title: Evaluation of a commercial real-time PCR kit for the detection of the Q80K polymorphism in plasma from HCV genotype 1a infected patients.
abstract: BACKGROUND
  Screening of the natural HCV NS3 polymorphism Q80K is required prior to simeprevir administration due to the reduced susceptibility of genotype 1 viruses carrying this amino acid variant. A simple, rapid and robust test for Q80K screening would be advisable in routine diagnostic laboratories.
  
  
  OBJECTIVE
  The aim of this study was to evaluate a commercial NS3 Q80K real-time PCR kit (Q80K Polymorphism Kit, Clonit srl, Milan, Italy).
  
  
  METHODS
  Forty-three plasma samples obtained from untreated HCV genotype 1a-infected patients and previously sequenced at a reference laboratory, were sent to two public clinical virology laboratories for blinded Q80K screening with the kit under evaluation. The sample panel included 25 cases with the wild type 80Q, 17 with the mutant 80K and 1 with the mutant 80L.
  
  
  RESULTS
  Laboratory 1 identified 22/25 (88.0%) 80Q and 17/17 (100.0%) 80K cases. Laboratory 2 identified 23/25 (92.0%) 80Q and 16/17 (94.2%) 80K cases. All of the unidentified cases were scored as negative, with no mutant/wild type miscalling. The 80L variant was scored as indeterminate by Laboratory 1 and as negative by Laboratory 2. Overall, sensitivity and specificity for detection of 80K were 97.1% (95% C.I., 82.9-99.8%) and 100.0% (90.2-100.0%), respectively. However, the system did not provide any result for 6/84 cases (7.1% failure rate), not including the 80L variant which is not expected to be detected as stated in the kit package insert. Global inter-laboratory concordance was 93.0%.
  
  
  CONCLUSIONS
  Despite good specificity, this Q80K detection system needs improvements in amplification success rate and robustness.
authors.affiliation: Department of Medical Biotechnology, University of Siena, Viale Bracci n.16, 53100 Siena, Italy. Electronic address: ilariavicenti@gmail.com.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Vicenti
authors.affiliation: Department of Molecular Medicine, Sapienza University of Rome, Italy. Electronic address: francesca.falasca@uniroma1.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Falasca
authors.affiliation: Hygiene Unit, IRCCS AOU San Martino-IST, Genoa, Italy; Department of Health Sciences, University of Genoa, Genoa, Italy. Electronic address: sticchi@unige.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Sticchi
authors.affiliation: Hygiene Unit, IRCCS AOU San Martino-IST, Genoa, Italy. Electronic address: bianca.bruzzone@hsanmartino.it.
authors.forename: Bianca
authors.initials: B
authors.lastname: Bruzzone
authors.affiliation: Department of Molecular Medicine, Sapienza University of Rome, Italy. Electronic address: ombretta.turriziani@uniroma1.it.
authors.forename: Ombretta
authors.initials: O
authors.lastname: Turriziani
authors.affiliation: Department of Medical Biotechnology, University of Siena, Viale Bracci n.16, 53100 Siena, Italy. Electronic address: maurizio.zazzi@gmail.com.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Zazzi
mesh_terms: D000998:Antiviral Agents / Q000627:therapeutic use; D024882:Drug Resistance, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D007558:Italy; D011110:Polymorphism, Genetic; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D000069616:Simeprevir / Q000627:therapeutic use; D017361:Viral Nonstructural Proteins / Q000097:blood* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D011933:Reagent Kits, Diagnostic; D000069616:Simeprevir; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: Drug resistance assay; HCV; Natural polymorphisms; Q80K; Simeprevir