Compact Targeted Next-Generation Sequencing for Rapid Diagnosis of Drug-Resistant Tuberculosis using Minimum Biocontainment

Background: Next-generation sequencing is a more rapid and robust technique than phenotypic drug susceptibility testing (pDST) for diagnosing drug-resistant tuberculosis (DR-TB). Targeted next-generation sequencing (tNGS) using a portable Oxford Nanopore Technology is an easy, tabletop, and portable option for comprehensive drug resistance profiling. Methods: Sputum samples/Mycobacterium tuberculosis (M. tb) isolates from 351 bacteriologically confirmed TB patients between January 2022 and June 2023 were used in the study. In this study, we assessed the feasibility of using DNA extracted directly from sputum or liquid culture using an extraction device with a rapid point-of-care test (POCT) for TB. A commercial Deeplex Myc-TB primer set was used for species-level identification, genotyping, and antibiotic resistance prediction of M. tb. Library preparation was done using a rapid barcoding kit from Oxford Nanopore Technology (ONT), and the run was done using MinION Mk1C (ONT). A bioinformatics pipeline was customized in-house for lineage and drug resistance prediction using tNGS data at ICMR-NIRT as part of this study. Results: tNGS performed using DNA extracted with Trueprep device shows successful runs. Samples with high bacillary load were used which showed good depth and coverage across genes. The distribution of single-nucleotide polymorphisms (SNPs) and coverage and depth of the reads from the runs in MinION, Oxford Nanopore Technology, were similar to previous studies when evaluated with our customized bioinformatic pipeline. Among the runs, the highest depth was obtained for inhA(1160.11) while rrl (89.52) had the lowest depth, comparable to a few previously published results. tNGS showed 99-100% concordance to whole genome sequencing (WGS) and pDST for the first line, second line, and newer/repurposed drugs. Discussion: Our study is the first to assess DNA extraction from direct sputum using a Trueprep AUTO extraction device, part of the POCT Truenat MTB that does not require a BSL3 or TB containment lab for the extraction procedure. With the availability of WHO-endorsed nucleic acid amplification technology (NAATs) across many low- and middle-income countries (LMICs), using DNA available from NAAT in tNGS to achieve comprehensive drug resistance profiling is promising. We propose a new diagnostic algorithm for LMIC programmatic settings to perform tNGS as a single follow-on molecular test toward comprehensive drug resistance profiling.