title: Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5'UTR.
abstract: BACKGROUND
  Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options.
  
  
  OBJECTIVE
  To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B.
  
  
  METHODS
  Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA.
  
  
  RESULTS
  Of 100 samples from 50 patients, ≥ 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold.
  
  
  CONCLUSIONS
  Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK. Electronic address: a.mccormick@westminster.ac.uk.
authors: Adele L
authors: AL
authors: McCormick
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Malcolm J
authors: MJ
authors: Macartney
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Ikran
authors: I
authors: Abdi-Abshir
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Wendy
authors: W
authors: Labbett
authors: Department of Infection and Population Health, University College London, UK.
authors: Colette
authors: C
authors: Smith
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Dianne
authors: D
authors: Irish
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Daniel P
authors: DP
authors: Webster
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
authors: Tanzina
authors: T
authors: Haque
mesh_terms: D020121:5' Untranslated Regions; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020121:5' Untranslated Regions; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C069130:nucleocapsid protein, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: DNA sequencing; Genotyping; Hepatitis C virus; LiPA; RealTime PCR; Subtyping