Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Havlicek J
PROVIDER: S-EPMC6531379 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tests based on bacteriophage replication enable rapid screening of Mycobacterium tuberculosis for drug resistance. We describe a novel broth-based colorimetric method for detecting phage replication. When clinical isolates were tested by this novel method, high concordance was observed with both the traditional phage assay and gene mutation analysis for detection of resistance to rifampin.
| S-EPMC1865820 | biostudies-literature
Project description:Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9 to 57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The ability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of Bactec MGIT 960, Wayne's test, and whole-genome sequencing (WGS) to detect PZA-resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both Bactec MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance, whereas Wayne's test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug-resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools for their capacity to detect heteroresistance in TB.
| S-EPMC8370246 | biostudies-literature
Project description: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.
| S-EPMC4313251 | biostudies-literature
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
Project description:Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.
| S-EPMC4657947 | biostudies-literature
Project description:Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.
| S-EPMC525196 | biostudies-literature
Project description:[This corrects the article DOI: 10.1371/journal.pone.0143444.].
| S-EPMC4894557 | biostudies-literature
Project description:BACKGROUND:Drug-resistant tuberculosis (TB), including resistance to both rifampicin (RIF) and isoniazid (INH) referred to as multidrug-resistant tuberculosis (MDR-TB), has become an increasing global threat in recent years. Effective management of patients infected with MDR-TB strains requires identifying such patients by performing conventional drug-susceptibility testing (DST) on bacteria isolated from sputum, a process that can take up to 2 months. This delay in diagnosis can result in worsening and continued transmission of MDR-TB. Molecular methods that rely upon nucleic acid amplification of specific alleles known to be associated with resistance to specific drugs have been helpful in shortening the time to detect drug resistant TB. METHODS:We investigated the utility of the REBA MTB-Rifa®, a commercially available line probe assay (LPA) for detecting rifampicin (RIF) resistance in the RIF resistance-determining region (RRDR) of the rpoB gene. Altogether, 492 Mycobacterium tuberculosis (M. tuberculosis) clinical isolates and additional 228 smear- and culture-positive sputum samples with confirmed M. tuberculosis were collected from subjects with suspected MDR-TB in South Korea. The results were compared with conventional phenotypic DST and sequencing of the rpoB gene. RESULTS:A total of 215 of the 492 isolates were resistant to RIF by conventional DST, and of which 92.1% (198/215) were MDR-TB strains. The REBA MTB-Rifa® assay identified RIF resistance in 98.1% (211/215) of these isolates but failed to identify resistance in four phenotypically RIF resistant isolates. These four isolates lacked mutations in the RRDR but three were confirmed to be MDR-TB strains by sequencing. The sensitivity and specificity of this test for clinical isolates was thus 98.1% (211/215) and 100% (277/277), respectively. When applied directly to 228 smear positive sputum samples, the sensitivity and the specificity of REBA MTB-Rifa® assay was 100% (96/96, 132/132), respectively. CONCLUSIONS:These findings support the use of the REBA MTB-Rifa® assay for rapid detection of RIF resistance on clinical isolates and smear positive sputum samples. The results also suggest that RIF resistance is a good surrogate marker of MDR-TB in South Korea and the need to add more probes to other LPAs which can cover newly identified mutations relevant to RIF resistance.
| S-EPMC3852947 | biostudies-literature
Project description:Pyrazinamide (PZA) has important sterilizing activity in tuberculosis (TB) chemotherapy. We describe trends, risk factors, and molecular epidemiology associated with PZA-resistant (PZA(r)) Mycobacterium tuberculosis in New York City (NYC). From 2001 to 2008, all incident culture-positive TB cases reported by the NYC Department of Health and Mental Hygiene (DOHMH) were genotyped by IS6110-based restriction fragment length polymorphism and spoligotype. Multidrug-resistant (MDR) isolates underwent DNA sequencing of resistance-determining regions of pncA, rpoB, katG, and fabG1. Demographic and clinical information were extracted from the NYC DOHMH TB registry. During this period, PZA(r) doubled (1.6% to 3.6%) overall, accounting for 44% (70/159) of the MDR population and 1.4% (75/5511) of the non-MDR population. Molecular genotyping revealed strong microbial phylogenetic associations with PZA(r). Clustered isolates and those from acid-fast bacillus (AFB) smear-positive cases had 2.7 (95% confidence interval [CI] = 1.71 to 4.36) and 2.0 (95% CI = 1.19 to 3.43) times higher odds of being PZA(r), respectively, indicating a strong likelihood of recent transmission. Among the MDR population, PZA(r) was acquired somewhat more frequently via primary transmission than by independent pathways. Our molecular analysis also revealed that several historic M. tuberculosis strains responsible for MDR TB outbreaks in the early 1990s were continuing to circulate in NYC. We conclude that the increasing incidence of PZA(r), with clear microbial risk factors, underscores the importance of routine PZA drug susceptibility testing and M. tuberculosis genotyping for the identification, control, and prevention of increasingly resistant organisms.
| S-EPMC4576106 | biostudies-literature
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