Toward Precision Detection of Pyrazinamide Resistance: Critical Concentration Assessment and Rapid Molecular Method Validation

This study aimed to evaluate the diagnostic performance of broth microdilution (BMD) and fluorescence PCR melting curve analysis (MeltPro MTB/PZA assay) for detecting pyrazinamide (PZA) resistance in rifampicin-resistant tuberculosis (RR-TB).RR-TB strains isolated from patients in TB prevention and control in stitutions and designated hospitals in Beijing from January to December 2009 were analyzed. PZA susceptibility was assessed using BMD, MeltPro MTB/PZ A assay (targeting pncA mutations), and whole-genome sequencing (WGS). The sensitivity, specificity, agreement rate, and Kappa value of BMD (at critical c oncentrations (CCs) of 100 μg/mL and 200 μg/mL)and MeltPro MTB/PZA assa y were evaluated using WGS as the reference standard. At CCs of 100 μg/mL and 200 μg/mL, BMD showed resistance rates of 63.6% (70/110) and 45.5% (50/110), respectively. Compared to WGS, BMD demonstrated sensitivities of 96 .1% and 92.2%, specificities of 64.4% and 94.9%, with κ values of 0.590 and 0.872, respectively. The MeltPro assay detected resistance in 44.5% (53/119) of strains, with 89.7% sensitivity, 98.4% specificity, and κ value of 0.882. The MeltPro MTB/PZA assay demonstrates high diagnostic accuracy for PZA resista nce screening in RR-TB patients, supporting its utility as a frontline detection t ool. BMD at 200 μg/mL showed better concordance with WGS. Additionally, f ive novel pncA mutations (Ile5Thr, Leu27Gln, Ser67stop, Pro69Arg, Trp119Ser) identified in this study expand the catalog of known resistance-associated vari ants and offer new targets for mechanistic investigations.