Direct identification of mycobacteria from clinical specimens by multiplex real-time PCR.

Abstract

To directly identify clinically relevant mycobacteria from clinical specimens, we have developed a multiplex real-time PCR assay with hydrolysis probes that can identify 20 mycobacterial species. The assay was initially evaluated using 248 strains, including both reference strains and clinical isolates. Then, the assay was implemented according to a scheme in our laboratory. The scheme based on the clinical differences between the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) consisted of three stepwise PCRs. MTC and NTM were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and step 3 PCRs. During a 2·5-year period, 1877 isolates of MTC (1142 directly recovered from clinical specimens) and 596 isolates of NTM (143 directly recovered from clinical specimens) were detected, and the species of 590 (99·0%) of the 596 NTM isolates were identified. Our experience shows that this is a new paradigm for rapidly and accurately identifying clinically relevant mycobacteria, in which a multiplex real-time PCR assay is directly applied to clinical specimens in a stepwise fashion. This study is the first report of a multiplex real-time PCR assay for identifying clinically important mycobacterial species directly from clinical specimens and its application in a clinical microbiology laboratory.