Detection of Mycobacterium ulcerans with IS2404 loop-mediated isothermal amplification and a fluorescent reporter probe.

An exquisitely sensitive quantitative PCR (qPCR) assay targeting the high-copy number insertion sequence, IS2404, is the gold standard diagnostic test for Mycobacterium ulcerans, the agent of the neglected tropical skin disease Buruli ulcer. Here, we designed and tested an alternative M. ulcerans diagnostic test, a fluorescent probe-based, loop-mediated isothermal amplification (P-LAMP) assay that also targets IS2404. Benchmarked against IS2404 qPCR, P-LAMP was equally specific and nearly as sensitive (analytical sensitivity of four vs two M. ulcerans genome copies). Clinical and environmental specimen validation against IS2404 qPCR showed P-LAMP had 100% sensitivity and specificity. P-LAMP was twice as fast as qPCR with an average time-to-positive at the limit-of-detection of 19 minutes. P-LAMP targeting IS2404 is a versatile assay that addresses the performance issues of previously described IS2404 LAMP formats. This study tackles a key research priority for Buruli ulcer and represents another avenue for the development of rapid and accessible molecular diagnostics for this neglected tropical disease.IMPORTANCEBuruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. Correct diagnosis is essential before appropriate treatment for Buruli ulcer can be started. Development of a portable, easy-to-use diagnostic test for M. ulcerans has been identified by the World Health Organization as a research priority. Buruli ulcer most commonly occurs in remote, rural areas; therefore, an ideal test is one that can be used at (or near) the point of care (community health centres) without the need for specialized laboratories. Here, we describe a molecular test using loop-mediated isothermal amplification (LAMP) to detect DNA specific to M. ulcerans and show that this new test has equivalent performance to the gold standard M. ulcerans PCR test currently used worldwide. Our new test is rapid (30 minutes to run), simple to perform, and could be further developed into a robust, portable format to provide accessible and affordable M. ulcerans diagnostics anywhere.