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)

doi:10.1128/AAC.45.2.495-501.2001

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Antimicrobial Agents and Chemotherapy, February 2001, p. 495-501, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.495-501.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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)

Atsuko Hachiya,¹ Saori Aizawa-Matsuoka,¹ Mari Tanaka,¹ Yukiko Takahashi,¹ Setsuko Ida,¹ Hiroyuki Gatanaga,¹^,² Yoshihiro Hirabayashi,¹ Asato Kojima,³ Masashi Tatsumi,⁴ and Shinichi Oka¹^,^*

Experimental Retroviral Section, Department of Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,² and Departments of Pathology³ and Veterinary Science,⁴ National Institute of Infectious Diseases, and AIDS Clinical Center, International Medical Center of Japan,¹ Tokyo, Japan

Received 10 February 2000/Returned for modification 11 April 2000/Accepted 7 November 2000

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 amountsof HIV-1 in culture supernatants, which enabled us to performthe phenotypic resistance assay. Determination of HIV-1 susceptibilityto various protease inhibitors (PI) and nucleoside reverse transcriptaseinhibitors was completed within 15 days in T-cell-tropic (X4)and macrophage-tropic (R5) viruses using fresh plasma samplescontaining at least 10⁴ copies/ml. The nucleotide sequence of the envelope V3 regionof HIV-1 in plasma was almost identical to that of the virus isolatedby MAGIC-5 cells, suggesting a lack of selection bias in our assay.The assay variability was confined to within five-fold in alldrugs examined. Accordingly, we used a 10-fold increase in the50% inhibitory concentration as the cutoff value for viral resistancein the present assay. HIV-1 resistant to lamivudine, which wasnot detected by conventional genotypic assays, was isolated. InHIV-1 with PI-associated primary amino acid substitutions, ourassay showed that drug resistance profiles correlated well withpreviously reported genotypic-assay data. Furthermore, our assayprovided comprehensive results regarding PI resistance in thepresence of multiple mutations. The novel assay successfully quantifiedthe level of resistance of clinical HIV-1 isolates to a batteryof anti-HIV drugs, indicating its clinical usefulness, particularlyin patients who failed to respond to antiretroviralchemotherapy.

^* Corresponding author. Mailing address: AIDS Clinical Center, International Medical Center of Japan, 1-21-1, Toyama, Shinjuku-ku,Tokyo 162-8655, Japan. Phone and fax: 81-3-5273-5193. E-mail:oka{at}imcj.hosp.go.jp.

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