MycoPLEX: Multiplexed fragment analysis for mycobacterial species and subspecies identification.

Accurate, rapid routine identification of mycobacteria from clinical material remains challenging due to the need for either laboratory-developed molecular or mass-spectrometric assays or a complex sequencing method. Furthermore, there are currently no FDA-cleared methods for definitive identification of nontuberculous mycobacteria (NTM), despite increasing recognition of these organisms as important opportunistic pathogens. To address these diagnostic gaps, we developed and validated MycoPLEX, a novel multiplexed fragment analysis assay for detecting unique mycobacterial gene targets from either solid or liquid culture media within three hours of specimen preparation. MycoPLEX identifies members of the Mycobacterium tuberculosis complex (MTBC) as well as NTM within the Mycobacterium avium complex (MAC), Mycobacterium kansasii, and Mycobacterium gordonae/paragordonae. Additionally, subspecies within the Mycobacterium abscessus are distinguished through detection of either intact (M. abscessus subsp. abscessus / bolletii) or truncated (M. abscessus subsp. massiliense) erythromycin ribosomal methylase gene, erm(41). The overall sensitivity and specificity of MycoPLEX were 100%, respectively (MTBC only: 100% and 100%; NTMs only: 100% and 100%, respectively). MycoPLEX is a practical screening test accessible to most clinical laboratory setups, enabling the rapid identification of clinically significant mycobacteria.