Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of PCR Sequencing.

An in-house-developed sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtie MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, sequencing detected mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel mutations without and mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in structural genes and 1 with a mutation (T-12C) at the promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.