Culture-free whole genome sequencing of Mycobacterium tuberculosis using ligand-mediated bead enrichment method
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Le résumé fourni par la source
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 from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n=17; bronchoalveolar lavage, BAL, 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% (IQR = 7.9-39.3). There was a positive correlation between load of bacilli on smears and genome coverage (p-value < 0.001). We detected 58 genes listed in the WHO 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 5/34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin which is not covered by Xpert MTB/RIF. This approach also allowed us to identify mixed infections in eight samples (BAL=4/8, pus=2/3 and sputum= 2/10) including samples that were infected with three or more different strains of Mtb . 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.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé, mais le titre doit être comparé manuellement.
- Titre Crossref
- Culture-free whole genome sequencing of <i>Mycobacterium tuberculosis</i> using ligand-mediated bead enrichment method
- Date Crossref
- 23/01/2024
- Éditeur
- openRxiv
- Type
- posted-content
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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Manipal Academy of Higher Education pays non établi dans la noticeUniversité ou école supérieure
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Institute of Bioinformatics pays non établi dans la noticeOrganisation à but non lucratif
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Mayo Clinic pays non établi dans la noticeÉtablissement de santé
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Mayo Clinic in Florida pays non établi dans la noticeÉtablissement de santé
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Kasturba Medical College Department of Microbiology pays non établi dans la noticeUniversité ou école supérieure
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National Institute of Mental Health and Neurosciences Center for Molecular Medicine pays non établi dans la noticeÉtablissement de santé
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Department of Laboratory Medicine and Pathology pays non établi dans la noticeStructure de recherche
Manipal Academy of Higher Education, Institute of Bioinformatics et Mayo Clinic, avec 4 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.