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. 2023 May 31;11(6):1467.
doi: 10.3390/microorganisms11061467.

Rapid Identification of Lineage and Drug Resistance in Clinical Samples of Mycobacterium tuberculosis

Jéssica Comín  1 , Jesús Viñuelas  2   3 , Carmen Lafoz  4 , Alberto Cebollada  5 , Daniel Ibarz  6 , María-José Iglesias  6   7   8 , Sofía Samper  1   6   7   8
Affiliations

Rapid Identification of Lineage and Drug Resistance in Clinical Samples of Mycobacterium tuberculosis

Jéssica Comín et al. Microorganisms. .

Abstract

Background: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required.

Methods: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples.

Results: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination.

Conclusion: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform.

Keywords: AmpliSeq technology; antimicrobial resistance; genomics; tuberculosis; tuberculosis diagnosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Description of the three protocols tested for DNA extraction directly from clinical samples. Samples containing more than 1 mL were worked in different vials and the pellets were collected together after the saline washing step. Method 1 was described by Votintseva et al. [1,2].
See this image and copyright information in PMC

References

    1. Votintseva A.A., Pankhurst L.J., Anson L.W., Morgan M.R., Gascoyne-Binzi D., Walker T.M., Quan T.P., Wyllie D.H., Del Ojo Elias C., Wilcox M., et al. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures. J. Clin. Microbiol. 2015;53:1137–1143. doi: 10.1128/JCM.03073-14. - DOI - PMC - PubMed
    1. Votintseva A.A., Bradley P., Pankhurst L., Del Ojo Elias C., Loose M., Nilgiriwala K., Chatterjee A., Smith E.G., Sanderson N., Walker T.M., et al. Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples. J. Clin. Microbiol. 2017;55:1285–1298. doi: 10.1128/JCM.02483-16. - DOI - PMC - PubMed
    1. Brown A.C., Bryant J.M., Einer-Jensen K., Holdstock J., Houniet D.T., Chan J.Z.M., Depledge D.P., Nikolayevskyy V., Broda A., Stone M.J., et al. Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples. J. Clin. Microbiol. 2015;53:2230–2237. doi: 10.1128/JCM.00486-15. - DOI - PMC - PubMed
    1. Doyle R.M., Burgess C., Williams R., Gorton R., Booth H., Brown J., Bryant J.M., Chan J., Creer D., Holdstock J., et al. Direct Whole-Genome Sequencing of Sputum Accurately Identifies Drug-Resistant Mycobacterium tuberculosis Faster than MGIT Culture Sequencing. J. Clin. Microbiol. 2018;56:e00666-18. doi: 10.1128/JCM.00666-18. - DOI - PMC - PubMed
    1. Bonnet I., Enouf V., Morel F., Ok V., Jaffré J., Jarlier V., Aubry A., Robert J., Sougakoff W. A Comprehensive Evaluation of GeneLEAD VIII DNA Platform Combined to Deeplex Myc-TB® Assay to Detect in 8 Days Drug Resistance to 13 Antituberculous Drugs and Transmission of Mycobacterium tuberculosis Complex Directly From Clinical Samples. Front. Cell. Infect. Microbiol. 2021;11:707244. doi: 10.3389/fcimb.2021.707244. - DOI - PMC - PubMed