BioProject

Diagnosis of drug-resistant tuberculosis (DR-TB) by next-generation sequencing is feasible since it is a more rapid and robust technique than phenotypic drug susceptibility testing (pDST). More...

Diagnosis of drug-resistant tuberculosis (DR-TB) by next-generation sequencing is feasible since it is a more rapid and robust technique than phenotypic drug susceptibility testing (pDST). Targeted next-generation sequencing (tNGS) using a portable Oxford Nanopore Technology is an easy, tabletop, point-of-care test (POCT) option for comprehensive drug resistance profiling. In this study, we have assessed the feasibility of using DNA extracted directly from sputum during routine nucleic acid amplification technology (NAAT) procedure(s) and developed an in-house bioinformatic pipeline for analysis of tNGS runs. Sputum samples/Mycobacterium tuberculosis (M. tb) isolates from 351 bacteriologically confirmed TB patients between January 2022 to June 2023 were used in the study. 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 developed in-house for lineage and drug resistance prediction using tNGS data at ICMR-NIRT as part of this study. DNA extraction by the cTAB method for LJ culture and Trueprep AUTO device for direct sputum showed good yields as observed by higher IQR for the concentration of DNA. LJ cultures gave 93.80% successful runs while direct sputum gave 60.58%. The distribution of single nucleotide polymorphisms (SNPs) and depth of reads among all the runs done using MinION, Oxford Nanopore Technology was comparable to previous studies when evaluated with our in-house bioinformatic pipeline. With the in-house pipeline, the highest depth was obtained for inhA (1160.11) while rrl (89.52) had the lowest depth comparable to few previously published results. tNGS showed 99-100% concordance to WGS and pDST for the first line, second line, and newer/repurposed drugs. Our study is the first to assess DNA extraction from direct sputum using a POCT device that does not require a BSL3 or TB containment lab for the entire procedure. With the availability of WHO-endorsed NAATs across many low middle-income countries (LMICs), the use of DNA from them in tNGS to achieve comprehensive drug resistance profiling is promising. We propose a new diagnostic algorithm for use in an LMIC programmatic setting to perform upfront tNGS toward comprehensive drug resistance profiling. Less...