Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis
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..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:81 |
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| Enthalten in: |
Current microbiology - 81(2023), 1 vom: 02. Dez. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gupta, Rakesh Kumar [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Anmerkungen: |
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
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| doi: |
10.1007/s00284-023-03519-2 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a 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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