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Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis

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Abstract

Multidrug-resistant tuberculosis (MDR-TB) requires treatment with fluoroquinolone (FLQ) drugs, however, the excessive use of FLQ has led to the rise of extensively drug-resistant TB. In 2019, ~ 20% of total MDR-TB cases were estimated to be resistant to FLQ drugs. In the present study, we developed and evaluated the utility of high-resolution melt curve analysis (HRM) for the rapid detection of FLQ-resistant Mycobacterium tuberculosis for the first time directly from sputum samples. A reference plasmid library was generated for the most frequently observed mutations of gyrA gene and was used to discriminate between mutant and wild-type samples in the FLQ-HRM assay. The developed assay was evaluated on n = 25 MDR M. tuberculosis clinical isolates followed by validation on archived sputum DNA (n = 88) using DNA sequencing as a gold standard. The FLQ-HRM assay showed a 100% sensitivity [95% Confidence Interval (CI): 71.5 to 100] and specificity (95% CI: 39.7 to 100) in smear-positive category, and a sensitivity of 88.9% (95% CI: 77.3 to 95.8) with 84.2% (95% CI: 60.4 to 96.6) specificity in smear-negative category. The assay showed a high level of concordance of ~ 90% (κ = 0.74) with DNA sequencing, however, we were limited by the absence of phenotypic drug susceptibility testing data. In conclusion, HRM is a rapid, cost-effective (INR 150/USD 1.83) and closed-tube method for direct detection of FLQ resistance in sputum samples including direct smear-negative samples.

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Data Availability

The data used or analysed during the present study are available from the corresponding author on reasonable request.

Code Availability

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Acknowledgements

Not applicable.

Funding

The work was supported by the intramural grant (71/2-Edu-16/4465) from PGIMER, Chandigarh and extramural grant from Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India (BT/PR20814/MED/29/1214/2017).

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Author notes

  1. Rakesh Kumar Gupta and Divya Anthwal have contributed equally to this work.

Authors and Affiliations

  1. Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh, India

    Rakesh Kumar Gupta, Divya Anthwal & Sagarika Haldar

  2. Department of Bioscience and Biotechnology, Banasthali Vidyapith, P.O. Banasthali Vidyapith, Rajasthan, 304022, India

    Rakesh Kumar Gupta, Divya Anthwal & Sangeeta Choudhary

  3. Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Mehrauli, New Delhi, India

    Manpreet Bhalla

  4. Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

    Jaya Sivaswami Tyagi

  5. Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3Rd Milestone, Faridabad-Gurgaon Expressway, PO box #04, Faridabad, India

    Sagarika Haldar

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  1. Rakesh Kumar Gupta
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Contributions

Conceptualization: SH; Methodology: RKG, DA; Formal analysis: RKG, DA; Investigation and data curation RKG, DA, MB; Writing—original draft preparation: RKG, DA; Writing—review and editing: MB, JST, SC, SH; Funding acquisition: SH; Resources: SH; Supervision: SC, JST, SH.

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Correspondence to Sagarika Haldar.

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An ethical approval was obtained from the Institutional Ethics Committee of the Postgraduate Institute of Medical Education and Research (PGIMER, INT/IEC/2018/1456), Chandigarh for the use of archived DNA samples.

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Gupta, R.K., Anthwal, D., Bhalla, M. et al. Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis. Curr Microbiol 81, 27 (2024). https://doi.org/10.1007/s00284-023-03519-2

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