Ann Lab Med.  2018 Jul;38(4):316-323. 10.3343/alm.2018.38.4.316.

Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeqâ„¢ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

• Park J
• Shin SY
• Kim K
• Park K
• Shin S
• Ihm C

• KMID: 2408070
• DOI: http://doi.org/10.3343/alm.2018.38.4.316

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.

Keyword

Ion semiconductor sequencing; Ion AmpliSeq TB panel; Multidrug-resistant; Mutations; Mycobacterium tuberculosis

MeSH Terms

Amikacin
Clinical Coding
Complement System Proteins
DNA
Drug Resistance*
Ethambutol
Fluoroquinolones
Isoniazid
Kanamycin
Levofloxacin
Mycobacterium tuberculosis*
Mycobacterium*
Ofloxacin
Pyrazinamide
Rifampin
Semiconductors*
Streptomycin
Amikacin
Complement System Proteins
DNA
Ethambutol
Fluoroquinolones
Isoniazid
Kanamycin
Ofloxacin
Pyrazinamide
Rifampin
Streptomycin

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