Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Gratz, Jean; Rahman, S. M. Mazidur; Ferdous, Sara Sabrina; Foongladda, Suporn; Boonlert, Duangjai; Огарков, О. Б.; Жданова, С. Н.; Kibiki, Gibson; Heysell, Scott K; Houpt, Eric R.

doi:10.1128/mbio.02273-14

Cited by 29 publications

(35 citation statements)

References 35 publications

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“…HRM has also been used successfully as a molecular DST for other drugs, including isoniazid (INH), fluoroquinolones, streptomycin, and pyrazinamide, in M. tuberculosis clinical isolates from sputum, with a sensitivity range of 74 to 100% and specificity range of 83 to 100% (9)(10)(11)(12)(13)(14)(15)(16). A microfluidic TaqMan array card that utilizes sequence-specific probes and HRM has also been reported for the detection of mutations involved in MDR and extensively drug-resistant TB (XDR-TB) in clinical isolates, with 96% accuracy (17). To the best of our knowledge, the HRM assay has not been evaluated for the diagnosis and screening of RIF and INH resistance directly from sputum samples.…”

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confidence: 99%

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

et al. 2017

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Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INHinhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% ( value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR] 400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.KEYWORDS DNA sequencing, high-resolution melt curve analysis, tuberculosis, molecular diagnostics, multidrug resistance, sputum T uberculosis (TB) is one of the world's deadliest transmittable diseases (1). In 2015, worldwide, 10.4 million people were estimated to be infected with TB, with 1.4 million people dying from the disease. Drug-resistant TB is a menace for TB control worldwide, and 60% of reported TB patients estimated to have multidrug-resistant TB (MDR-TB) were not detected in 2015 (1). India holds the dubious distinction of harboring 27% of the global TB burden, with 2.5% of its new TB cases and 16% of previously treated cases having MDR-TB (1). Conventional Mycobacterium tuberculosis culturebased systems for first-line and second-line drug susceptibility testing (DST) are timeconsuming, while commercial liquid culture-based DST reduces turnaround times but

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confidence: 99%

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

et al. 2017

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“…The paired sputum and isolate were characterized genotypically using a TAC [6] version 2. We incorporated wild-type probes for each target and included additional resistance-associated probes for rpoB and gyrA to now interrogate all the major (>3% sensitivity) mutations described by the TB Drug Resistance Database [10] or ReseqTB.org.…”

Section: Methodsmentioning

confidence: 99%

Performance of TaqMan array card to detect TB drug resistance on direct specimens

et al. 2017

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Culture based phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis (TB) is time consuming therefore rapid genotypic methods are increasingly being utilized. We previously developed and evaluated on TB isolates a rapid genotypic TaqMan array card (TAC) that detects mutations in several resistance-associated genes using dozens of primer pairs, probes, and high resolution melt analysis, with >96% accuracy versus Sanger sequencing. In this study we examined the performance of TAC on sputum, comparing results between 71 paired sputum and TB isolates of which 62 were MDR-TB. We also adapted the TAC to include wild-type probes and broadened coverage for rpoB and gyrA mutations. TAC was 89% successful at detecting wild-type or mutations within inhA, katG, rpoB, eis, gyrA, rplC, and pncA on smear positive sputa and 33% successful on smear negative sputa. The overall accuracy of these detections as compared to the TAC results of the paired isolate was 95% ± 7 (average sensitivity 98% ± 3; specificity 92% ± 14). Accuracy of sputum TAC results versus phenotypic DST for isoniazid, rifampin, ofloxacin/moxifloxacin, and pyrazinamide was 85% ± 12. This was similar to that of the isolate TAC results (accuracy 88% ± 13), thus inaccuracies primarily reflected intrinsic genotypic-phenotypic discordance. The TAC is a rapid, modular, comprehensive, and accurate TB DST for the major first and second line TB drugs and could be used for supplemental testing of GeneXpert resistant smear positive sputum.

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“…Selected references in the reference list demonstrate this finding (Morgan, Paillart, & Thompson, 1998;Santos & Rivera, 2013;Stensvold & Nielsen, 2012). The need for comprehensive screening assays in both outbreak settings and routine clinical investigation has therefore been recognised and the latter developed and used in a number of settings (Liu et al, 2013(Liu et al, , 2014Pholwat et al, 2015). To date, a single screening assay has not been available for mass event settings but such assays are under development.…”

Section: Gastrointestinal Tacsmentioning

confidence: 99%

Low-Density TaqMan® Array Cards for the Detection of Pathogens

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Rolfe

Gleadall

et al. 2015

Methods in Microbiology

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Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

Cited by 29 publications

References 35 publications

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Performance of TaqMan array card to detect TB drug resistance on direct specimens

Low-Density TaqMan® Array Cards for the Detection of Pathogens

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