title: Quantitative analysis of influenza A (H3N2) E119V and R292K variants in clinical specimens by real-time reverse transcription polymerase chain reaction.
abstract: BACKGROUND
  Because influenza virus isolates after cell culture are required to determine their susceptibility to neuraminidase inhibitors, the differences in normal or low-susceptibility variant population frequencies between clinical samples and isolates have not been considered.
  
  
  OBJECTIVE
  To identify variations in low-susceptibility populations in clinical samples after initiation of oseltamivir and zanamivir therapy by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).
  
  
  METHODS
  We measured the populations of the low-susceptibility influenza A H3N2 variants E119V and R292K by qRT-PCR using 305 nasal aspiration samples collected over time from 13, 16, and 11 patients treated with no neuraminidase inhibitors, oseltamivir, and zanamivir, respectively. The variant population in the isolates was also determined when the population of low-susceptibility variants in the clinical samples increased following treatment. Moreover, the susceptibility of all isolates was measured.
  
  
  RESULTS
  The E119V variant was detected in only one patient during oseltamivir therapy, exhibiting decreased susceptibility to oseltamivir. Prior to treatment, R292K variants were detected in all clinical samples; however, they comprised only a small fraction of the total population. The proportion of the R292K variant in clinical samples increased for 6/27 (22.2%) patients treated with oseltamivir or zanamivir, whereas an increase in the proportion of the R292K variant in virus isolates was observed in only one patient.
  
  
  CONCLUSIONS
  Discrepancies in the proportion of R292K variants between clinical samples and isolates should be suspected in clinical settings. qRT-PCR is useful for quantitative analysis of drug-resistant influenza virus and for immediate notification of the result.
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: smasatoki@yahoo.co.jp.
authors.forename: Masatoki
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: Ken_Honzumi@yahoo.co.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Honzumi
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: ts3005@fmu.ac.jp.
authors.forename: Toshiko
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: don@fmu.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: Department of Pediatrics, Fukushima South Aizu Hospital, 14-1 Nagata-aza-Kazashita, Minamiaizu 967-0006, Japan. Electronic address: nabe5658@fmu.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: watarikko0421@gmail.com.
authors.forename: Kyohei
authors.initials: K
authors.lastname: Miyazaki
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: kyuki@fmu.ac.jp.
authors.forename: Yukihiko
authors.initials: Y
authors.lastname: Kawasaki
authors.affiliation: Department of Pediatrics, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Electronic address: mhosoya@fmu.ac.jp.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Hosoya
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000627:therapeutic use; D012634:Bodily Secretions / Q000821:virology; D002648:Child; D002675:Child, Preschool; D024882:Drug Resistance, Viral; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics* / Q000302:isolation & purification*; D007251:Influenza, Human / Q000188:drug therapy / Q000821:virology*; D008297:Male; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics*; D020125:Mutation, Missense; D009296:Nasal Cavity / Q000821:virology; D053139:Oseltamivir / Q000627:therapeutic use; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D053243:Zanamivir / Q000627:therapeutic use
chemical_list: D000998:Antiviral Agents; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D050505:Mutant Proteins; D053139:Oseltamivir; D053243:Zanamivir
keywords: Antiviral therapy; Neuraminidase inhibitor; Oseltamivir; Polymerase chain reaction; Resistant virus; Zanamivir