Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis.
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 inMycobacterium tuberculosisin sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions ofrpoB,katG, and aninhApromoter and used as positive controls in HRM. The assay was first validated in 25 MDRM. tuberculosisclinical isolates. The assay was evaluated on DNA isolated from 99M. tuberculosisculture-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 INH-inhAassays 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% (κ 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.
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
Divya
D
Anthwal
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
Rakesh Kumar
RK
Gupta
Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Mehrauli, New Delhi, India.
Manpreet
M
Bhalla
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
Shinjini
S
Bhatnagar
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India jayatyagi.aiims@gmail.com sagarikahaldar@thsti.res.in.|Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
Jaya Sivaswami
JS
Tyagi
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India jayatyagi.aiims@gmail.com sagarikahaldar@thsti.res.in.
Sagarika
S
Haldar
D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D054872:Fatty Acid Synthesis Inhibitors; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D044366:Transition Temperature; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D054872:Fatty Acid Synthesis Inhibitors; D007538:Isoniazid; D012293:Rifampin
DNA sequencing; high-resolution melt curve analysis; molecular diagnostics; multidrug resistance; sputum; tuberculosis