Dataset Information

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

ABSTRACT: Pyrazinamide (PZA) is a key antibiotic for the treatment of drug susceptible tuberculosis. PZA-resistance is mainly mediated by mutations in the pncA gene; however the current gold standard is a phenotypic drug susceptibility test requiring a well-adjusted pH-value for reliable results. Our melting curve assay detects a non-wild type genotype in selected pncA regions in at least 3750 gene copies/mL within 2.5 hours. The prototype assay was further evaluated by analyzing 271 Mycobacterium tuberculosis complex isolates from Swaziland originating from a previously published drug resistance survey and including 118 isolates with pncA mutations. Sensitivity was 83% (95% CI 75-89%) and specificity was 100% (95% CI 98-100%). Under consideration of further improvements with regard to the target range our melting curve assay has the potential as a rapid rule-in test for PZA susceptibility (wild type pncA), however false resistant results (mutant pncA, but PZA susceptible) cannot be ruled out completely.

SUBMITTER: Havlicek J

PROVIDER: S-EPMC6531379 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Havlicek Juliane J Dachsel Beatrice B Slickers Peter P Andres Sönke S Beckert Patrick P Feuerriegel Silke S Niemann Stefan S Merker Matthias M Labugger Ines I

Diagnostic microbiology and infectious disease 20181230 2

Pyrazinamide (PZA) is a key antibiotic for the treatment of drug susceptible tuberculosis. PZA-resistance is mainly mediated by mutations in the pncA gene; however the current gold standard is a phenotypic drug susceptibility test requiring a well-adjusted pH-value for reliable results. Our melting curve assay detects a non-wild type genotype in selected pncA regions in at least 3750 gene copies/mL within 2.5 hours. The prototype assay was further evaluated by analyzing 271 Mycobacterium tubercu ...[more]

PMID: 30733004

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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.

Project description:Tuberculosis (TB) remains a significant global health challenge, exacerbated by the emergence of drug-resistant strains, such as those resistant to pyrazinamide (PZA). The current scarcity of affordable and precise quantitative diagnostic tests for PZA resistance underscores the urgent need for more accessible diagnostic tools. We evaluated PZA susceptibility in 264 TB-positive samples by quantifying pyrazinoic acid (POA) production, using both the MODS-Wayne qualitative assay and our newly developed quantitative approach (MODS-WQ). The MODS-WQ was assessed in 7H9 medium (MODS-WQ7H9) or citrate buffer (MODS-WQCB), with POA levels measured via spectrophotometry against a calibration curve. PZA susceptibility determinations were based on a composite reference standard. Associations between POA levels and pyrazinamidase mutations were explored. The composite standard detected PZA resistance in 23.5% of the samples, which accounts for 62.8% of the multidrug-resistant (MDR) samples. The MODS-WQ established specific POA cutoffs of 123.25 µM for MODS-WQ7H9 and 664.7 µM for MODS-WQCB, with sensitivities of 81.3% and 92.3% and specificities of 77.2% and 95.9%, respectively. Notably, samples with mutations in the pyrazinamidase metal-binding site exhibited significantly lower POA levels compared with mutations in the enzyme periphery. Furthermore, a significant correlation was found between POA production and PZA resistance, Bactec Growth Index, and minimum inhibitory concentration (MIC) values. This study presents a novel, direct, and accessible susceptibility test for PZA resistance that quantifies POA, enhancing the detection capabilities for this condition. The citrate-buffered MODS-WQ assay demonstrated high sensitivity and specificity for quantifying POA, confirming that POA production is a reliable indicator of PZA resistance.ImportancePZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

Dataset Information

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

Publications

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

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