Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan

Abstract

BackgroundPyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan.MethodsWe performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations.ResultsA total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%).ConclusionMutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

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

“…In this study, we detected 32 new mutations. [38][39][40][41][42] In addition, 89 of the100mutations were found in only one strain, and the remaining 11 mutations, which contained 35 strains, were shared mutations. Mutations in the promoter region were associated with PZA resistance.…”

Rapid Pyrazinamide Drug Susceptibility Testing using a Closed-Tube PCR Assay of the Entire pncA gene

“…Finally, the assay focuses on any mutation in the entire pncA gene, which may result in identification of mutations not associated with PZA resistance 26,35,36 . The next version of the assay could be expanded to include targets from the rpsA gene, since mutations in this gene have been associated with PZA resistance and to develop an algorithm to relate specific mutations to fluorescent signatures for pncA mutations not associated with PZA resistance 37,38 .…”

Structural Dynamics Behind Clinical Mutants of PncA-Asp12Ala, Pro54Leu, and His57Pro of Mycobacterium tuberculosis Associated With Pyrazinamide Resistance

“…All the PZA-resistant samples were subjected to genomic DNA extraction via sonication (Buck et al, 1992;Kirschner et al, 1993;Khan et al, 2019b). The Mycobacterium Growth Indicator Tube (MGIT) that contained a fresh culture from which 1 µL was taken and transferred to a microcentrifuge tube.…”