Heliyon (Jan 2025)
Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China
Abstract
Background: Diseases caused by M. tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) have similar clinical symptoms but require different treatments. Rapid and accurate identification of MTB and NTM is essential for proper patient management and treatment. Methods: To develop and assess a multiplex real-time fluorescence PCR (Multiplex PCR) method for rapid identification of MTB, M. avium complex (MAC), M. chelonae-M. abscessus group (MCAG), and M. kansasii in clinical samples. The specificity and limit of detection (LOD) were tested using standard strains and clinical isolates. The accuracy of the Multiplex PCR was validated with DNA from 228 known clinical samples confirmed by Targeted next-generation sequencing (tNGS). Additionally, 901 consecutive clinical samples were assessed to evaluate the Multiplex PCR's diagnostic performance in detecting mycobacteria in pulmonary and extrapulmonary samples. Results: LOD of the four mycobacteria ranged from 11.7 to 360.0 CFU/mL in water, 43.8–922.0 CFU/mL in sputum, and 53.2–859.3 CFU/mL in sputum mixed infection, with 98.7 % sample detection accuracy and 100 % strains identification accuracy. Based on the composite reference standard, the sensitivity of the Multiplex PCR for detecting tuberculosis in pulmonary and extrapulmonary samples was comparable to Xpert (p > 0.05) and higher than Culture, especially in extrapulmonary samples (P 0.05) but higher than CapitalBio RT-PCR (P < 0.05). Overall, the Multiplex PCR showed significantly higher sensitivity for mycobacterial diseases compared to the other three methods (P < 0.01), with a specificity of 96 %. Conclusions: The Multiplex PCR demonstrated excellent diagnostic performance in both pulmonary and extrapulmonary samples, offering a low-cost, rapid identifying tool for major pathogenic mycobacteria in eastern China.
