Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group

Abstract

A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 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%o 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 FM. The ZDV 50% inhibitory concentrations (IC5o) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 pM and from 0.15 to >5.0 ,IM, 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. Anmong 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.

Virologic and Immunologic Determinants of Heterosexual Transmission of Human Immunodeficiency Virus Type 1 in Africa

“…Correlation between this method and standard measurement of CD4 + T cells by flow cytometry range from 0.87 to 0.95. 29,30 The ACTG consensus method was used to isolate HIV-1 from the PBMCs of subjects as described by Japour et al 31 …”

HIV-1 Genotypic Resistance Patterns Predict Response to saquinavir–ritonavir Therapy in Patients in Whom Previous Protease Inhibitor Therapy Had Failed

“…Genotypic and phenotypic methods of measuring drug resistance are increasingly available to clinicians (31)(32)(33)(34)(35)(36)(37). However, the role of these tests in clinical practice has not been fully assessed.…”

Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

“…Although the molecular mechanisms of resistance are well studied, the pharmacodynamics of resistance are less well understood. Resistance is typically measured as a change in IC 50 (Table S1) relative to WT virus (27)(28)(29)(30). Antiretroviral drugs are used at concentrations above IC 50 , however, and inhibition at clinical concentrations can only be predicted from IC 50 if the shape of the dose-response curve is known.…”