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

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

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Academic research paper on topic "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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From Innovation to Application

I'PLOS

NEGLECTED TROPICAL DISEASES

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

Marcus Beissner1*9, Dominik Symank19, Richard Odame Phillips2, Yaw Ampem Amoako2, Nana-Yaa Awua-Boateng3, Fred Stephen Sarfo2, Moritz Jansson1, Kristina Lydia Huber1, Karl-Heinz Herbinger1, Florian Battke4, Thomas Löscher1, Ohene Adjei2, Gisela Bretzel1

1 Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians University, Munich, Germany, 2 Komfo Anokye Teaching Hospital (KATH), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana, 3 Kumasi Centre for Collaborative Research (KCCR), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana, 4 Dr. Battke SCIENTIA GmbH, Taufkirchen, Germany

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).

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).

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 (TibMol-Biol, 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

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 RealTime 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: © 2012 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 № 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.

* E-mail: beissner@lrz.uni-muenchen.de

. These authors contributed equally to this work.

Table 1. M. ulcerans cultures subjected to the 16S rRNA RT/IS2404 qPCR assay.

M. ulcerans Strain Source Origin3 16S rRNA RT-qPCRb \S2404 qPCRc \S2404 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].

aM. 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]. bResults of the 16S rRNA RT-qPCR of mycobacterial RNA extracts. cResults of the IS2404 qPCR of mycobacterial DNA extracts.

dResults 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.

doi:10.1371/journal.pntd.0001756.t001

with a hydrolysis probe (Table 3) that was re-designed by DNAsis Max for thermo-dynamic 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 All-

Prep 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).

Sensitivity

The analytical sensitivity was determined as lower limit of detection (LOD, lowest template concentration rendering amplification of 95% of samples) [10] for

Table 2. Specificity of 16S rRNA and IS2404 qPCR assays.

Bacterial Species Source9 Originb 16S rRNAd \S2404e

M. abscessus M. africanum NRZ NRZ Human isolatep Human isolatep - -

M. avium M. bovis NRZ MD7 Human isolatep p Cattle isolate - -

M. chelonae NRZ NRZ MD7 Human isolatep Human isolatec - -

M. fortuitum M. gordonae NRZ NRZ ГЧТЛЛ Human isolatec Human isolatec - -

M. gordonae M. kansasii DiTM NRZ ГЧТЛЛ Human isolatep Human isolatep - -

M. leprae M. malmoense M. marinum DiTM NRZ NRZ Human isolatec Human isolatep - -

M. microti M. scrofulaceum NRZ MD7 Mouse isolatep Human isolatep - -

M. smegmatis NRZ NRZ MD7 Human isolatep Human isolatep - -

M. szulgai M. tuberculosis M. ulcerans NRZ NRZ ГЧТЛЛ Human isolatep Human isolatep - -

M. xenopi DiTM NRZ MVP Human isolatec Human isolatec + +

E. coli P. acnes MVP Human isolatep Human isolatec - -

Staph. aureus Staph. epidermidis Str. pyogenes MVP MVP MVP Human isolatec 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. aDNA 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. bThe respective primary patient isolates were considered as ppathogenic bacteria or as ccommensals/contaminants of clinical samples. dResults of the 16S rRNA RT-qPCR of DNA extracts; ''+'' indicates a positive and ''-'' a negative test result. eResults of the IS2404 qPCR of DNA extracts; ''+'' indicates a positive and ''-'' a negative test result. doi:10.1371/journal.pntd.0001756.t002

Inclusion criteria Exclusion criteria

- Written informed consent provided - Denial to participate

- Participant >5 years of age - Participant <5 years of age

- New case (no history of BUD in the past) - Recurrence or secondary lesion

- Ulcerative lesion with undermined edge - Ulcer with scarred edge

- Duration of disease < 3 months - Duration of disease > 3 months

- Antibiotic treatment not started - Antibiotic treatment started

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. doi:10.1371/journal.pntd.0001756.g001

Inclusion criteria Exclusion criteria

- Written informed consent provided - Denial to participate

- Participant >5 years of age - Participant <5 years of age

- New case - Recurrence or secondary lesion

- Wound has not completely healed - Completely healed wound

- Antibiotic treatment completed (56 doses) - Antibiotic treatment not completed

- No dose missed within 56 days - compliance not ensured

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.

doi:10.1371/journal.pntd.0001756.g002

both qPCR components using 10-fold serial dilutions of cloned IS'2404 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).

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.

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, mycobac-teriological 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).

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,

Table З. Primers and probes.

Primer/Probea Sequence (5 -3 ) Target Geneb Nucleotide Position0 Amplicon Sized

MU^S TF MU^S TR MU^S TP CGA TCT GCC CTG CAC TTC CCA CAC CGC AAA AGC TT 6 FAM-CAC AGG ACA TGA ATC CCG TGG TC-BBQe 16s rRNA 4414800-4414817 4414718-4414734 4414740-4414762 100 bp

IS2404 TF AAA GCA CCA CGC AGC ATC T

IS2404 TR AGC GAC CCC AGT GGA TTG

IS2404 TP2 б FAM-CCG TCC AAC GCG ATC GGC A-BBQe

IS2404 9бб85-9ббб7 59 bp

9бб27-9бб44 9ббб4-9бб4б

T13f TGC ACA CAG GCC ACA AGG GA 16S rRNA 4413906-4413925 935 bp

T39f CG AAC GGG TGA GTA ACA CG 4414822-4414840

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].

aTF, forward primer; TR, reverse primer; TP2, hydrolysis probe (TibMolBiol, Berlin, Germany).

b16S rRNA, gene for the ribosomal 16S RNA detected as 16S cDNA; IS2404, insertion sequence 2404.

cNucleotide 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]. dbp, base pairs.

e6 FAM, 6-Carboxyfluorescein fluorescent dye; BBQ, BlackBerry Quencher.

fPrimers 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).

doi:10.1371/journal.pntd.0001756.t003

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 NA following gDNA wipeout before reverse transcription

Internal positive control To exclude false negative results due to inhibition TaqMan exogenous internal TaqMan exogenous internal positive control (IPC)d 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. a16S rRNA RT PCR, reverse transcriptase real-time PCR targeting the 16S ribosomal RNA of M. ulcerans. bIS2404 qPCR, quantitative real-time PCR targeting the insertion sequence (IS) 2404 of M. ulcerans. cgDNA, genomic DNA wipeout was conducted using DNAses provided in the QuantiTect Reverse Transcription Kit (Qiagen). dTaqMan exogenous internal positive control (Applied Biosystems, Carlsbad, CA). ecDNA, complementary DNA obtained through reverse transcription of M. ulcerans RNA by QuantiTect Reverse Transcription Kit (Qiagen). doi:10.1371/journal.pntd.0001756.t004

Table 5. Intra- and inter-assay variability of the 16S rRNA RT/IS2404 qPCR assay.

qPCR Target3 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 DCt max.d CV max.e Ct-rangef CVg DCt max.h CV max.'

16S rRNA 1 0.23 0.50 0.12 0.48 0.17 0.42 0.23 0.49 0.55 1.33 0.75 1.33

2 0.09 0.19 0.16 0.30 0.19 0.35 0.24 0.53

3 0.12 0.18 0.06 0.20 0.20 0.32 0.31 0.55

4 0.15 0.22 0.17 0.25 0.12 0.22 0.75 1.15

5 0.07 0.10 0.15 0.20 0.16 0.20 0.71 0.92

IS2404 1 0.12 0.53 0.13 0.54 0.10 0.42 0.35 0.65 0.61 2.67 0.80 2.66

2 0.18 0.65 0.15 0.48 0.18 0.57 0.71 2.35

3 0.02 0.07 0.23 0.60 0.11 0.28 0.80 2.13

4 0.18 0.39 0.14 0.28 0.10 0.22 0.80 1.76

5 0.31 0.58 0.25 0.42 0.22 0.38 0.58 1.09

6 0.15 0.23 0.31 0.47 0.20 0.32 0.31 0.58

7 0.35 0.48 0.15 0.33 0.08 0.29 0.74 1.10

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 ml) 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 ml) 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). a16S rRNA, target of the 16S rRNA RT-qPCR; IS2404, target of the IS2404 qPCR. bCt-range, range of Ct-values of samples tested in the same dilution.

cCV, coefficient of variation of copy numbers from samples tested in quadruplicate of the same dilution. dDCt max., maximum Ct-variation of all samples tested within one run. eCV max., maximum CV of all samples tested within one run.

fCt-range, range of Ct-values of samples tested in the same dilution within three runs.

gCV of samples in the same dilution tested within three runs.

hDCt max. of all samples tested within three runs.

iCV max. of all samples tested within three runs.

doi:10.1371/journal.pntd.0001756.t005

Standard curve and limit of detection of the 16S rRNA RT-qPCR

= -3,4* + 41,6 Q? n firi7 s

6 300 3E+3 3E*4 3E+5 3E+6

Copy number of 16S rRNA gene/cDNA

• 16S rRNA gene standards X Limit oi detection A Clinical samples — trend tine

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 ml) 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). doi:10.1371/journal.pntd.0001756.g003

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 ml) 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. doi:10.1371/journal.pntd.0001756.g004

Table 6. Study participants, clinical information, and diagnostic results.

Clinical Data Molecular Viability Assay" Routine Diagnostics'1

No.c BUD Pat'entd Duration (Weeks)e Category of Lesionf IS2404 [Ct]g Bacillary Loadh 16S rRNA' MICk pcr1

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.

aResults 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.

bRoutine diagnostics were conducted following standardized procedures at KCCR [3]. cNo., consecutive number of study participants.

dYes, IS2404 qPCR confirmed BUD patients; No, IS2404 negative study participants. eDuration of disease before presentation of study participants in weeks. fCategory of lesion according to the World Health Organization's clinical criteria [1]. gResults of the IS2404 qPCR with corresponding cycle threshold (Ct)-values.

hThe 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. 'Results of the 16S rRNA RT-qPCR.

kMIC, 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 10AFB/1 field. lPCR, conventional, single target gel-based IS2404 DRB PCR. doi:10.1371/journal.pntd.0001756.t006

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 J) may limit the applicability.

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

References

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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.

Mycobacterium ulcerans infection. Clin Infect Dis 54(4): 519-526.

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Protocol S2 Simultaneous RNA/DNA extraction from swab samples and reverse transcription of whole transcriptome RNA from M. ulcerans. (PDF)

Protocol S3 Combined 16S rRNA RT/

IS2404 qPCR assay.

Protocol S4 16S rRNA RT/IS2404

qPCR run protocol.

Table S1 GenBank accession numbers. (DOC)

Acknowledgments

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 Rusch-Gerdes and Dr. Elvira Richter (National Reference Center for Myco-bacteria, Borstel, Germany), as well as Dr. Soeren Schubert (Max von Pettenkofer-Insti-tute, Ludwig-Maximilians University, Munich, Germany) for providing (myco-) bacterial DNA extracts. The manuscript contains parts of the doctoral thesis of Dominik Symank.

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