Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates-Chennai, India, 2013-2016.

Sembulingam Tamilzhalagan,Narayanan Shivaram Gomathi,Patricia J Hall,Patrick K Moonan,Sriram Selvaraju,Srikanth Prasad Tripathy,Soumya Swaminathan,Diya Surie,Sivakumar Shanmugam,Mukesh Kumar Sathyanarayanan,Sakthi Suba,Lavanya Jayabal,Ashok Selvaraj,Uma Devi Ranganathan,Chittibabu Suganthi,Kuldeep Singh Sachdeva

doi:10.3389/fmicb.2021.720436

Abstract

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

Highlights

India contributes 27% of the global burden of drug-resistant tuberculosis (DR TB) (World Health Organization, 2020)
Available molecular drug susceptibility tests (DSTs) detect well-characterized mutations associated with antibiotic resistance
The WHO recommends commercially available molecular assays that target wellcharacterized mutations associated with anti-TB antibiotic resistance in addition to conventional, culture-based phenotypic DST for detection of mutations associated with DR TB (World Health Organization, 2013, 2016, 2018a,b)

Summary

Introduction

India contributes 27% of the global burden of drug-resistant tuberculosis (DR TB) (World Health Organization, 2020). WGS Detects Missed Drug-Resistant TB (World Health Organization, 2020). Available molecular drug susceptibility tests (DSTs) detect well-characterized mutations associated with antibiotic resistance. The WHO recommends commercially available molecular assays that target wellcharacterized mutations associated with anti-TB antibiotic resistance in addition to conventional, culture-based phenotypic DST for detection of mutations associated with DR TB (World Health Organization, 2013, 2016, 2018a,b). Despite widespread use of these tests, DR TB may be missed when specific mutations underlying drug resistance are not targeted by commercial assays or well characterized in clinically-resistant M. tuberculosis isolates (Campbell et al, 2011; Variava and Martinson, 2018). Whole-genome sequencing (WGS) can detect DR TB resistance-associated mutations, hetero-resistance, and novel mutations, but global implementation has been limited (Brown et al, 2015; Farhat et al, 2016)

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