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

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.

Evaluation of target-enrichment accompanied by next-generation sequencing for detection of four pathogens in bloodstream infections / 军事医学

Objective To establish an Ampliseq method that combines target-enrichment and the next-generation sequencing for simultaneous detection of Staphylococcus aureus, S.epidermidis, Klebsiella pneumoniae and Listeria monocytogenes in order to provide a fast and accurate means to detect pathogens in bloodstream infections.Methods A method to evaluate the LOD,specificity and sensitivity by constructing simulated samples spiked in known pathogens was established.Results and Conclusion Target-enrichment Ampliseq showed good sensitivity and specificity,and the limit of detection(LOD)was as low as 101CFU/ml.The sensitivity was 95.38%,the specificity was 95.45%and the Kappa value was between 0.839 -1.000.This method can detect S.aureus,S.epidermidis,K.pneumoniae and L.monocytogenes simultaneously in one reaction within 15 hours.

Association between Mutation and Expression of TP53 as a Potential Prognostic Marker of Triple-Negative Breast Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: TP53, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the role of TP53 status as a prognostic and predictive marker of triple-negative breast cancer (TNBC). MATERIALS AND METHODS: We performed p53 immunohistochemistry (IHC), nCounter mRNA expression assay, and DNA sequencing to determine the relationship between TP53 alteration and clinical outcomes of TNBC patients. RESULTS: Seventy-seven of 174 TNBC patients were found to harbor a TP53 mutation. Patients with missense mutations showed high protein expression in contrast to patients with deletion mutations (positivity of IHC: wild type vs. missense vs. deletion mutation, 53.6% vs. 89.8% vs. 25.0%, respectively; p < 0.001). TP53 mRNA expression was influenced by mutation status (mRNA expression [median]: wild type vs. missense vs. deletion mutation, 207.36± 132.73 vs. 339.61±143.21 vs. 99.53±99.57, respectively; p < 0.001). According to survival analysis, neither class of mutation nor protein or mRNA expression status had any impact on patient prognosis. In subgroup analysis, low mRNA expression was associated with poor prognosis in patients with a TP53 missense mutation (5-year distant recurrence-free survival [5Y DRFS]: low vs. high, 50.0% vs. 87.8%; p=0.009), while high mRNA expression with a TP53 deletion mutation indicated poor prognosis (5Y DRFS: low vs. high, 91.7% vs. 75.0%; p=0.316). CONCLUSION: Association between TP53 mutation and expression indicates a potential prognostic marker of TNBC; hence both DNA sequencing and mRNA expression analysis may be required to predict the prognosis of TNBC patients.