Pilot evaluation of a compact targeted next-generation sequencing with minimum biocontainment for rapid diagnosis of drug-resistant tuberculosis

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 Oxford Nanopore Technology (ONT) is an easy, tabletop, and portable option for comprehensive drug resistance profiling. Sputum sample/ Mycobacterium tuberculosis ( M. tb ) isolates from 351 bacteriologically confirmed TB patients between January 2022 and June 2023 were used in the study. We assessed the feasibility of using DNA isolated with an extraction device from a routine rapid point-of-care test, thereby requiring minimal biocontainment. A commercial Deeplex Myc-TB primer set was used with M. tb for tNGS. Library preparation was done with a rapid barcoding kit from ONT, and run using MinION Mk1C (ONT). tNGS performed with the DNA extracted using the Trueprep device showed successful runs with lineage and DR prediction for 252 samples. As part of this study, a bioinformatics pipeline was customized in-house and validated at ICMR-NIRT and has been made publicly available on GitHub. Among the runs, the highest depth was obtained for inhA (1160.11), while rrl (89.52) had the lowest depth. tNGS showed comparable sensitivity and specificity with a sensitivity of 95% for RIF, 88% for INH, and 100% for FQ, AMG, and LZD. The agreement between tNGS and pDST by Cohen's kappa was between 0.90 and 1.00 for RIF, FQ, AMG, STR, and LZD, and less than 0.8 for PZA and ETH. Our pilot evaluation study was conducted to assess the feasibility of implementing tNGS using an ONT device from direct unprocessed sputum using a Trueprep AUTO extraction device for DNA extraction independent of a BSL3 or TB containment laboratory. A conceptual diagnostic algorithm for low- and middle-income country to perform tNGS as a single follow-on molecular test under programmatic setting, together with an in-house bioinformatic pipeline, to ensure equity access to rapid DR-TB testing has been proposed. Importance Our study highlights the importance of using point-of-care test DNA directly for targeted next-generation sequencing (tNGS), thus involving minimum biocontainment. The testing is rapid and promises tNGS in different tiers of tuberculosis laboratory under programmatic setting in low-resource high-burden setting.