Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test

Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP),a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR),IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%;100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%;cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level

Enrolment criteria for the post-treatment study population.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g002" target="_blank">Figure 2</a> describes enrolment criteria for IS<i>2404</i> PCR confirmed BUD patients with incomplete wound healing (collection of swab samples feasible) who presented at Agogo Presbyterian Hospital, Ghana (<i>n</i> = 7), following completion of 56 doses of rifampicin and streptomycin administered within eight weeks. None of the eligible study participants was excluded.</p

Standard curve and limit of detection of the IS<i>2404</i> qPCR.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g004" target="_blank">Figure 4</a> shows mean Ct-values of calibration standards and clinical samples plotted versus the quantified copy number of IS<i>2404</i>. Cloned IS<i>2404</i> templates were used as standards (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t005" target="_blank">Table 5</a>). Log 10 fold serial dilutions (<i>n</i> = 8) were prepared ranging from 2E+8 to 20 copies of the IS<i>2404</i> (PCR template: 2 µl) and were subjected to the IS<i>2404</i> 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 IS<i>2404</i> to the assay. The LOD was 2 copies of the target sequence.</p

Study participants, clinical information, and diagnostic results.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t006" target="_blank">Table 6</a> shows suspected BUD cases with ulcerative lesions enrolled in the pre-treatment cohort (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g001" target="_blank">Figure 1</a>), clinical information, and diagnostic results. Swab samples from 24 suspected BUD cases were subjected to 16S rRNA RT/IS<i>2404</i> 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 IS<i>2404</i> dry-reagent-based (DRB) PCR (swab 3 in Cell Lysis Solution [Qiagen]). 18 patients were laboratory confirmed by IS<i>2404</i> qPCR and 15 out of those were RNA positive; the quantification by IS<i>2404</i> 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 IS<i>2404</i> qPCR negative study participants were also RNA negative. Direct correlation of AFB enumeration with IS<i>2404</i> qPCR quantification is not feasible due to inhomogeneous distribution of <i>M. ulcerans</i> in different clinical samples. NA, not applicable; Neg, negative test result; Pos, positive test result.</p>a<p>Results of the 16S rRNA RT/IS<i>2404</i> qPCR viability assay. Clinical swab samples in PANTA were directly processed at KCCR, and <i>M. ulcerans</i> DNA and cDNA were transported to DITM and subjected to qPCR.</p>b<p>Routine diagnostics were conducted following standardized procedures at KCCR <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Beissner1" target="_blank">[3]</a>.</p>c<p>No., consecutive number of study participants.</p>d<p>Yes, IS<i>2404</i> qPCR confirmed BUD patients; No, IS<i>2404</i> negative study participants.</p>e<p>Duration of disease before presentation of study participants in weeks.</p>f<p>Category of lesion according to the World Health Organization's clinical criteria <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-World1" target="_blank">[1]</a>.</p>g<p>Results of the IS<i>2404</i> qPCR with corresponding cycle threshold (Ct)-values.</p>h<p>The bacillary load in the respective swab samples (No. 2) was estimated on the basis of IS<i>2404</i> quantification given an IS<i>2404</i> copy number of 209 copies per <i>M. ulcerans</i> genome <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Fyfe1" target="_blank">[9]</a>. For bacterial numbers <10 ranges were estimated.</p>i<p>Results of the 16S rRNA RT-qPCR.</p>k<p>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.</p>l<p>PCR, conventional, single target gel-based IS<i>2404</i> DRB PCR.</p

Clinical samples and laboratory results of primary and secondary BUD lesions.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001747#pntd-0001747-t001" target="_blank">Table 1</a> shows samples collected from the primary and secondary lesions in July 2010 and 2011 and the corresponding laboratory results. “Neg” indicates a negative test result, “pos” indicates a positive test result.</p>a<p>MIC, microscopic examination of acid fast bacilli (AFB) following Ziehl-Neelsen staining conducted at CHR, DITM, and INH (samples from secondary lesion only).</p>b<p>IS<i>2404</i> PCR, conventional, single-step, gel-based IS<i>2404</i> polymerase-chain-reaction conducted at DITM.</p>c<p>IS<i>2404</i> qPCR, real-time quantitative IS<i>2404</i> polymerase-chain-reaction conducted at DITM.</p>d<p>16S RT qPCR, <i>Mycobacterium ulcerans</i>–specific reverse-transcription real-time quantitative polymerase-chain-reaction targeting the ribosomal 16S RNA of <i>M. ulcerans</i> conducted at DITM.</p>e<p>CUL, mycobacterial culture on Löwenstein-Jensen medium conducted at IML red, synlab, Asklepios Gauting, Germany.</p>f<p>FNA, fine-needle aspiration.</p>g<p>CLS, Puregene cell lysis solution, Qiagen, Germany.</p>h<p>ND, not done.</p>i<p>AFB, acid fast bacilli.</p>j<p>PANTA, transport medium for viable mycobacteria containing Polymyxin B, Amphotericin, Nalidixic acid, Trimethoprim, and Azlocillin.</p>k<p>No growth, culture result negative, no growth of acid fast bacilli.</p

Scar of secondary nodule at the back of the right thigh, September 2011.

<p>Scar of secondary nodule at the back of the right thigh, September 2011.</p

Specificity of 16S rRNA and IS<i>2404</i> qPCR assays.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t002" target="_blank">Table 2</a> shows DNA extracts from closely related mycobacterial species and bacteria potentially contaminating the human skin subjected to the combined 16S rRNA RT/IS<i>2404</i> qPCR viability assay and the corresponding test results. Mycobacterial species were selected according to their respective genetic contiguousness to <i>M. ulcerans</i> Agy99 (GenBank accession no. CP000325.1) within the 16S rRNA gene sequences as determined by BLASTN analysis (GenBank, NCBI) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Benson1" target="_blank">[13]</a>. <i>M.</i>, <i>Mycobacterium</i>; <i>E.</i>, <i>Escherichia</i>; <i>P., Propionibacterium</i>; <i>Staph</i>., <i>Staphylococcus</i>; <i>Str</i>., <i>Streptococcus</i>. While in-silico analysis revealed that the combined 16S rRNA RT/IS<i>2404</i> assay will also amplify mycolactone-producing mycobacteria (MPM) other than <i>M. ulcerans</i> (e.g., <i>M. pseudoshottsii</i>, <i>M. liflandii</i>, and the environmental <i>M. marinum</i> [GenBank accession No. NR_042988.1, AY500838.1, and AF456241.1, respectively]), these MPM species were not included in specificity testing.</p>a<p>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.</p>b<p>The respective primary patient isolates were considered as ^ppathogenic bacteria or as ^ccommensals/contaminants of clinical samples.</p>d<p>Results of the 16S rRNA RT-qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p>e<p>Results of the IS<i>2404</i> qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p

Primers and probes.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t003" target="_blank">Table 3</a> 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 IS<i>2404</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Fyfe1" target="_blank">[9]</a>.</p>a<p>TF, forward primer; TR, reverse primer; TP2, hydrolysis probe (TibMolBiol, Berlin, Germany).</p>b<p>16S rRNA, gene for the ribosomal 16S RNA detected as 16S cDNA; IS<i>2404</i>, insertion sequence <i>2404</i>.</p>c<p>Nucleotide positions are provided for the first (IS<i>2404</i>) or single (16S rRNA) copy of the respective amplicon in <i>M. ulcerans</i> Agy99 (GenBank accession no. CP000325.1) as determined by BLASTN analysis within GenBank (NCBI) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Benson1" target="_blank">[13]</a>.</p>d<p>bp, base pairs.</p>e<p>6 FAM, 6-Carboxyfluorescein fluorescent dye; BBQ, BlackBerry Quencher.</p>f<p>Primers T13 (forward) and T39 (reverse) were used for the amplification of a 935-bp region of the <i>M. ulcerans</i> 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 <i>M. ulcerans</i> 16S rRNA gene (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t001" target="_blank">Table 1</a>).</p

Primary nodule at the left costal arch, June 2010.

<p>Primary nodule at the left costal arch, June 2010.</p