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Updated: Sep 14, 2025
Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples
Published on: June 6, 2025
Operational Feasibility of "TB Concentration & Transport" and "TB DNA Extraction" Kits for Biosafe Dry Sputum
Keerti Chauhan1, Rakesh Kumar Gupta1, Divya Anthwal1
1Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Background:
We recently described the utility of the "TB Concentration & Transport" kit for biosafe, ambient temperature transport of dried sputum on Trans-Filter, and the "TB DNA Extraction" kit for DNA extraction from Trans-Filter for the early diagnosis of drug-resistant tuberculosis (TB). This study aimed to assess the feasibility and compatibility of these kits with line probe assay (LPA) under National Tuberculosis Elimination Programme (NTEP) settings.
Methods:
Patients with presumptive pulmonary TB, multidrug-resistant (MDR) TB, or extensively drug-resistant TB (N = 8491) who attended Designated Microscopy Centers (DMCs, n = 13) under National Reference Laboratories (NRLs) at Bhopal, New Delhi, Chennai, and Bhubaneswar were screened by smear microscopy. The performance of Trans-Filter extracted DNA-based LPA (Kit-LPA) was assessed against Direct-LPA on smear-positive sputum (n = 681), and feedback was obtained from scientists (n = 10) and laboratory technicians (n = 42) regarding logistics, kit usage, training, and troubleshooting. A scoring questionnaire was used to assess (i) the TB Concentration & Transport kit versus conventional sputum transport and (ii) the TB DNA Extraction kit versus Hain GenoLyse kit (statistical significance of the scores was calculated using paired t test).
Results:
Kit-LPA showed a sensitivity and specificity in the range of 89%-96% and 99%-100%, respectively, for rifampicin and isoniazid resistance detection and was comparable to Direct-LPA (concordance = 99%-100%; κ = 0.94-0.97). Overall scores indicated that (i) sputum transport on Trans-Filters was more convenient as compared to conventional sputum transport (P < .0001) and (ii) Trans-Filter extracted DNA was easily amalgamated with LPA testing for MDR-TB detection.
Conclusions:
These findings provide evidence for incorporation of these kits into the NTEP. Their use can facilitate sputum transport from DMCs to NRLs and provide universal drug susceptibility testing to people with TB residing in remote areas.
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