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

Marcus Beissner,Moritz Jansson,Florian Battke,Kristina Lydia Huber,Gisela Bretzel,Malkin Bauer,Michael Frimpong,Kossi Badziklou,Franz Xaver Wiedemann,Agata Rhomberg,Abiba Banla Kere,Karl-Heinz Herbinger,Thomas Löscher,Dominik Symank,Fred Stephen Sarfo,Ebekalisai Piten,Richard Odame Phillips,Issaka Maman

doi:10.1371/journal.pntd.0004219

Abstract

BackgroundAs 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 FindingsFollowing 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/SignificanceBoth 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.

Highlights

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, is an infectious disease affecting skin, soft tissues and sometimes the bones
The results from this study suggest that the assay provides a valuable alternative to other PCR tests as currently used for laboratory confirmation of BUD
According to McNemar test, there was no significant difference between DRB PCR or conventional gel-based PCR (cPCR) (100% concordant results) compared with quantitative realtime PCR (qPCR) (ORcrude = 1.60; 95%-CI: 0.81–3.20; P-value = 0.15), between Conventional IS2404 LAMP (cLAMP) compared with qPCR (ORcrude = 1.76; 95%-CI: 0.89–3.50; P-value = 0.08) and between DRB PCR or cPCR compared with cLAMP (ORcrude = 1.10; 95%-CI: 0.57–2.11; P-value = 0.76)

Summary

Introduction

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, is an infectious disease affecting skin, soft tissues and sometimes the bones. Application of the 100% M. ulcerans specific diagnostic reference standard for clinical samples, i.e. amplification of the multicopy insertion sequence (IS) 2404 by dry-reagent-based (DRB) PCR, conventional gel-based PCR (cPCR), or quantitative realtime PCR (qPCR) requires fully equipped molecular biology units with highly-skilled personnel and is mostly restricted to tertiary (reference) level laboratories or national research centres [4,5,6,7,8,9]. As the major burden of BUD exists in (remote) rural areas of endemic countries and up to one-third of BUD cases are diagnosed in advanced category III stages [10–. 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. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagentbased LAMP assay (DRB-LAMP) employing lyophilized reagents

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Results

Discussion

Conclusion