Rapid Sequencing of the Mycobacterium tuberculosis
            <i>pncA</i>
            Gene for Detection of Pyrazinamide Susceptibility

Streicher, Elizabeth M.; Maharaj, Kashmeel; York, Talita; Heerden, Carel van; Barnard, Marinus; Diacon, Andreas H.; Mendel, Carl M.; Bosman, M; Hepple, Juli A.; Pym, Alexander S.; Warren, Robin M.; Helden, Paul D. van

doi:10.1128/jcm.02438-14

Cited by 19 publications

(19 citation statements)

References 10 publications

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“…Another four (44%) PZA-resistant strains did not show any nucleotide change suggesting that the mechanism of resistance was not conferred by mutations in pncA, but could involve decreasing the drug delivery into the bacterial cell or increasing the efflux pump. Resistance may also involve mutations in other genes that were not tested here such as rpsA [55]. Based on these results, sequence analysis of pncA may not be appropriate for detecting PZA resistance.…”

Section: Discussionmentioning

confidence: 98%

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

Suthum

Samosornsuk

2020

J Infect Dev Ctries

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Introduction: Multidrug-resistant tuberculosis (MDR-TB) is commonly found in Thailand especially in the public health region 5, the Western region of Thailand. This study’s aim was to characterize katG, inhA, rpoB and pncA genes in Mycobacterium tuberculosis. Methodology: One hundred strains of Mycobacterium tuberculosis (MTB) were isolated from sputum samples of MDR-TB risk patients in the laboratory of the Office of Disease Prevention and Control 5th Ratchaburi province, Thailand from January to December 2015. Drug susceptibility testing (DST) was performed using a BACTEC MGIT 960 system. Furthermore, the genes katG, inhA, rpoB and pncA were characterized by DNA sequencing. Results: Of a total of 100 MTB samples which underwent drug susceptibility testing, 42% showed isoniazid (INH) and rifampicin (RIF) resistance, and a further 25% showed INH mono-resistance (25%). The most common gene mutations found using DNA sequencing were katG_Ser315Thr (70%), rpoB_Ser531leu (81%) and pncA_Ile31Thr (84%). The common mutation of pncA_Ile31Thr substitution was detected in 26 of 91 (29%) pyrazinamide (PZA) susceptible isolates. Conclusion: Using DNA sequencing to screen for gene mutations conferring drug resistance may be feasible and use less time than using DST to detect resistance patterns.

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Section: Discussionmentioning

confidence: 98%

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

Suthum

Samosornsuk

2020

J Infect Dev Ctries

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“…Review of the current literature showed that the levels of pncA mutation concordance in PZA-resistant strains ranged from 72% to 99%, suggesting that pncA is a good indicator for detecting PZA resistance (25). The diagnostic performance of the use of the pncA sequence to predict PZA resistance has been previously evaluated (10,15,26,27), but those retrospective evaluations were conducted on clinical isolates or AFB smear-positive specimens only. In our current study, apart from using archived MTBC clinical isolates collected over a 14-year period to demonstrate test accuracy, we also extended our findings by prospectively evaluating our in-housedeveloped pncA sequencing for genotypic identification of PZA resistance in both AFB smear-positive and AFB smear-negative respiratory specimens.…”

Section: Discussionmentioning

confidence: 99%

“…Other molecular approaches such as pncA sequencing and use of the commercial Genoscholar PZA-TB II assay (NIPRO Corporation, Japan) have been evaluated for analyzing pncA mutations in previous studies (13)(14)(15). However, evaluation studies of these platforms included only purified isolates or specimens subjected to genolysis with confirmed smear-positive and MTBC culture results that were positive for acid-fast bacilli (AFB) (13)(14)(15). In response to the current threat of MDR-TB pandemic, a rapid and highly accurate PZA resistance diagnostic tool is, therefore, urgently needed (16).…”

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confidence: 99%

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

et al. 2019

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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.

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“…DNA sequencing of the pncA, panD and rpsA genes were done to identify PZA resistance, as previously described [12, 13, 16]. No restriction fragment length polymorphism (RFLP) genotyping was performed.…”

Section: Methodsmentioning

confidence: 99%

Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis

et al. 2016

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Rapid Sequencing of the Mycobacterium tuberculosis pncA Gene for Detection of Pyrazinamide Susceptibility

Cited by 19 publications

References 10 publications

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

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

Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis

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