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Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility.

ABSTRACT: We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.

SUBMITTER: Streicher EM

PROVIDER: S-EPMC4313251 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility.

Streicher Elizabeth M EM Maharaj Kashmeel K York Talita T Van Heerden Carel C Barnard Marinus M Diacon Andreas A Mendel Carl M CM Bosman Marlein E ME Hepple Juli A JA Pym Alexander S AS Warren Robin M RM van Helden Paul D PD

Journal of clinical microbiology 20140827 11

We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide. ...[more]

PMID: 25165081

Similar Datasets

Association between pncA gene mutations, pyrazinamidase activity, and pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

Project description:We determined MICs for, confirmed the presence of pncA mutations in, and performed pyrazinamidase testing on colonies (subclones) obtained from seven isolates that exhibited differential pyrazinamide (PZA) susceptibility. Six of the seven strains were found to exhibit characteristics resulting from the mixture of strains possessing different properties. In addition, our analysis revealed large pncA-spanning deletions (1,565 bp, 4,475 bp, and 6,258 bp) in three strains that showed high PZA resistance.

| S-EPMC4136016 | biostudies-literature

Rapid detection of Mycobacterium tuberculosis and pyrazinamide susceptibility related to pncA mutations in sputum specimens through an integrated gene-to-protein function approach.

Project description:Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.

| S-EPMC3911477 | biostudies-literature

Role of pncA and rpsA gene sequencing in detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates from southern China.

Project description:We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3' end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.

| S-EPMC3911430 | biostudies-literature

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

Project description:Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

| S-EPMC163747 | biostudies-other

Gene sequencing for routine verification of pyrazinamide resistance in Mycobacterium tuberculosis: a role for pncA but not rpsA.

Project description:Pyrazinamide (PZA) is an important component of first-line therapy for the treatment of tuberculosis. Here, we evaluate targeted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine sequencing of pncA, but not rpsA, is effective for verification of PZA susceptibility results.

| S-EPMC3486241 | biostudies-literature

Non-<i>pncA</i> Gene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Project description:Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB). pncA gene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-S TB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-type pncA gene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy: panD or rpsA mutations identified by whole-genome sequencing (WGS) (n = 7), heteroresistance (n = 8), and mixed populations with Mycobacterium avium (n = 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-R clinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymous pncA mutation (Thr87Met), whereas a nonsynonymous rpsA mutation was found in 17 isolates. None of these isolates carried a nonsynonymous panD mutation, while all 15 of the MDRPZA-R isolates harbored a nonsynonymous pncA mutation. Our findings indicate that it is necessary to consider the occurrence of panD mutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymous rpsA mutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role of rpsA in PZA resistance.

| S-EPMC5442549 | biostudies-literature

Pyrazinamide Resistance and pncA Mutation Profiles in Multidrug Resistant Mycobacterium Tuberculosis.

Project description:PurposePyrazinamide (PZA) is a critical component of standardized chemotherapy for tuberculosis (TB) and is recommended for the treatment of multidrug-resistant (MDR) TB. We aimed to characterize mutations in pncA of M. tuberculosis and evaluate their diagnostic accuracy for PZA susceptibility in China. We also combined genotypic methods with phenotypic susceptibility testing and pyrazinamidase (PZAse) activity to confirm PZA-resistant M. tuberculosis isolates.ResultsAn evaluation of 82 MDR M. tuberculosis strains revealed that 28.0% (23/82) were phenotypically resistant to 100 mg/L PZA and 15.9% (13/82) showed resistance to 300 mg/L PZA. Mutations in pncA were detected at 33 unique sites, and the majority were point mutations. No evident mutation hotspots or mutations affecting multiple amino acids were found, but the association between pncA mutations and PZA resistance was significant under 100 and 300 mg/L. The sensitivity of pncA mutation detection for predicting PZA susceptibility was 82.6% (19/23), and the specificity was 61.0% (36/59), based on 100 mg/L PZA, whereas the sensitivity was 84.6% (11/13) and the specificity was 55.1% (38/69), based on 300 mg/L PZA. All mutations identified in the highly PZA-resistant (300 mg/L) strains had an 80% loss relative to PZAse activity. No evident PZAse activity loss was observed in one synonymous mutation strain and the loss exceed 60% in all other strains.ConclusionThe association between pncA mutation and PZA resistance was significant. Relatively, the molecular method have shown better reliability than the phenotypic method for the detection of PZA resistance. This provides a theoretical basis for the clinical diagnosis of drug-resistant TB.

| S-EPMC9440668 | biostudies-literature

Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms.

Project description:A gene (pncA) with mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis complex members was characterized in 67 pyrazinamide-resistant and 51 pyrazinamide-susceptible isolates recovered from diverse geographic localities and anatomic sites and typed by IS6110 profiling. All pyrazinamide-susceptible organisms had identical pncA alleles. In striking contrast, 72% of the 67 resistant organisms had pncA mutations that altered the primary amino acid sequence of pyrazinamidase. A total of 17 previously undescribed mutations were found, including upstream mutations, missense changes, nucleotide insertions and deletions, and termination mutations. The mutations were arrayed along virtually the entire length of the gene. These data are further evidence that most drug resistance in M. tuberculosis is due to simple mutations occurring in chromosomally encoded genes rather than to acquisition of resistance genes by horizontal transfer events.

| S-EPMC163764 | biostudies-other

Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis.

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| S-EPMC6531379 | biostudies-literature

Correlation between pyrazinamide activity and pncA mutations in Mycobacterium tuberculosis isolates in Taiwan.

Project description:A total of 76 clinical Mycobacterium tuberculosis isolates from Taiwan were tested for pyrazinamidase activity, pyrazinamide susceptibility, and pncA mutations. Frequency of resistance to PZA rose with increases in resistance to first-line drugs. Of 17 pyrazinamide-resistant strains, 7 (3 of which had not been previously described) possessed mutations in the pncA gene.

| S-EPMC253789 | biostudies-literature

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