Detection Of Pyrazinamide Resistance Research Articles

Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries

Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (DST) in clinical TB isolates from two countries of the Balkan Peninsula. Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel. Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.

Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.

Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

ABSTRACTRoutine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Pyrazinamide Drug Resistance in Mycobacterium Tuberculosis: A Minireview

Pyrazinamide (PZA) is a nicotinamide analog that is used as a frontline drug to treat tuberculosis (TB). It has special place in modern TB therapy, as it appears to kill a population of semi dormant tubercle bacilli persisting in the body. It is important drug because of its sterilizing activity against semi dormant tubercle bacilli. Although PZA has a very high in vivo activity, it’s in vitro activity is not apparent unless an acidic environment is available, which makes PZA drug susceptibility testing (DST) more difficult by conventional methods. Being a nicotinamide analogue, PZA needs to be converted into its active bactericidal form pyrazinoic acid (POA) by the mycobacterial enzyme pyrazinamidase (PZase).PZase is encoded by pncA, and mutations in this gene have been demonstrated as the major mechanism of PZA resistance.PZA-resistant Mycobacterium tuberculosis strains are usually correlated well with defective PZase activity. The rapid detection of PZA resistance is of utmost importance for an effective treatment and also to avoid further spread of MDR strains. Analysis of the pncAgene provides rapid and useful information regarding PZA susceptibility in M. tuberculosis and may therefore contribute to early optimization of treatment. The diversity of methods currently used in clinical laboratories for the detection of PZA resistance in M. tuberculosis isolates causes inconsistent results of PZA DST and such inconsistent results of PZA DST have been reported by a number of laboratories by various methods, including the qualitative BACTEC test. Therefore, it is essential to elucidate the genetic basis of clinical resistance and to correlate phenotypic and molecular resistance data.

Genome sequencing reveals novel deletions associated with secondary resistance to pyrazinamide in MDR Mycobacterium tuberculosis.

Detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates presents significant challenges in settings with no dominant clonal lineages, such as Australia. We assessed the utility of WGS versus standard PCR amplification assays for the characterization of pyrazinamide resistance in MDR-TB isolates identified in New South Wales, Australia, over an 8 year period. PCR amplicon sequencing was used to identify molecular markers associated with antibiotic resistance in pyrazinamide-resistant MDR-TB isolates recovered by the New South Wales Mycobacterium Reference Laboratory between 2007 and 2014. WGS was subsequently performed on two isolates for which pncA amplification failed. WGS identified two novel genomic deletions associated with in vitro resistance to pyrazinamide in MDR-TB. One isolate also carried a second deletion involving the genes dfrA and thyA associated with resistance to para-aminosalicylic acid. Steadily decreasing sequencing costs are increasing the appeal of WGS as an alternative approach for detecting complex patterns of pyrazinamide resistance in MDR-TB.

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

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.

Detection of pncA mutation associated with pyrazinamide resistance in Mycobacterium tuberculosis by high-resolution melting cure analysis

To evaluate the application of a high-resolution melting (HRM) curve analysis for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis (M. tuberculosis). Clinical isolates of M. tuberculosis were collected from Shenzhen from 2007 - 2009 and pncA mutations were characterized by sequence analysis. HRM analysis method was established and used to determine pncA gene mutation in 49 pncA mutant isolates and 78 wild type isolates. BCATEC MGIT 960 was used as the standard method to evaluate application of the HRM curve. Among 49 pncA mutant isolates, 48 isolates were characterized as mutant and 1 isolate with double point mutations misclassified as wild type by using HRM detection. In 78 wild type isolates, HRM found 6 mutant isolates. Using the BCATEC MGIT 960 to detect pyrazinamide drug susceptibility, 62 isolates were found resistant and 65 were susceptible. Among the 62 resistant strains by HRM results 53 strains were resistant, and among the 65 susceptible stains by HRM results 64 were susceptible. Compared to the results of BACTEC MGIT 960, the sensitivity and specificity of HRM were 85.5% (53/62) and 98.5% (64/65), respectively. The Youden index was found to be 0.840. The results showed a good correlation between HRM curve analysis and BACTEC MGIT 960. HRM curve analysis is a rapid and accurate method that can be used as a screening test for identification of resistance to pyrazinamide.

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.

Development and Evaluation of a Line Probe Assay for Rapid Identification of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Strains

Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA. The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis. The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.

Novel pncA Mutations in Pyrazinamide-Resistant Isolates of Mycobacterium tuberculosis.

The gene coding for pyrazinamidase (PZase) activity, pncA, in Mycobacterium tuberculosis was recently cloned, and several mutations which correlate with in vitro resistance to pyrazinamide (PZA) have been identified. During the development of a clinical molecular assay for the detection of PZA resistance, two previously unreported mutations in isolates of PZA-resistant M. tuberculosis were identified. The assay that uses automated DNA sequencing is relatively rapid and allows the detection of any mutations present in the coding region of the pncA gene. The new mutations are both point mutations resulting in amino acid substitutions; 241T --> G results in F80V, and 511G --> C results in A171P. The identification of these mutations accentuates the utility of automated DNA sequencing in the clinical laboratory.

Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography.

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.