title: Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 using CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5).
abstract: We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 10(4) copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.
authors.affiliation: AIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Hachiya
authors.forename: S
authors.initials: S
authors.lastname: Aizawa-Matsuoka
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: Y
authors.initials: Y
authors.lastname: Takahashi
authors.forename: S
authors.initials: S
authors.lastname: Ida
authors.forename: H
authors.initials: H
authors.lastname: Gatanaga
authors.forename: Y
authors.initials: Y
authors.lastname: Hirabayashi
authors.forename: A
authors.initials: A
authors.lastname: Kojima
authors.forename: M
authors.initials: M
authors.lastname: Tatsumi
authors.forename: S
authors.initials: S
authors.lastname: Oka
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D015704:CD4 Antigens / Q000235:genetics*; D002999:Clone Cells; D005434:Flow Cytometry; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D019713:Receptors, CCR5 / Q000096:biosynthesis*; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D019380:Anti-HIV Agents; D015704:CD4 Antigens; D007202:Indicators and Reagents; D019713:Receptors, CCR5; D019718:Receptors, CXCR4