Detection of <i>Mycobacterium ulcerans</i> with IS <i>2404</i> loop-mediated isothermal amplification and a fluorescent reporter probe

An exquisitely sensitive quantitative PCR (qPCR) assay targeting the high-copy number insertion sequence, IS<i>2404</i>, is the gold standard diagnostic test for <i>Mycobacterium ulcerans</i>, the agent of the neglected tropical skin disease Buruli ulcer. Here, we designed and tested an alternative <i>M. ulcerans</i> diagnostic test, a fluorescent probe-based, loop-mediated isothermal amplification (P-LAMP) assay that also targets IS<i>2404</i>. Benchmarked against IS<i>2404</i> qPCR, P-LAMP was equally specific and nearly as sensitive (analytical sensitivity of four vs two <i>M. ulcerans</i> genome copies). Clinical and environmental specimen validation against IS<i>2404</i> 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 IS<i>2404</i> is a versatile assay that addresses the performance issues of previously described IS<i>2404</i> 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 <i>Mycobacterium ulcerans</i>. Correct diagnosis is essential before appropriate treatment for Buruli ulcer can be started. Development of a portable, easy-to-use diagnostic test for <i>M. ulcerans</i> 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 <i>M. ulcerans</i> and show that this new test has equivalent performance to the gold standard <i>M. ulcerans</i> 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 <i>M. ulcerans</i> diagnostics anywhere.