title: Identification of hepatitis C virus genotype 3 by a commercial assay challenged by natural polymorphisms detected in Spain from patients with diverse origins.
abstract: BACKGROUND
  Hepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals.
  
  
  OBJECTIVE
  Given the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5' untranslated region (5' UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS).
  
  
  METHODS
  For the 24 indeterminate samples, the 5' UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed.
  
  
  RESULTS
  HCV genotyping based on 5' UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5' UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples.
  
  
  CONCLUSIONS
  The observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options.
authors.affiliation: Microbiology Service, Germans Trias i Pujol University Hospital, Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Spain; Centro de Investigación Biomédica en Red (CIBER) en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Saludes
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institute of Research (VHIR-HUVH), Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Quer
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institute of Research (VHIR-HUVH), Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Roche Diagnostics SL, Sant Cugat del Vallès, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Gregori
authors.affiliation: Microbiology Service, Germans Trias i Pujol University Hospital, Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Spain.
authors.forename: Elisabet
authors.initials: E
authors.lastname: Bascuñana
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institute of Research (VHIR-HUVH), Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Damir
authors.initials: D
authors.lastname: García-Cehic
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institute of Research (VHIR-HUVH), Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Juan Ignacio
authors.initials: JI
authors.lastname: Esteban
authors.affiliation: Microbiology Service, Germans Trias i Pujol University Hospital, Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Ausina
authors.affiliation: Microbiology Service, Germans Trias i Pujol University Hospital, Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Spain; Centro de Investigación Biomédica en Red (CIBER) en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain. Electronic address: emartro.igtp.germanstrias@gencat.cat.
authors.forename: Elisa
authors.initials: E
authors.lastname: Martró
mesh_terms: D020121:5' Untranslated Regions; D000328:Adult; D000368:Aged; D016000:Cluster Analysis; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D010802:Phylogeny; D013030:Spain; D055815:Young Adult
chemical_list: D020121:5' Untranslated Regions
keywords: Genotype 3; Genotyping; HCV; Indeterminate; UDPS