%0 Journal Article
%T Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.
%A Vasanthaiah S
%A Verma R
%A Kumar A
%A Bandari AK
%A George J
%A Rastogi M
%A Manjunath GK
%A Sharma J
%A Kumar A
%A Subramani J
%A Chawla K
%A Pandey A
%J Open Forum Infect Dis
%V 11
%N 7
%D 2024 Jul
%M 38957687
%F 4.423
%R 10.1093/ofid/ofae320
%X <B>UNASSIGNED: </B>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.<BR><B>UNASSIGNED: </B>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.<BR><B>UNASSIGNED: </B>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.<BR><B>UNASSIGNED: </B>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.