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. 2025 Apr 11;12(7):ofaf207.
doi: 10.1093/ofid/ofaf207. eCollection 2025 Jul.

Operational Feasibility of "TB Concentration & Transport" and "TB DNA Extraction" Kits for Biosafe Dry Sputum Transport for Direct Detection of Drug Resistance Using Line Probe Assay: A Field Study in India

Keerti Chauhan  1 , Rakesh Kumar Gupta  1 , Divya Anthwal  1 , Nikita Panwalkar  2 , Prabha Desikan  2 , Ritu Singhal  3 , Manpreet Bhalla  3 , Vithal Prasad Myneedu  3 , Khalid Umar Khayyam  3 , Siva Kumar Shanmugam  4 , K Silambu Chelvi  4 , Padmapriyadarsini Chandrasekaran  4 , Sidhartha Giri  5 , Jyotirmayee Turuk  5 , Dasarathi Das  5 , Sanghamitra Pati  5 , Abhinav Goyal  6 , Ashawant Gupta  6 , Nalini Kant Gupta  6 , Manjula Singh  7 , Jaya Sivaswami Tyagi  8 , Sagarika Haldar  1
Affiliations

Operational Feasibility of "TB Concentration & Transport" and "TB DNA Extraction" Kits for Biosafe Dry Sputum Transport for Direct Detection of Drug Resistance Using Line Probe Assay: A Field Study in India

Keerti Chauhan et al. Open Forum Infect Dis. .

Abstract

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.

Keywords: LPA; MDR-TB; biosafe sputum transport; diagnosis; tuberculosis.

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Conflict of interest statement

Potential conflicts of interest. A. Gu. and N. K. G. manufactured the ‘TB Concentration & Transport’ kits, ‘TB DNA extraction’ kits and ‘TBDetect microscopy’ kits and A. Go. participated in the training of laboratory technicians at DMCs and NRLs who used the kits. These authors were not involved in conducting the study and analyzing the results. D. A., R. K. G., V. P. M., A. G., N. K. G., S. H., and J. S. T. are joint inventors in an Indian Provisional Patent application named “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155). All other authors report no potential conflicts of interest.

Figures

Figure 1.
Figure 1.
Workflow of the present study. Abbreviations: DMC, Designated Microscopy Center; LED-FM, Light-emitting diode fluorescent microscopy; LPA, Line probe assay; NALC-NaOH, N-acetyl L-cysteine- sodium hydroxide; NRL/IRL, National Reference Laboratory/Intermediate Reference Laboratory; TB, Tuberculosis; MDR-TB, Multi drug resistant-TB; XDR-TB, Extensively drug resistant-TB.
Figure 2.
Figure 2.
Workflow of final analysis (Kit-LPA vs Direct-LPA) for drug resistance detection. Abbreviations: Direct-LPA, line probe assay from direct sputum; DMC, designated microscopy center; Kit-LPA, kit-extracted DNA amalgamated with line probe assay; LPA, line probe assay.
Figure 3.
Figure 3.
Concordance/discordance between Kit-LPA and Direct-LPA for detection of rifampicin and isoniazid resistance in a combined analysis of all 4 sites. Abbreviations: INH, isoniazid; Kit-LPA, kit-extracted DNA amalgamated with line probe assay; LPA, line probe assay; MUT, mutant; RIF, rifampicin; WT, wild-type.
Figure 4.
Figure 4.
Scoring feedback on sputum transport using the TB Concentration & Transport kit vs conventional sputum cold transport method (A) and the TB DNA Extraction kit vs regular line probe assay (LPA) DNA extraction method (GenoLyse kit) (B). *Statistically significant.
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