Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates.

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. 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). 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. 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.

Park J ，Shin SY ，Kim K ，Park K ，Shin S ，Ihm C ... -  《-》

Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing.

The increasing burden of multidrug resistant (MDR)-TB, defined by resistance to rifampin (RFP) and isoniazid (INH), and extensively drug resistant-TB, defined by MDR-TB with additional resistance to fluoroquinolones (FQs) and more than one second-line injectable drug, is a serious impediment to global TB control. We evaluated the feasibility of full-length gene analysis including inhA, katG, rpoB, pncA, rpsL embB, eis, and gyrA using a semiconductor NGS with the Ion AmpliSeq TB panel to directly analyse 34 sputum specimens confirmed by phenotypic DST: INH, RFP, ethambutol (EMB), pyrazinamide (PZA), amikacin, kanamycin, streptomycin (SM), FQs including ofloxacin, moxifloxacin, and levofloxacin. The molecular drug resistance profiles showed "very good" and "substantial" strength of agreement for the phenotypic DST results of RFP and EMB, PZA, SM, FQs resistance with specificities of 96%, and 88%, 97%, 100% and sensitivities of 100%, and 88%, 60%, 67%, respectively. The strength of agreement for the detection of resistance to INH was "substantial", compared between katG mutation and phenotypic INH only. Ion semiconductor NGS could make possible detection of several uncommon or novel amino acid changes in the full coding regions of these eight genes. However, molecular drug resistant profile should be complemented and validated by subsequent phenotypic DST studies at the same time.

Park J ，Jang W ，Kim M ，Kim Y ，Shin SY ，Park K ，Kim MS ，Shin S ... -  《-》

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea.

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

Jnawali HN ，Hwang SC ，Park YK ，Kim H ，Lee YS ，Chung GT ，Choe KH ，Ryoo S ... -  《-》

Application of next-generation sequencing to detect variants of drug-resistant Mycobacterium tuberculosis: genotype-phenotype correlation.

Ko DH ，Lee EJ ，Lee SK ，Kim HS ，Shin SY ，Hyun J ，Kim JS ，Song W ，Kim HS ... -  《Annals of Clinical Microbiology and Antimicrobials》

Detection of multidrug resistance in Mycobacterium tuberculosis.

Sekiguchi J ，Miyoshi-Akiyama T ，Augustynowicz-Kopeć E ，Zwolska Z ，Kirikae F ，Toyota E ，Kobayashi I ，Morita K ，Kudo K ，Kato S ，Kuratsuji T ，Mori T ，Kirikae T ... -  《-》