Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS 2404 Real-Time qPCR Assay

Abstract

Introduction 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 [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. Development and Validation Ethical Approval 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 [8] 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). 10.1371/journal.pntd.0001756.t001 Table 1 M. ulcerans cultures subjected to the 16S rRNA RT/IS2404 qPCR assay. M. ulcerans Strain Source Origina 16S rRNA RT-qPCRb IS2404 qPCRc IS2404 qPCR – Wipeout Controld K4s-C1 DITM Human isolate – Kamerun Positive Positive Negative K4s-C2 DITM Human isolate – Kamerun Positive Positive Negative K4s-C3 DITM Human isolate – Kamerun Positive Positive Negative K5d-C1 DITM Human isolate – Kamerun Positive Positive Negative K5d-C2 DITM Human isolate – Kamerun Positive Positive Negative K5d-C1 DITM Human isolate – Kamerun Positive Positive Negative K5d-C2 DITM Human isolate – Kamerun Positive Positive Negative K5d-C3 DITM Human isolate – Kamerun Positive Positive Negative K5d-C4 DITM Human isolate – Kamerun Positive Positive Negative K5s-C1 DITM Human isolate – Kamerun Positive Positive Negative K5s-C2 DITM Human isolate – Kamerun Positive Positive Negative K5s-C3 DITM Human isolate – Kamerun Positive Positive Negative K5s-C4 DITM Human isolate – Kamerun Positive Positive Negative K5s-C5 DITM Human isolate – Kamerun Positive Positive Negative K7b-C1 DITM Human isolate – Kamerun Positive Positive Negative K7b-C2 DITM Human isolate – Kamerun Positive Positive Negative K7b-C3 DITM Human isolate – Kamerun Positive Positive Negative K7b-C4 DITM Human isolate – Kamerun Positive Positive Negative K7s-C1 DITM Human isolate – Kamerun Positive Positive Negative K7s-C2 DITM Human isolate – Kamerun Positive Positive Negative K12S-C1 DITM Human isolate – Kamerun Positive Positive Negative 941328-C1 DITM Human isolate – Ghana Positive Positive Negative 07-C1 DITM Human isolate – Ghana Positive Positive Negative DS1-C1 DITM Human isolate – Ghana Positive Positive Negative 97680-C1 DITM Human isolate – Ghana Positive Positive Negative G.A.P.001-C1 KCCR Human isolate – Ghana Positive Positive Negative G.A.P.033-C1 KCCR Human isolate – Ghana Positive Positive Negative G.A.P.071-C1 KCCR Human isolate – Ghana Positive Positive Negative G.A.P.078-C1 KCCR Human isolate – Ghana Positive Positive Negative Table 1 shows 29 M. ulcerans cultures that were available at the Department of Infectious Diseases and Tropical Medicine (DITM) and the Kumasi Centre for Collaborative Research (KCCR) for development and technical validation of the 16S rRNA RT/IS2404 qPCR viability assay and the corresponding test results. Sequence analysis of 16S rRNA genes from the listed strains revealed 100% nucleotide concordance of the corresponding genomic regions amplified by the 16S rRNA RT-qPCR; no SNPs or mutations were detected, suggesting a high selectivity of the assay. Sequencing primers are described in Table 3 [11]. a M. ulcerans cultures were available from previous studies from Kamerun (n = 21) and Ghana (n = 4) at DITM [8] or were available at KCCR (n = 4) from the present study. All strains were of human origin (BUD patients) and confirmed by conventional IS2404 PCR and sequencing of rpoB- and rpsL-genes that revealed the M. ulcerans Agy99 wild-type sequences (GenBank accession no. CP000325.1) [11], [12]. b Results of the 16S rRNA RT-qPCR of mycobacterial RNA extracts. c Results of the IS2404 qPCR of mycobacterial DNA extracts. d Results of the IS2404 qPCR of genomic DNA (gDNA) wipeout controls (see Protocols S2 and S3); a positive result indicates gDNA contamination of RNA extracts following DNAse digestions, and a negative result indicates RNA extracts free of gDNA. 10.1371/journal.pntd.0001756.t002 Table 2 Specificity of 16S rRNA and IS2404 qPCR assays. Bacterial Species Sourcea Originb 16S rRNAd IS2404 e M. abscessus NRZ Human isolatep − − M. africanum NRZ Human isolatep − − M. avium NRZ Human isolatep − − M. bovis NRZ Cattle isolatep − − M. chelonae NRZ Human isolatep − − M. fortuitum NRZ Human isolatec − − M. gordonae NRZ Human isolatec − − M. gordonae DITM Human isolatec − − M. kansasii NRZ Human isolatep − − M. leprae DITM Human isolatep − − M. malmoense NRZ Human isolatec − − M. marinum NRZ Human isolatep + − M. microti NRZ Mouse isolatep − − M. scrofulaceum NRZ Human isolatep − − M. smegmatis NRZ Human isolatep − − M. szulgai NRZ Human isolatep − − M. tuberculosis NRZ Human isolatep − − M. ulcerans DITM Human isolatep + + M. xenopi NRZ Human isolatec − − E. coli MVP Human isolatec − − P. acnes MVP Human isolatep − − Staph. aureus MVP Human isolatec − − Staph. epidermidis MVP Human isolatec − − Str. pyogenes MVP Human isolatep − − Table 2 shows DNA extracts from closely related mycobacterial species and bacteria potentially contaminating the human skin subjected to the combined 16S rRNA RT/IS2404 qPCR viability assay and the corresponding test results. Mycobacterial species were selected according to their respective genetic contiguousness to M. ulcerans Agy99 (GenBank accession no. CP000325.1) within the 16S rRNA gene sequences as determined by BLASTN analysis (GenBank, NCBI) [13]. M., Mycobacterium; E., Escherichia; P., Propionibacterium; Staph., Staphylococcus; Str., Streptococcus. While in-silico analysis revealed that the combined 16S rRNA RT/IS2404 assay will also amplify mycolactone-producing mycobacteria (MPM) other than M. ulcerans (e.g., M. pseudoshottsii, M. liflandii, and the environmental M. marinum [GenBank accession No. NR_042988.1, AY500838.1, and AF456241.1, respectively]), these MPM species were not included in specificity testing. a DNA extracts that were not available at the DITM were provided by the National Reference Center (NRZ) for Mycobacteria, Borstel, Germany, and the Max von Pettenkofer-Institute (MVP), Ludwig-Maximilians University, Munich, Germany. b The respective primary patient isolates were considered as ppathogenic bacteria or as ccommensals/contaminants of clinical samples. d Results of the 16S rRNA RT-qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result. e Results of the IS2404 qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result. 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). 10.1371/journal.pntd.0001756.g001 Figure 1 Enrolment criteria for the pre-treatment study population. 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. 10.1371/journal.pntd.0001756.g002 Figure 2 Enrolment criteria for the post-treatment study population. 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) [3]. 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. [9] were used in combination with a hydrolysis probe (Table 3) that was re-designed by DNAsis Max for thermodynamic reasons. 10.1371/journal.pntd.0001756.t003 Table 3 Primers and probes. Primer/Probea Sequence (5′–3′) Target Geneb Nucleotide Positionc Amplicon Sized MU16S TFMU16S TRMU16S TP CGA TCT GCC CTG CAC TTC CCA CAC CGC AAA AGC TT6 FAM-CAC AGG ACA TGA ATC CCG TGG TC-BBQe 16S rRNA 4414800–44148174414718–44147344414740–4414762 100 bp IS2404 TFIS2404 TRIS2404 TP2 AAA GCA CCA CGC AGC ATC T AGC GAC CCC AGT GGA TTG6 FAM-CCG TCC AAC GCG ATC GGC A-BBQe IS2404 96685–9666796627–9664496664–96646 59 bp T13fT39f TGC ACA CAG GCC ACA AGG GACG AAC GGG TGA GTA ACA CG 16S rRNA 4413906–44139254414822–4414840 935 bp Table 3 indicates primers and probes designed for the 16S rRNA RT-qPCR, the primers described by Fyfe et al., and a re-designed hydrolysis probe used for the amplification, detection, and quantification of IS2404 [9]. a TF, forward primer; TR, reverse primer; TP2, hydrolysis probe (TibMolBiol, Berlin, Germany). b 16S rRNA, gene for the ribosomal 16S RNA detected as 16S cDNA; IS2404, insertion sequence 2404. c Nucleotide positions are provided for the first (IS2404) or single (16S rRNA) copy of the respective amplicon in M. ulcerans Agy99 (GenBank accession no. CP000325.1) as determined by BLASTN analysis within GenBank (NCBI) [13]. d bp, base pairs. e 6 FAM, 6-Carboxyfluorescein fluorescent dye; BBQ, BlackBerry Quencher. f Primers T13 (forward) and T39 (reverse) were used for the amplification of a 935-bp region of the M. ulcerans 16S rRNA gene, encompassing the region amplified by qPCR primers MU16S TF and MU16S TR, to generate single copy replicates. Furthermore, these primers were used for sequencing of the M. ulcerans 16S rRNA gene (Table 1). 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). 10.1371/journal.pntd.0001756.t004 Table 4 Controls applied in 16S rRNA RT/IS2404 qPCR. Control Purpose Material 16S rRNA RT-qPCRa IS2404 qPCRb gDNA wipeout controlc To exclude DNA contamination of RNA extracts Aliquot of each RNA extract following gDNA wipeout before reverse transcription NA Internal positive control To exclude false negative results due to inhibition TaqMan exogenous internal positive control (IPC)d TaqMan exogenous internal positive control (IPC)d Positive run control To ensure adequate performance of PCR M. ulcerans cDNAe Cloned IS2404 standard Negative no template control To exclude contamination during PCR set up H2O H2O Negative extraction control To exclude contamination during extraction procedure NA Extract treated in the same way as samples Table 4 indicates controls applied in 16S rRNA RT/IS2404 qPCR. NA, not applicable. a 16S rRNA RT PCR, reverse transcriptase real-time PCR targeting the 16S ribosomal RNA of M. ulcerans. b IS2404 qPCR, quantitative real-time PCR targeting the insertion sequence (IS) 2404 of M. ulcerans. c gDNA, genomic DNA wipeout was conducted using DNAses provided in the QuantiTect Reverse Transcription Kit (Qiagen). d TaqMan exogenous internal positive control (Applied Biosystems, Carlsbad, CA). e cDNA, complementary DNA obtained through reverse transcription of M. ulcerans RNA by QuantiTect Reverse Transcription Kit (Qiagen). 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). 10.1371/journal.pntd.0001756.t005 Table 5 Intra- and inter-assay variability of the 16S rRNA RT/IS2404 qPCR assay. qPCR Targeta Standard No. Run No. 1 Run No. 2 Run No. 3 Intra-Assay Variability Inter-Assay Variability Ct-rangeb CVc Ct-rangeb CVc Ct-rangeb CVc ΔCt max.d CV max.e Ct-rangef CVg ΔCt max.h CV max.i 16S rRNA 12345 0.230.090.120.150.07 0.500.190.180.220.10 0.120.160.060.170.15 0.480.300.200.250.20 0.170.190.200.120.16 0.420.350.320.220.20 0.23 0.49 0.550.240.310.750.71 1.330.530.551.150.92 0.75 1.33 IS2404 1234567 0.120.180.020.180.310.150.35 0.530.650.070.390.580.230.48 0.130.150.230.140.250.310.15 0.540.480.600.280.420.470.33 0.100.180.110.100.220.200.08 0.420.570.280.220.380.320.29 0.35 0.65 0.610.710.800.800.580.310.74 2.672.352.131.761.090.581.10 0.80 2.66 Table 5 shows intra- and inter-assay variability of the 16S rRNA RT/IS2404 qPCR assay. 16S rRNA RT-qPCR: 16S rRNA gene standards (935 bp) were generated by conventional PCR according to Talaat et al. [12]. Quantification of PCR products was conducted by Picogreen fluorometry (Invitrogen) and copy numbers were calculated based on the known mass of one amplicon. Serial standards were prepared from PCR products in 5 Log dilutions ranging from 3E+6 (standard no. 1) to 3E+2 copies (standard no. 5) of the 16S rRNA amplicon (PCR template: 2 µl) and were subjected to the 16S rRNA RT-qPCR in quadruplicate on one 96-well plate to assess intra-assay variability. The runs were repeated on three days to determine the inter-assay variability between runs 1 through 3. The intra- and inter-assay variability of the 16S rRNA RT-qPCR was low with maximum coefficients of variation (CV) of 0.49 (intra-assay) and 1.33 (inter-assay). IS2404 qPCR: Cloned IS2404 replicates (1,047 bp, complete sequence; M. ulcerans Agy99) were used as standards. Quantification of IS2404 templates was conducted by Picogreen fluorometry (Invitrogen) and copy numbers were calculated based on the known mass of one template. Serial standards were prepared in 7 Log dilutions ranging from 2E+8 to 2E+2 copies of the IS2404 (PCR template: 2 µl) and were subjected to the IS2404 qPCR in quadruplicate on one 96-well plate to assess intra-assay variability. The runs were repeated on three days to determine the inter-assay variability between runs 1 through 3. The intra- and inter-assay variability of the IS2404 qPCR was low with maximum CV of 0.65 (intra-assay) and 2.66 (inter-assay). a 16S rRNA, target of the 16S rRNA RT-qPCR; IS2404, target of the IS2404 qPCR. b Ct-range, range of Ct-values of samples tested in the same dilution. c CV, coefficient of variation of copy numbers from samples tested in quadruplicate of the same dilution. d ΔCt max., maximum Ct-variation of all samples tested within one run. e CV max., maximum CV of all samples tested within one run. f Ct-range, range of Ct-values of samples tested in the same dilution within three runs. g CV of samples in the same dilution tested within three runs. h ΔCt max. of all samples tested within three runs. i CV max. of all samples tested within three runs. Sensitivity The analytical sensitivity was determined as lower limit of detection (LOD, lowest template concentration rendering amplification of 95% of samples) [10] 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). 10.1371/journal.pntd.0001756.g003 Figure 3 Standard curve and limit of detection of the 16S rRNA RT-qPCR. 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). 10.1371/journal.pntd.0001756.g004 Figure 4 Standard curve and limit of detection of the IS2404 qPCR. 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). 10.1371/journal.pntd.0001756.t006 Table 6 Study participants, clinical information, and diagnostic results. Clinical Data Molecular Viability Assaya Routine Diagnosticsb No.c BUD Patientd Duration (Weeks)e Category of Lesionf IS2404 [Ct]g Bacillary Loadh 16S rRNAi MICk PCRl 1 No NA NA Neg [NA] NA Neg 0 Neg 2 Yes 6 III Pos [15,04] >1000 Pos +1 Pos 3 Yes 4 III Pos [26,80] 584 Pos +1 Pos 4 Yes 9 III Pos [32,93] 6–10 Pos 0 Neg 5 Yes 4 I Pos [35,94] 1–5 Neg 0 Neg 6 Yes 8 II Pos [36,72] 1–5 Neg 0 Neg 7 Yes 2 I Pos [36,74] 1–5 Neg 0 Neg 8 Yes 10 I Pos [27,05] 497 Pos +1 Pos 9 No NA NA Neg [NA] NA Neg 0 Neg 10 Yes 3 I Pos [30,61] 42 Pos +1 Pos 11 Yes 8 II Pos [33,89] 6–10 Pos 0 Neg 12 Yes 9 I Pos [33,68] 6–10 Pos 0 Neg 13 Yes 3 III Pos [29,27] 106 Pos +1 Pos 14 Yes 3 I Pos [27,98] 261 Pos +1 Pos 15 Yes 1 I Pos [26,85] 571 Pos +1 Pos 16 Yes 2 I Pos [33,07] 6–10 Pos 0 Pos 17 Yes 2 II Pos [31,44] 24 Pos +1 Pos 18 Yes 3 II Pos [21,85] >1000 Pos +2 Pos 19 Yes 4 III Pos [22,98] >1000 Pos +1 Pos 20 Yes 3 I Pos [23,47] >1000 Pos +2 Pos 21 No NA NA Neg [NA] NA Neg 0 Neg 22 No NA NA Neg [NA] NA Neg 0 Neg 23 No NA NA Neg [NA] NA Neg 0 Neg 24 No NA NA Neg [NA] NA Neg 0 Neg Table 6 shows suspected BUD cases with ulcerative lesions enrolled in the pre-treatment cohort (Figure 1), clinical information, and diagnostic results. Swab samples from 24 suspected BUD cases were subjected to 16S rRNA RT/IS2404 qPCR viability assay (swab 1 in PANTA), microscopic examination and enumeration of acid fast bacilli (AFB) following Ziehl-Neelsen staining (swab 2, direct smear), and conventional IS2404 dry-reagent-based (DRB) PCR (swab 3 in Cell Lysis Solution [Qiagen]). 18 patients were laboratory confirmed by IS2404 qPCR and 15 out of those were RNA positive; the quantification by IS2404 qPCR revealed a bacillary load (1–2 bacilli per sample) below the lower limit of detection of the RNA assay for samples from three RNA negative patients. All samples from six IS2404 qPCR negative study participants were also RNA negative. Direct correlation of AFB enumeration with IS2404 qPCR quantification is not feasible due to inhomogeneous distribution of M. ulcerans in different clinical samples. NA, not applicable; Neg, negative test result; Pos, positive test result. a Results of the 16S rRNA RT/IS2404 qPCR viability assay. Clinical swab samples in PANTA were directly processed at KCCR, and M. ulcerans DNA and cDNA were transported to DITM and subjected to qPCR. b Routine diagnostics were conducted following standardized procedures at KCCR [3]. c No., consecutive number of study participants. d Yes, IS2404 qPCR confirmed BUD patients; No, IS2404 negative study participants. e Duration of disease before presentation of study participants in weeks. f Category of lesion according to the World Health Organization's clinical criteria [1]. g Results of the IS2404 qPCR with corresponding cycle threshold (Ct)-values. h The bacillary load in the respective swab samples (No. 2) was estimated on the basis of IS2404 quantification given an IS2404 copy number of 209 copies per M. ulcerans genome [9]. For bacterial numbers <10 ranges were estimated. i Results of the 16S rRNA RT-qPCR. k MIC, microscopic detection and enumeration of AFB was conducted at KCCR including external quality assurance by DITM. The following scale was applied: 0 = negative, +1 = 10–99 AFB/100 fields, +2 = 1–10 AFB/1 field, +3 = more than 10 AFB/1 field. l PCR, conventional, single target gel-based IS2404 DRB PCR. All seven post-treatment swab samples were IS2404 qPCR positive and 16S rRNA negative. Specificity 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) [3] 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. Future Application 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 Advantages 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. Disadvantages 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. Conclusions 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 Genes or DNA sequences of mycobacterial strains used in this study were retrieved from GenBank (NCBI) [13]. The respective sequences and accession numbers are summarized in Table S1. Supporting Information Protocol S1 Preparation of PANTA transport medium and stabilisation of M. ulcerans RNA/DNA in swab samples and culture suspensions. (PDF) Click here for additional data file. Protocol S2 Simultaneous RNA/DNA extraction from swab samples and reverse transcription of whole transcriptome RNA from M. ulcerans. (PDF) Click here for additional data file. Protocol S3 Combined 16S rRNA RT/IS2404 qPCR assay. (PDF) Click here for additional data file. Protocol S4 16S rRNA RT/IS2404 qPCR run protocol. (XLS) Click here for additional data file. Table S1 GenBank accession numbers. (DOC) Click here for additional data file.