Dataset Information

Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.

ABSTRACT:

Background

Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background.

Methods

We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform.

Results

Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF.

Conclusions

We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.

SUBMITTER: Vasanthaiah S

PROVIDER: S-EPMC11218775 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Publications

Culture-Free Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i> Using Ligand-Mediated Bead Enrichment Method.

Vasanthaiah Shruthi S Verma Renu R Kumar Ajay A Bandari Aravind K AK George John J Rastogi Mona M Manjunath Gowrang Kasaba GK Sharma Jyoti J Kumar Abhishek A Subramani Janavi J Chawla Kiran K Pandey Akhilesh A

Open forum infectious diseases 20240615 7

<h4>Background</h4>Direct whole genome sequencing (WGS) of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background.<h4>Methods</h4>We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extractio ...[more]

PMID: 38957687

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