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

Abstract

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis 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 pncA 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 Realtime 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 pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD 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 pncA structural genes and 1 with a mutation (T-12C) at the pncA 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 pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

<p>Pyrazinamide Resistance and Mutation Patterns Among Multidrug-Resistant <em>Mycobacterium tuberculosis</em> from Henan Province</p>

“…Moreover, out of 35 strains with shared mutations, 28 strains belonged to the Beijing genotype, six strains belonged to the T1 genotype, and one strain belonged to the T2 genotype. Sixteen strains with shared mutations had the same spoligotype and were 11,13,15 therefore, panD and rpsA mutations were analysed in this study. In 2011, Shi et al reported that rpsA could be another PZA resistance-related gene.…”

“…Specifically, the PZA resistance rate is 2-7.5% in non-MDR-TB patients and 36-85% in MDR-TB patients. 15,16 Therefore, the reliable prediction of PZA resistance before treatment facilitates the development of more effective treatments. As the most populous region in China, Henan has the largest number of patients with TB and MDR-TB in the country, making it a hotspot for TB prevention.…”

Drug resistance and epidemiology characteristics of multidrug-resistant tuberculosis patients in 17 provinces of China

“…The low pH medium and the stringent inoculum size of the assays often lead to inconsistent and nonreproducible results which deter clinicians from relying on these tests for guidance. Hence those data were unavailable in this study[34]. Also, we should point out that demographic pattern and epidemiological link associated with drug resistance development are multifactorial.…”

Hindsight Is 2019—the Year in Clinical Microbiology

“…There is a considerable body of literature on the impact of an MTBC gene with pyrazinamidase/nicotinamidase activity (pncA) on PZA resistance. Sequencing pncA to evaluate for mutations demonstrated very good sensitivity for detecting PZA resistance in a retrospective cohort of isolates with defined phenotypic AST profiles, as well as in a small prospective evaluation [9]. Of note, sequencing results were all available within 4 days during the prospective study compared to 35 days for phenotypic testing of the same isolates performed by the same laboratory.…”