Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates.
10.3343/alm.2018.38.4.316
- Author:
Joonhong PARK
1
;
So Youn SHIN
;
Kyungjong KIM
;
Kuhn PARK
;
Soyoung SHIN
;
Chunhwa IHM
Author Information
1. Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.
- Publication Type:Original Article
- Keywords:
Ion semiconductor sequencing;
Ion AmpliSeq TB panel;
Multidrug-resistant;
Mutations;
Mycobacterium tuberculosis
- MeSH:
Amikacin;
Clinical Coding;
Complement System Proteins;
DNA;
Drug Resistance*;
Ethambutol;
Fluoroquinolones;
Isoniazid;
Kanamycin;
Levofloxacin;
Mycobacterium tuberculosis*;
Mycobacterium*;
Ofloxacin;
Pyrazinamide;
Rifampin;
Semiconductors*;
Streptomycin
- From:Annals of Laboratory Medicine
2018;38(4):316-323
- CountryRepublic of Korea
- Language:English
-
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.
