A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

Kurt Van Baelen, Evelien Rondelez, Veerle Van Eygen, Kevin Ariën, Marleen Clynhens, Peggy Van den Zegel, Bart Winters, Lieven J Stuyver

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Abstract

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Original languageEnglish
JournalJournal of Virological Methods
Volume161
Issue number2
Pages (from-to)231-9
Number of pages9
ISSN0166-0934
DOIs
Publication statusPublished - Nov-2009
Externally publishedYes

Keywords

  • Drug Resistance, Multiple, Viral
  • HIV Infections
  • HIV Integrase
  • HIV Integrase Inhibitors
  • HIV Reverse Transcriptase
  • HIV-1
  • Humans
  • Microbial Sensitivity Tests
  • Polymorphism, Genetic
  • Pyrrolidinones
  • Quinolones
  • RNA, Viral
  • Reproducibility of Results
  • Ribonuclease H, Human Immunodeficiency Virus
  • Sensitivity and Specificity
  • Sequence Analysis, DNA

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