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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 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 104 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.

* 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.

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.


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