Direct detection of Fluoroquinolones resistance in sputum samples from tuberculosis patients by High Resolution Melt curve analysis
Author(s): Rakesh Kumar Guptaa, Divya Anthwala, Manpreet Bhallab, Jaya Sivaswami Tyagic, Sagarika Haldara,d
Institute(s): Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarha; Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Mehrauli, New Delhib; Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhic; Center for Bio-design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad – Gurgaon Expressway, PO box #04, Faridabadd
Abstract: Tuberculosis (TB) remains one of the most serious infectious diseases worldwide causing 1.3 million deaths in 2017. TB treatment due to multidrug-resistant TB (MDR-TB) requires the use of fluoroquinolones (FLQ) and excessive use of FLQ has led to the rise of extensively drug-resistant TB (XDR-TB). In 2017, 22% MDR-TB cases were resistant to FLQ drugs. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of FLQ resistance in M. tuberculosis in sputum samples. A reference plasmid library was generated for most frequently observed mutations of gyrA gene and used as the positive-controls in HRM, then evaluated in 20 MDR-M. tuberculosis clinical isolates. The assay was validated on DNA isolated from 70 M. tuberculosis culture-positive sputum samples (including 56 smear-negative samples) using DNA sequencing as gold standard. Mutants were discriminated from wild-type by comparing melting-curve patterns with those of control plasmids. The FLQ-HRM assay had a sensitivity and specificity of 100% and 96.4%, respectively and also detected the natural polymorphism (S95T mutation) in 39/70 samples. HRM is a rapid, cost-effective (INR 180/USD2.8) and closed-tube method for direct detection of FLQ resistance in sputum samples especially for smear-negative sputum samples.