Development of a Rapid Automated Point-of-Care Test for Mycobacterium tuberculosis Detection from Tongue Swabs and Sputum Specimens on the DASH® Rapid PCR System.

Mycobacterium tuberculosis (MTB) disease is a major global health threat with most tuberculosis (TB) cases occurring in low-and middle-income countries (LMIC) with limited healthcare infrastructure. Near-point-of-care testing which can be deployed at peripheral clinical settings is needed to start treatment earlier and thereby improve treatment outcomes. Here we report the development and preliminary characterization of an MTB detection assay that utilizes tongue swab or sputum specimens for The DASH® Rapid PCR System which employs cartridge-based automated sequence specific capture sample prep combined with dual target qPCR multicopy MTB insertion sequences IS6110 and IS1081 amplification and detection. MTB is resistant to conventional bacterial lysis techniques; therefore, we evaluated two pre-cartridge lysing techniques, mechanical lysis and sonication, and selected sonication for all subsequent studies. The DASH MTB assay demonstrated a limit of detection of 2.5 MTB cells/swab with no detection of 10 non-tuberculosis Mycobacterium strains. Clinical testing of 100 (49 positive and 51 negative) de-identified blinded sputa from South African symptomatic clinic attendees yielded an overall test sensitivity of 96% (100% for smear positive samples and 88% for smear negative samples) and specificity of 88% when compared to sputum culture. In a separate study of 110 tongue swab specimens (70 positive and 40 negative) from South African symptomatic clinic attendees, the sensitivity was 93% and the specificity was 100%. We further demonstrated that the test is compatible with peripheral LMIC settings via external battery operation and cartridge stability at 45°C for up to one year.