Abstract
Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZAR) primarily related to pncA mutations. However, discordance between phenotypic resistance and conventional pncA-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among Mycobacterium tuberculosis (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZAR strains were genetically assessed by Sanger sequencing of PZAR-associated customary genes. Five pncA-wild-type PZAR strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZAR strains harbored mutations in pncA and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZAR strains lacked pncA mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating clpC1 mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (pncA alone) to 100%. This is the first report from southern China that identifies novel clpC1 mutations in wild-type pncA PZAR Mtb isolates. Our findings underscore the limitations of pncA-targeted diagnostics and support the integration of WGS and clpC1 analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.