Sequencing data for discordant samples.

In India’s National Tuberculosis Elimination Program (NTEP), diagnosing tuberculosis (TB) and Drug-Resistant TB (DR-TB) involves collecting two sputum specimens from presumptive TB patients. Mycobacterium tuberculosis (MTB) is detected using the Truenat MTB - RIF assay at peripheral sites, with Trueprep for DNA extraction. In the case of a MTB positive, the second specimen is sent to a linked reference laboratory for Line Probe Assay (LPA) testing, requiring DNA extraction again. This study aimed to validate Trueprep-extracted DNA for First Line (FL) and Second Line (SL) LPA testing to assess real-time programmatic field conditions and DNA transportation across diverse Indian geographies. Four hundred MTB-positive Trueprep extracted DNA with ≥104 cfu/ml value from the first specimen, accompanied by the second sputum specimen, were transported from nine strategically selected Truenat sites (regions: South- 2, North- 2, East- 2, West- 3) to reference laboratories in a cool chain. The MTB-positive Trueprep-extracted DNA and the Genolyse-extracted DNA from the second specimen were subjected to FL and SL LPA testing, and the results were compared. Whole genome sequencing was performed for discordance resolution among mismatched Trueprep and Genolyse DNA results. Interpretable results were obtained in 397 (99.25%) of the 400 Trueprep-extracted DNA samples for FL LPA testing and 363 (90.75%) samples for SL-LPA testing. In comparison, Genolyse extracted DNA yielded 371 (99.46%) and 362 (97.05%) valid FL and SL LPA results, respectively, from 373 smear-positive specimens. Among 400 specimens, 160 were 105, 122 of 106, 101 of 104, 15 of 107, and 2 of 108 cfu/ml value. High concordance (97.7% for FL, 99.5% for SL LPA) was observed between Trueprep and Genolyse DNA. This study validates Trueprep-extracted DNA for reliable FL and SL-LPA testing in field settings, potentially streamlining NTEP’s diagnostic process, reducing reference laboratory workload, eliminating duplicate DNA extractions, and reducing the turnaround time (TAT).