Whole genome enrichment approach for rapid detection of Mycobacterium tuberculosis and drug resistance-associated mutations from direct sputum sequencing. (March 2020)

• Record Type: Journal Article
• Authors: Soundararajan, Lakshmi; Kambli, Priti; Priyadarshini, Sushri; Let, Biswajit; Murugan, Sakthivel; Iravatham, Chitra; Tornheim, Jeffrey A.; Rodrigues, Camilla; Gupta, Ravi; Ramprasad, V.L.
• Is Part Of: Tuberculosis. Volume 121(2020)
• Publication Date: 2020-03
• Subjects: 616.995
• DOI: 10.1016/j.tube.2020.101915
• Languages: English
• ISSNs: 1472-9792

Abstract

Abstract: Tuberculosis is the leading cause of death among infectious diseases worldwide. Detection of Mycobacterium tuberculosis ( Mtb ), using routine culture-based methods is time consuming resulting in delayed diagnosis and poor treatment outcomes. Currently available molecular tests provide faster diagnosis but are able to screen only limited hot-spot mutations. Whole genome sequencing from direct sputum offers a potential solution, however, due to the presence of other microbes and host DNA its use in diagnostic testing remains challenging. In this study, we present a targeted Mtb -enrichment assay for lineage-4 coupled with an improved analysis pipeline that uses 1657 bacterial taxa as background for reducing non- Mtb genome from sputum DNA. This method drastically improved the recovery of Mtb DNA from sputum ( Mtb alignment increased from 3% to >65%) as compared to non-enrichment-based sequencing. We obtained >99% Mtb genome coverage as compared to 49% in non-enriched sputum sequencing. We were able to identify Mtb positive samples from controls with 100% accuracy using Mpt64 gene coverage. Our method not only achieved 100% sensitivity to resistance variants profiled by line probe assay (LPA), but also outperformed LPA in determining drug resistance based on phenotypic drug susceptibility tests for 6 anti-tuberculosis drugs (accuracy of 97.7% and 92.8% by enriched WGS and LPA, respectively).