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

Date Published: November 13, 2015

Publisher: Public Library of Science

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

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.

Partial Text: Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, is an infectious disease affecting skin, soft tissues and sometimes the bones. The major endemic foci occur in rural areas of Sub-Saharan Africa where BUD mainly affects children below the age of 15 years.

BUD belongs to the currently five neglected diseases in line for the IDM (innovative and intensified disease management) approach demanding a major scaling up of active detection, treatment, monitoring and surveillance. Development of diagnostic tests that bring health services closer to where NTDs are is considered a research priority. LAMP, a technology that features cost effectiveness, robustness and modest needs in terms of equipment, has recently been selected by the WHO as one of the promising tools for decentralized diagnostics [13–14, 41].



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