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Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper

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Abstract

In 2019, amongst half a million new rifampicin-resistant tuberculosis (TB) cases, 78% were multi drug-resistant TB (MDR-TB). Access to rapid and Universal-Drug susceptibility testing (DST) to patients in remote areas is a major challenge to combat drug-resistant TB. To overcome this challenge, we had recently reported the development of ‘TB Concentration & Transport kit’ for bio-safe ambient temperature transport of dried sputum on filter-paper (Trans-Filter). The present study was conducted to evaluate the utility of DNA extracted from sputum on Trans-Filter in a Multiplex PCR-based sequencing assay (Mol-DSTseq) for diagnosing drug-resistant TB. The developed Mol-DSTseq assays were standardized on Mycobacterium tuberculosis clinical isolates (n = 98) and further validated on DNA extracted from sputum on Trans-Filter (n = 100). Using phenotypic DST as gold standard, the Mol-DSTseq assay showed 100% (95% Confidence Interval [CI] 79.4–100%) and 73.3% (95% CI 54.1–87.7%) sensitivity for detecting rifampicin and isoniazid resistance with a specificity of 85.1% (95% CI 66.2–95.8%) and 100% (95% CI:82.3–100%), respectively. For fluoroquinolones and aminoglycosides, the Mol-DSTseq assay showed a sensitivity of 78.5% (95% CI 49.2–95.3%) and 66.6% (95% CI 9.4–99.1%) with a specificity of 88.2% (95% CI 72.5–96.7%) and 100% (95% CI 93.1–100%), respectively. The Mol-DSTseq assays exhibited a high concordance of ~ 83–96% (κ value: 0.65–0.81) with phenotypic DST for all drugs. In conclusion, the ‘TB Concentration and Transport kit’ was compatible with Mol-DSTseq assays and has the potential to provide ‘Universal-DST’ to patients residing in distant areas in high burden countries, like India for early initiation of anti-tubercular treatment.

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Acknowledgements

SH acknowledges the Intramural grant (71/2-Edu-16/4465) from PGIMER, Chandigarh. DA and RKG are thankful for Senior Research Fellowship through Indian Council of Medical Research (ICMR), Government of India. JST acknowledges the award of Senior Scientist Fellowship by the National Academy of Sciences (India).

Funding

The work was supported by the Intramural Grant (71/2-Edu-16/4465) from PGIMER, Chandigarh.

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Author notes

  1. Divya Anthwal and Shaina Jamwal have contributed equally to this work.

Authors and Affiliations

  1. Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

    Divya Anthwal, Shaina Jamwal, Rakesh Kumar Gupta & Sagarika Haldar

  2. Department of Bioscience and Biotechnology, Banasthali Vidyapith, P.O. Banasthali Vidyapith, Tonk, Rajasthan, India

    Divya Anthwal, Rakesh Kumar Gupta & Sangeeta Choudhary

  3. Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Mehrauli, New Delhi, India

    Ritu Singhal, Ajoy Kumar Verma, Manpreet Bhalla, Vithal Prasad Myneedu & Rohit Sarin

  4. Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

    Jaya Sivaswami Tyagi

  5. Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad – Gurgaon Expressway, PO box #04, Faridabad, India

    Sagarika Haldar

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  1. Divya Anthwal
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Contributions

Conceptualization: SH; Methodology: DA, SJ, SH; Formal analysis: DA, SJ; Investigation and data curation: DA, SJ, RKG, RS, AKV, MB, VPM, RS; writing—original draft preparation: DA, SJ, SH; writing—review and editing: DA, SJ, RKG, RS, AKV, MB, VPM, RS, SC, JST, SH; funding acquisition: SH; resources: SH; supervision: SC, JST, SH.

Corresponding author

Correspondence to Sagarika Haldar.

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Conflict of interest

DA, RKG, AKV, VPM, RS, JST and SH 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).

Ethical Approval

All archived samples were used after ethical clearance from the Institutional Ethics Committee at Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh (PGIMER: INT/IEC/2018/1456).

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All authors have read and agreed to publish this version of the manuscript.

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The data used or analyzed during the present study are available from the corresponding author on reasonable request.

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Supplementary Information

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284_2022_2780_MOESM1_ESM.tif

Supplementary Fig. 1. Limit of Detection (LOD) and electropherograms of respective target genes of (A) Triplex MDR-TB assay; and (B) Duplex XDR-TB assay. 106 to 10 indicates M. tuberculosis genome equivalents, L stands for DNA Ladder and red circle highlights the LOD of respective assay (TIF 22431 kb)

284_2022_2780_MOESM2_ESM.tif

Supplementary Fig. 2 Workflow of sample analysis (Mol-DSTseq assay vs. MGIT) for drug resistance detection. *M. tb: Mycobacterium tuberculosis, confirmed by AFB-positive smear and SD BIOLINE TB Ag MPT64 kit Rapid test (in MGIT-culture) (TIF 19823 kb)

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Anthwal, D., Jamwal, S., Gupta, R.K. et al. Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper. Curr Microbiol 79, 110 (2022). https://doi.org/10.1007/s00284-022-02780-1

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