding controls (Table 4, Protocols S3 and S4).
Intra- and Inter-Assay Variability
Intra- and inter-assay variability was assessed by testing of each sample in quadruplicate within one 96-well plate, repeated on three different days (Table 5).
The analytical sensitivity was determined as lower limit of detection (LOD, lowest template concentration rendering amplification of 95% of samples)  for both qPCR components using 10-fold serial dilutions of cloned IS2404 templates (GenExpress, Berlin, Germany) with known copy numbers (IS2404 qPCR) and exactly quantified M. ulcerans whole genome DNA extracts from cultures (16S rRNA RT-qPCR). The LOD was two (IS2404) and six templates (16S rRNA gene), respectively (Figures 3 and 4).
Figure 3 shows Ct-values of clinical samples plotted versus quantified 16S rRNA copy numbers. Standards for the 16S rRNA RT-qPCR were generated by conventional PCR amplification (Table 5). Log 10 fold serial dilutions (n = 5) were prepared ranging from 3E+6 to 300 copies of the 16S rRNA gene (PCR template: 2 µl) and were subjected to the assay in quadruplicate to generate a calibration curve. The regression line was y = −3.4x+41.68 with a coefficient of correlation >0.99 and the efficiency was E = 0.97. M. ulcerans whole genome extracts were quantified by means of IS2404 qPCR and the analytical sensitivity was determined as limit of detection (LOD) by subjecting 10 aliquots of a dilution series containing 30, 15, 10, 8, 6, 3, or 2 copies of the 16S rRNA gene to the assay. The LOD was 6 copies of the target sequence. The copy number (n = 1) of the 16S rRNA gene per M. ulcerans genome was determined by copy number variation assay (unpublished data).
Figure 4 shows mean Ct-values of calibration standards and clinical samples plotted versus the quantified copy number of IS2404. Cloned IS2404 templates were used as standards (Table 5). Log 10 fold serial dilutions (n = 8) were prepared ranging from 2E+8 to 20 copies of the IS2404 (PCR template: 2 µl) and were subjected to the IS2404 qPCR in quadruplicate to generate a calibration curve. The regression line was y = −3.35x+39.10 with a coefficient of correlation >0.99 and the efficiency was E = 0.97. The analytical sensitivity was determined as limit of detection (LOD) by subjecting 10 aliquots of a dilution series containing 10, 5, 4, 3, 2, or 1 copy of the IS2404 to the assay. The LOD was 2 copies of the target sequence.
M. ulcerans DNA and rRNA was detected in all culture extracts. Out of 24 pre-treatment swab samples, 18 (75.0%; 95%-CI: 57.7%–92.3%) had a positive IS2404 qPCR result, 12 out of those were also positive in routine DRB PCR, and rRNA was detected in 15 out of these 18 samples (83.3%; 95%-CI: 66.1%–100%); quantification of the three negative samples revealed a bacillary load below the LOD of the 16S rRNA RT-qPCR (Table 6).
All seven post-treatment swab samples were IS2404 qPCR positive and 16S rRNA negative.
Analysis of DNA extracts revealed 100% specificity for the combined assay. M. marinum (human isolate) was amplified by 16S rRNA RT-qPCR; however, simultaneous IS2404 qPCR was negative (Table 2).
Bacillary Survival Times
To investigate the effect of sample transport on bacillary survival, mycobacteriological transport media (PANTA and LTM)  were spiked with viable M. ulcerans and stored at 4°C and 31°C. RNA was detectable in both media for >4 weeks (4°C and 31°C).
After heat-inactivation of M. ulcerans–spiked PANTA-samples, RNA positivity decreased significantly within 12 h, whereas DNA was still detectable after seven days.
The assay will support clinicians in classification of secondary lesions and selection of adequate clinical management strategies and provides a powerful tool for clinical research evaluating novel treatment regimens (Box 1).
Box 1. Advantages and Disadvantages of the Molecular Viability Assay
- Provides a rapid, sensitive, and specific tool to detect viable bacilli in clinical samples of BUD patients, thus offering an alternative to cultures.
- Supports classification of secondary BUD lesions and monitoring of treatment success.
- Current test format requires well equipped laboratory with real-time PCR facilities.
- Costs per test (approximately 14 €) may limit the applicability.
Through analysis of sequential samples collected during antimycobacterial treatment, the assay will be employed to determine the proportional decrease of bacterial viability over time and to establish laboratory-based evidence for optimal time-points to collect follow-up samples for treatment monitoring.
Whereas the current format of the assay is restricted to reference laboratories, sample collection on FTA cards in combination with isothermal dry-reagent-based reverse transcription and amplification formats would facilitate processing of samples also at a peripheral level and at lower costs.
The novel combined 16S rRNA RT/IS2404 qPCR assay proved to be highly sensitive, specific, and efficient in detecting viable M. ulcerans in clinical samples under field conditions. The assay is applicable for classification of secondary lesions and monitoring of treatment success and provides a powerful tool for clinical research.
GenBank Accession Numbers
Preparation of PANTA transport medium and stabilisation of M. ulcerans RNA/DNA in swab samples and culture suspensions.
Simultaneous RNA/DNA extraction from swab samples and reverse transcription of whole transcriptome RNA from M. ulcerans.
Combined 16S rRNA RT/IS2404 qPCR assay.
16S rRNA RT/IS2404 qPCR run protocol.
GenBank accession numbers.
The authors thank Erna Fleischmann, Carolin Mengele and Kerstin Helfrich (DITM), and Mabel Peprah and Michael Frimpong (KCCR) for excellent technical assistance. The authors thank Dr. Sabine Rüsch-Gerdes and Dr. Elvira Richter (National Reference Center for Mycobacteria, Borstel, Germany), as well as Dr. Soeren Schubert (Max von Pettenkofer-Institute, Ludwig-Maximilians University, Munich, Germany) for providing (myco-) bacterial DNA extracts. The manuscript contains parts of the doctoral thesis of Dominik Symank.
- 1. World Health Organization (2008) Buruli ulcer: progress report, 2004–2008. Wkly Epidemiol Rec 83: 145–156.
- 2. World Health Organization (2008) Meeting of the WHO Technical Advisory Group on Buruli ulcer, 3 April 2008, Geneva, summary report. Geneva: World Health Organization.
- 3. Beissner M, Herbinger KH, Bretzel G (2010) Laboratory diagnosis of Buruli ulcer disease. Future Microbiol 5 (3) 363–370. doi: 10.2217/fmb.10.3
- 4. Ruf MT, Chauty A, Adeye A, Ardant MF, Koussemou H, et al. (2011) Secondary Buruli ulcer skin lesions emerging several months after completion of chemotherapy: paradoxical reaction or evidence for immune protection? PLoS Negl Trop Dis 5 (8) e1252 doi:10.1371/journal.pntd.0001252. doi: 10.1371/journal.pntd.0001252
- 5. Nienhuis WA, Stienstra Y, Abass KM, Tuah W, Thompson WA, et al. (2011) Paradoxical responses after start of antimicrobial treatment in Mycobacterium ulcerans infection. Clin Infect Dis 54 (4) 519–526. doi: 10.1093/cid/cir856
- 6. Martinez AN, Lahiri R, Pittman TL, Scollard D, Truman R, et al. (2009) Molecular determination of Mycobacterium leprae viability by use of real-time PCR. J Clin Microbiol 47 (7) 2124–2130. doi: 10.1128/jcm.00512-09
- 7. Desjardin LE, Perkins MD, Wolski K, Haun S, Teixeira L, et al. (1999) Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am J Respir Crit Care Med 160: 203–210. doi: 10.1164/ajrccm.160.1.9811006
- 8. Bretzel G, Siegmund V, Racz P, van Vloten F, Ngos F, et al. (2005) Post-surgical assessment of excised tissue from patients with Buruli ulcer disease: progression of infection in macroscopically healthy tissue. Trop Med Int Health 10 (11) 1199–1206. doi: 10.1111/j.1365-3156.2005.01507.x
- 9. Fyfe JA, Lavender CJ, Johnson PD, Globan M, Sievers A, et al. (2007) Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples. Appl Environ Microbiol 73 (15) 4733–4740. doi: 10.1128/aem.02971-06
- 10. Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (2010) A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods 50 (4) S1–S5. doi: 10.1016/j.ymeth.2010.01.005
- 11. Beissner M, Awua-Boateng NY, Thompson W, Nienhuis WA, Klutse E, et al. (2010) A genotypic approach for detection, identification, and characterization of drug resistance in Mycobacterium ulcerans in clinical samples and isolates from Ghana. Am J Trop Med Hyg 83 (5) 1059–1065. doi: 10.4269/ajtmh.2010.10-0263
- 12. Talaat AM, Reimschuessel R, Trucksis M (1997) Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis. Vet Microbiol 58 (2–4) 229–237. doi: 10.1016/s0378-1135(97)00120-x
- 13. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2008) GenBank. Nucleic Acids Res 36 (Database issue) D25–D30. doi: 10.1093/nar/gkm929