title: Natural prevalence of resistance-associated variants in hepatitis C virus NS5A in genotype 3a-infected people who inject drugs in Germany.
abstract: BACKGROUND
  People who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking.
  
  
  OBJECTIVE
  The aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naïve PWID living in Germany.
  
  
  METHODS
  The full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol.
  
  
  RESULTS
  With the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%).
  
  
  CONCLUSIONS
  A protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID.
authors: Institute for Virology, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany.
authors: Andreas
authors: A
authors: Walker
authors: LVR-Hospital Essen, Department of Addictive Behavior and Addiction Medicine, Faculty of Medicine, University of Duisburg-Essen, Germany.
authors: Holger
authors: H
authors: Siemann
authors: Institute of Virology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany.
authors: Svenja
authors: S
authors: Groten
authors: Institute of Virology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany.
authors: R Stefan
authors: RS
authors: Ross
authors: LVR-Hospital Essen, Department of Addictive Behavior and Addiction Medicine, Faculty of Medicine, University of Duisburg-Essen, Germany.
authors: Norbert
authors: N
authors: Scherbaum
authors: Institute for Virology, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany. Electronic address: joerg.timm@med.uni-duesseldorf.de.
authors: Jörg
authors: J
authors: Timm
mesh_terms: D024882:Drug Resistance, Viral; D055030:Drug Users; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D005858:Germany / Q000453:epidemiology; D016174:Hepacivirus / Q000145:classification / Q000187:drug effects* / Q000235:genetics*; D006526:Hepatitis C / Q000453:epidemiology* / Q000821:virology*; D006801:Humans; D008297:Male; D010802:Phylogeny; D015995:Prevalence; D062486:Public Health Surveillance; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Antiviral treatment; Genotype 3a; Hepatitis C virus; NS5A