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Background  The reduced amounts of  Mycobacterium leprae  ( M .  leprae ) among paucibacillary (PB) patients reflect the need to further optimize methods for leprosy diagnosis. An increasing number of reports have shown that droplet digital polymerase chain reaction (ddPCR) is a promising tool for diagnosis of infectious disease among samples with low copy number. To date, no publications have investigated the utility of ddPCR in the detection of  M .  leprae . The aim of this study was to develop and evaluate a ddPCR assay for the diagnosis of PB leprosy.    Methodology  The two most sensitive DNA targets for detection of  M .  leprae  were selected from electronic databases for assessment of sensitivity and specificity by quantitative polymerase chain reaction (qPCR) and ddPCR. Control patients (n = 59) suffering from other dermatological diseases were used to define the cut-off of the duplex ddPCR assay. For comparative evaluation, qPCR and ddPCR assays were performed in 44 PB patients and 68 multibacillary (MB) patients.    Principal findings   M .  leprae -specific repetitive element ( RLEP ) and  groEL  (encoding the 65 kDa molecular chaperone GroEL) were used to develop the ddPCR assay by systematically analyzing specificity and sensitivity. Based on the defined cut-off value, the ddPCR assay showed greater sensitivity in detecting  M .  leprae  DNA in PB patients compared with qPCR (79.5% vs 36.4%), while both assays have a 100% sensitivity in MB patients.    Conclusions/Significance  We developed and evaluated a duplex ddPCR assay for leprosy diagnosis in skin biopsy samples from leprosy patients. While still costly, ddPCR might be a promising diagnostic tool for detection of PB leprosy.

Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens