A simplified pyrazinamidase test for Mycobacterium tuberculosis pyrazinamide antimicrobial susceptibility testing

Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) treatment by eradicating the persisting Mycobacterium tuberculosis complex (MTBC). Due to cost and technical challenges, end TB strategies are hampered by the lack of a simple and reliable culture-based PZA antimicrobial susceptibility testing (AST) for routine use. We initially developed a simplified chromogenic pyrazinamidase (PZase) test in the TB reference laboratory using a training set MTBC isolates with various drug-resistant profiles, and validated its performance using consecutive BACTEC MGIT 960 (MGIT)-culture-positive culture in 10 clinical laboratories. The pncA gene Sanger sequencing results were used as the reference, and compared to the MGIT-PZA AST. Differential diagnosis of Mycobacterium bovis was conducted using patented in-house real-time PCR. Of the 106 training isolates, the PZase test and MGIT-PZA AST showed 100.0% and 99.1% concordance as compared to Sanger sequencing, respectively. We found 32.1% (34/106) isolates harbored pncA mutations, including one isolate with silent mutation S65S. For validation, 1,793 clinical isolates were tested including 150 duplicate isolates from specimens of the same cases and 16 isolates with uncharacterized drug resistance (UDR)-associated mutations. Excluding duplicated and UDR isolates, we identified 2.6% (43/1,627) PZA-resistant isolates, including 1.3% (21/1,627) M. bovis isolates. The kappa values were 0.851-1.000. In addition, the accuracy of the PZase test conducted by 10 laboratories was 98.5%-100.0%. Our simplified PZase test demonstrated high concordance with Sanger sequencing and MGIT-PZA AST. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB.

We developed and validated a simple pyrazinamidase (PZase) test for pyrazinamide (PZA) antimicrobial susceptibility testing (AST). Our results demonstrated that the PZase test had high agreement with the pncA gene sequencing and MGIT-PZA AST. Integrating PZase test into routine AST is effortless and represents an improvement in laboratory services for ending TB.