A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes.
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.
Van Baelen
Kurt
K
Virco BVBA, Generaal De Wittelaan L11 B3, Mechelen, Belgium. kvbaele1@its.jnj.com
Rondelez
Evelien
E
Van Eygen
Veerle
V
Ariën
Kevin
K
Clynhens
Marleen
M
Van den Zegel
Peggy
P
Winters
Bart
B
Stuyver
Lieven J
LJ
D024921:Drug Resistance, Multiple, Viral / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D019427:HIV Integrase / Q000235:genetics*; D019428:HIV Integrase Inhibitors / Q000627:therapeutic use; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D011110:Polymorphism, Genetic; D011760:Pyrrolidinones / Q000627:therapeutic use; D015363:Quinolones / Q000627:therapeutic use; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D000068898:Raltegravir Potassium; D015203:Reproducibility of Results; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D015363:Quinolones; D012367:RNA, Viral; D000068898:Raltegravir Potassium; C509700:elvitegravir; D019427:HIV Integrase; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus