Ann Lab Med 2018; 38(4): 316-323  https://doi.org/10.3343/alm.2018.38.4.316
Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeqTM TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates
Joonhong Park, M.D.1*, So Youn Shin, M.D.2*, Kyungjong Kim, Ph.D.2, Kuhn Park, M.D.3, Soyoung Shin, M.D.1, and Chunhwa Ihm, M.D.4
Department of Laboratory Medicine1, College of Medicine, The Catholic University of Korea, Seoul; Korean Institute of Tuberculosis2, Cheongju; Department of Thoracic and Cardiovascular Surgery3, College of Medicine, The Catholic University of Korea, Seoul; Department of Laboratory Medicine4, Eulji University Hospital, Daejeon, Korea
Corresponding author: Chunhwa Ihm
Department of Laboratory Medicine, Eulji University Hospital, Eulji University School of Medicine, 95 Dunsanseo-ro, Seo-gu, Daejeon 35233, Korea
Tel: +82-42-611-3478
Fax: +82-42-611-3464
E-mail: haneul@eulji.ac.kr
*These authors contributed equally to this work.
Received: July 30, 2017; Revised: December 11, 2017; Accepted: February 13, 2018; Published online: July 1, 2018.
© Korean Society for Laboratory Medicine. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: We examined the feasibility of a full-length gene analysis for the drug resistance- related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates.
Methods: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA).
Results: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA.
Conclusions: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.
Keywords: Ion semiconductor sequencing, Ion AmpliSeq TB panel, Multidrug-resistant, Mutations, Mycobacterium tuberculosis



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