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

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.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.