Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates

A. J. Japour, D. L. Mayers, V. A. Johnson, D. R. Kuritzkes, Laurel A Beckett, J. M. Arduino, J. Lane, R. J. Black, P. S. Reichelderfer, R. T. D'Aquila, C. S. Crumpacker, L. Abrams, F. McCutchan, D. Burke, L. Gardner, C. Roberts, R. Chung, C. Hicks, E. Shellie

Research output: Contribution to journalArticle

251 Scopus citations

Abstract

A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seronegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV- 1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1,000 50% tissue culture infective doses per 106 cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 μM. The ZDV 50% inhibitory concentrations (IC50) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 μM and from 0.15 to >5.0 μM, respectively. Use of this consensus protocol reduced interlaboratory variability for ZDV IC50 determinations with reference HIV- 1 isolates. Among eight laboratories, the coefficient of variation ranged from 0.85 to 1.25 with different PBMC protocols and was reduced to 0.39 to 0.98 with the standardized assay. Among the clinical HIV-1 isolates assayed by the standardized drug susceptibility assay, the median ZDV IC50 increased gradually with more ZDV therapy. This protocol provides an efficient and reproducible means to assess the in vitro susceptibility to antiretroviral agents of virtually all clinical HIV-1 isolates.

Original languageEnglish (US)
Pages (from-to)1095-1101
Number of pages7
JournalAntimicrobial Agents and Chemotherapy
Volume37
Issue number5
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology (medical)

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    Japour, A. J., Mayers, D. L., Johnson, V. A., Kuritzkes, D. R., Beckett, L. A., Arduino, J. M., Lane, J., Black, R. J., Reichelderfer, P. S., D'Aquila, R. T., Crumpacker, C. S., Abrams, L., McCutchan, F., Burke, D., Gardner, L., Roberts, C., Chung, R., Hicks, C., & Shellie, E. (1993). Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. Antimicrobial Agents and Chemotherapy, 37(5), 1095-1101.