title: Evaluation of the Roche prototype 454 HIV-1 ultradeep sequencing drug resistance assay in a routine diagnostic laboratory.
abstract: BACKGROUND
  Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings.
  
  
  OBJECTIVE
  To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory.
  
  
  METHODS
  50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals.
  
  
  RESULTS
  The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen.
  
  
  CONCLUSIONS
  The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications.
authors: Department of Virology, Royal Free London NHS Foundation Trust, London, United Kingdom. Electronic address: ana.garcia@nhs.net.
authors: A
authors: A
authors: Garcia-Diaz
authors: A
authors: A
authors: Guerrero-Ramos
authors: A L
authors: AL
authors: McCormick
authors: M
authors: M
authors: Macartney
authors: T
authors: T
authors: Conibear
authors: M A
authors: MA
authors: Johnson
authors: T
authors: T
authors: Haque
authors: D P
authors: DP
authors: Webster
mesh_terms: D024882:Drug Resistance, Viral; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009154:Mutation; D010949:Plasma / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D012189:Retrospective Studies
chemical_list: D012367:RNA, Viral
keywords: 454; HIV; Resistance; UDS