title: Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.
abstract: BACKGROUND
  The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.
  
  
  METHODS
  We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.
  
  
  CONCLUSIONS
  Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
authors.affiliation: Laboratory Branch, Division of HIV/ADS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. JJohnson1@cdc.gov
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Jin-Fen
authors.initials: JF
authors.lastname: Li
authors.forename: Xierong
authors.initials: X
authors.lastname: Wei
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lipscomb
authors.forename: Diane
authors.initials: D
authors.lastname: Bennett
authors.forename: Ashley
authors.initials: A
authors.lastname: Brant
authors.forename: Mian-Er
authors.initials: ME
authors.lastname: Cong
authors.forename: Thomas
authors.initials: T
authors.lastname: Spira
authors.forename: Robert W
authors.initials: RW
authors.lastname: Shafer
authors.forename: Walid
authors.initials: W
authors.lastname: Heneine
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics*; D005784:Gene Amplification; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D019380:Anti-HIV Agents; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides