Toward precision detection of pyrazinamide resistance: critical concentration assessment and rapid molecular method validation.

This study evaluated broth microdilution (BMD) and fluorescence PCR melting curve analysis (MeltPro MTB/PZA, targeting pncA mutations) for detecting pyrazinamide (PZA) resistance-associated mutations in rifampicin-resistant tuberculosis (RR-TB). PZA susceptibility was assessed in RR-TB isolates from patients at TB prevention and control institutions and at designated hospitals in Beijing. Whole-genome sequencing (WGS) was used as a genotypic comparator to assess the agreement of BMD at critical concentrations (CCs) of 100 and 200 μg/mL and of the MeltPro assay. BMD showed resistance rates of 63.6% (70/110) at 100 μg/mL and 45.5% (50/110) at 200 μg/mL. Relative to WGS-defined genotypes (wild-type vs. mutation), BMD at 100 μg/mL showed 96.1% agreement for isolates with mutations and 64.4% for wild-type (κ = 0.590); at 200 μg/mL, agreement was 92.2% for mutations and 94.9% for wild-type (κ = 0.872). The MeltPro assay showed 89.7% agreement for isolates with mutations and 98.4% for wild-type (κ = 0.882). These findings indicate that using BMD at 200 μg/mL as the critical concentration achieves closer agreement with WGS-defined genotypes than 100 μg/mL, and that the MeltPro MTB/PZA assay shows high concordance with WGS for rapid detection of pncA mutations in RR-TB isolates. Further validation using phenotypic reference methods and clinical outcomes is warranted.