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Introduction <p>Fluoroquinolones (FQs) are key components of World Health Organization (WHO)-recommended regimens for multidrug-resistant tuberculosis (MDR-TB). Accurate detection of FQ resistance is essential for optimizing treatment. This study evaluated the concordance between the Second-Line Line Probe Assay (SL-LPA) and Liquid Culture Drug Susceptibility Testing (LC-DST) for detecting FQ resistance in <i>Mycobacterium tuberculosis</i> isolates.</p> Methods <p>In this retrospective study, 1402 non-duplicate clinical isolates of MDR TB were tested using SL-LPA and LC-DST at a reference laboratory. Genotypic resistance was identified through mutations in the <i>gyrA</i> and <i>gyrB</i> genes&#xa0;identified by SL-LPA, while phenotypic resistance was determined using MGIT-based LC-DST at critical concentrations for fluoroquinolones. Targeted nanopore sequencing was performed on a subset of isolates with discordant molecular and phenotypic results to investigate resistance-associated mutations.</p> Results <p>SL-LPA detected FQ resistance in 907 (64.7%) isolates, whereas LC-DST identified resistance in 852 (60.8%) isolates. Using LC-DST as the reference standard, SL-LPA showed a sensitivity of 93.2%, specificity of 98.6%, positive predictive value of 99.2%, and negative predictive value of 88.7%. Overall concordance between the two methods was observed in 1292 (92.2%) isolates. Discordant results occurred in 110 (7.8%) isolates, mainly involving low-level resistance mutations or inferred resistance due to missing wild-type bands on SL-LPA. Nanopore sequencing of 15 discordant isolates identified high-confidence mutations (Asp94Tyr, Asp94Gly, Asp94Asn) and interim or low-confidence mutations (Ala90Val, Ser91Pro, Asp94Ala, <i>gyrB</i> Asn499Asp, Asp461Asn).</p> Conclusion <p>SL-LPA demonstrates excellent specificity and positive predictive value for detecting FQ resistance; however, discordance associated with low-confidence mutations and heteroresistance highlights the importance of integrating molecular assays with phenotypic DST and sequencing to improve MDR-TB resistance detection and guide treatment decisions.</p>

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Assessing fluoroquinolone resistance in Mycobacterium tuberculosis using nanopore sequencing: concordance with established diagnostic methods

  • 期刊论文

  • 发表日期：

    2026年4月22日

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  • Rashmi Ratnam,
  • Parul Jain,
  • Faisal Abbas,
  • Bhoopendra Pandey,
  • Manu Singh,
  • Urmila Singh,
  • Vijay Kumar,
  • Amita Jain,
  • Ram Awadh Singh Kushwaha

摘要

Introduction

Fluoroquinolones (FQs) are key components of World Health Organization (WHO)-recommended regimens for multidrug-resistant tuberculosis (MDR-TB). Accurate detection of FQ resistance is essential for optimizing treatment. This study evaluated the concordance between the Second-Line Line Probe Assay (SL-LPA) and Liquid Culture Drug Susceptibility Testing (LC-DST) for detecting FQ resistance in Mycobacterium tuberculosis isolates.

Methods

In this retrospective study, 1402 non-duplicate clinical isolates of MDR TB were tested using SL-LPA and LC-DST at a reference laboratory. Genotypic resistance was identified through mutations in the gyrA and gyrB genes identified by SL-LPA, while phenotypic resistance was determined using MGIT-based LC-DST at critical concentrations for fluoroquinolones. Targeted nanopore sequencing was performed on a subset of isolates with discordant molecular and phenotypic results to investigate resistance-associated mutations.

Results

SL-LPA detected FQ resistance in 907 (64.7%) isolates, whereas LC-DST identified resistance in 852 (60.8%) isolates. Using LC-DST as the reference standard, SL-LPA showed a sensitivity of 93.2%, specificity of 98.6%, positive predictive value of 99.2%, and negative predictive value of 88.7%. Overall concordance between the two methods was observed in 1292 (92.2%) isolates. Discordant results occurred in 110 (7.8%) isolates, mainly involving low-level resistance mutations or inferred resistance due to missing wild-type bands on SL-LPA. Nanopore sequencing of 15 discordant isolates identified high-confidence mutations (Asp94Tyr, Asp94Gly, Asp94Asn) and interim or low-confidence mutations (Ala90Val, Ser91Pro, Asp94Ala, gyrB Asn499Asp, Asp461Asn).

Conclusion

SL-LPA demonstrates excellent specificity and positive predictive value for detecting FQ resistance; however, discordance associated with low-confidence mutations and heteroresistance highlights the importance of integrating molecular assays with phenotypic DST and sequencing to improve MDR-TB resistance detection and guide treatment decisions.