Faster, clearer tests for pyrazinamide resistance in RR-TB

Drug resistance in tuberculosis is a major obstacle to effective treatment, and pyrazinamide (PZA) is an important drug used in many TB regimens. Detecting whether a strain is resistant to PZA is therefore critical for guiding therapy, but testing can be challenging. To address this, Xiaowei Dai and colleagues set out to compare laboratory approaches that could deliver reliable, rapid answers about PZA resistance in rifampicin-resistant tuberculosis (RR-TB). The team analyzed RR-TB strains collected from patients in TB prevention and control institutions and designated hospitals in Beijing between January and December 2009. They compared a traditional microbiological test, broth microdilution (BMD), with a rapid molecular test, the MeltPro MTB/PZA assay, which looks for mutations in the pncA gene known to cause PZA resistance. Whole-genome sequencing (WGS) served as the reference standard to judge how well each test detected resistance. The work was designed to show which approach, or which settings of the approaches, most closely matched WGS and could therefore be used confidently in clinical screening.

The researchers assessed PZA susceptibility using three methods: BMD, the MeltPro MTB/PZA assay (targeting pncA mutations), and whole-genome sequencing (WGS). For BMD they evaluated two critical concentrations (CCs): 100 μg/mL and 200 μg/mL. At those CCs, BMD classified 63.6% (70/110) and 45.5% (50/110) of isolates as resistant, respectively. Using WGS as the reference, BMD at 100 μg/mL showed 96.1% sensitivity and 64.4% specificity with a κ value of 0.590; at 200 μg/mL it showed 92.2% sensitivity and 94.9% specificity with a κ value of 0.872. The MeltPro MTB/PZA assay detected resistance in 44.5% (53/119) of strains and, against WGS, achieved 89.7% sensitivity, 98.4% specificity, and a κ value of 0.882. In addition, the study identified five novel pncA mutations—Ile5Thr, Leu27Gln, Ser67stop, Pro69Arg, Trp119Ser—adding to the known catalog of resistance-associated variants.

These findings point toward practical options for more precise detection of PZA resistance in RR-TB. The MeltPro MTB/PZA assay demonstrated high diagnostic accuracy and strong agreement with WGS, supporting its use as a frontline screening tool when rapid results are needed. Broth microdilution at the higher CC of 200 μg/mL matched WGS more closely than the lower CC, suggesting that choosing the right BMD threshold matters for concordant results. The discovery of five novel pncA mutations expands the set of genetic changes laboratories should consider when interpreting molecular tests and provides new leads for laboratory studies into how those changes cause resistance. Altogether, the work led by Xiaowei Dai offers evidence that combining rapid molecular screening with carefully calibrated microbiological testing and genetic confirmation can improve confidence in PZA resistance calls for RR-TB patients.