Abstract
Resistance to pyrazinamide (PZA) poses significant challenges to tuberculosis (TB) management, and prediction of susceptibility to PZA has been challenging. This study examined PZA resistance-associated gene mutations in 125 multidrug-resistant clinical isolates of Mycobacterium tuberculosis (MTB) from Wenzhou, China. Phenotypic drug-susceptibility testing (pDST) for PZA was performed on clinical isolates using the MGIT 960 system to accurately determine resistance. Subsequently, whole-genome sequencing (WGS) was conducted on the 125 isolates to identify genetic mutations linked to PZA resistance, focusing specifically on the pncA, rpsA, and panD genes. To provide a rapid diagnostic alternative to traditional methods, the MeltPro MTB/PZA assay was also utilized to detect mutations in pncA. pDST revealed a PZA resistance rate of 59.20%, with 29.41% observed in strains resistant only to isoniazid and rifampicin, 77.61% in pre-extensively drug-resistant TB (pre-XDR-TB), and 100% in extensively drug-resistant TB (XDR-TB). Among the isolates, WGS identified mutations primarily in the pncA (64.00%) and rpsA (6.40%), with panD mutations not detected. PZA resistance was strongly associated with pncA mutations, present in 97.30% of PZA-resistant strains. WGS demonstrated 97.30% sensitivity and 84.31% specificity compared to pDST, while MeltPro MTB/PZA showed 91.89% sensitivity and 86.27% specificity. Compared to WGS, MeltPro MTB/PZA showed 92.50% positive percent agreement and 97.78% negative percentage agreement, highlighting its diagnostic value. In conclusion, PZA resistance in multidrug-resistant tuberculosis (MDR-TB) is primarily due to pncA mutations. MeltPro MTB/PZA assay offers a reliable, rapid alternative for PZA resistance prediction, supporting timely treatment adaptations for improved TB patient care. IMPORTANCE: This study underscores the pressing need for reliable diagnostic methods to address high PZA resistance rates in TB cases, particularly in MDR-TB strains. By confirming that pncA mutations are the principal drivers of PZA resistance, we highlight the diagnostic potential of the MeltPro MTB/PZA assay as a rapid and effective alternative to conventional culture-based methods. Demonstrating sensitivity and specificity comparable to WGS and pDST, this assay offers a practical, accessible approach for timely PZA resistance prediction. It supports more tailored and effective MDR-TB treatment strategies, which are essential for optimizing patient care in both well-resourced and constrained settings.