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.
Zidovudine susceptibility was assessed for 525 clinical human immunodeficiency virus type 1 isolates, before and after reducing the number of replicates and zidovudine concentrations in the standardized consensus peripheral blood mononuclear cell culture assay. We conclude that omitting the 0.001 M concentration and using duplicate rather than triplicate wells are valid and cost-effective modifications of this expensive assay.Phenotypic zidovudine resistance in human immunodeficiency virus type 1 (HIV-1) isolates from patients on long-term zidovudine treatment has been shown to independently predict clinical decline (2). However, a limitation of the standardized consensus assay for in vitro drug susceptibility of HIV-1 isolates in peripheral blood mononuclear cells (PBMC) (3, 4) is its high cost. This study formally validates a cost-effective modification to the assay that involves reducing the number of replicate wells and the number of zidovudine concentrations at which viral inhibition is assessed.Zidovudine susceptibilities were determined on 525 HIV-1 isolates obtained from the National Institute of Allergy and Infectious Diseases AIDS Clinical Trials Group (ACTG) trial 116B/117 and from the laboratories of the U.S. Department of Defense (DoD) and the University of Minnesota Medical School. These determinations were made by using the PBMCbased assay validated by the ACTG and DoD (3, 4). For each HIV-1 isolate tested, triplicate quantitative p24 antigen determinations were performed with day 7 culture supernatants obtained from acutely infected PBMC. The zidovudine concentrations tested in triplicate were 0, 0.001, 0.01, 0.1, 1.0, and 5.0 M. The zidovudine concentration that inhibits virus replication by 50% (IC 50 ) was estimated by fitting a median-effect equation model (1) to the p24 antigen results. This study assessed the precision of IC 50 estimation after reducing the number of replicates per concentration from three to two and the number of zidovudine concentrations from six to five. In addition, the classification ability of the modified assay was assessed by using the following two phenotypic resistance classifications:(i) highly zidovudine resistant or not, as defined by an IC 50 value Ն1.0 M or Ͻ1.0 M, respectively; and (ii) zidovudine sensitive or not, as defined by an IC 50 value of Յ0.1 M or Ͼ0.1 M, respectively.The median-effect equation (1) relates the proportion of inhibition of HIV-1 replication to the zidovudine concentration. Estimation of IC 50 for each isolate was carried out by fitting the median-effect equation by using the nonlinear least squares procedure in the software package SAS (5). The proportion of virus inhibition (fraction affected) for each replicate was defined as 1 Ϫ (p24 antigen level/average p24 antigen level of the 0 M replicates). To examine the effect of removing replicates and zidovudine concentrations, IC 50 values were recalculated after removal of one replicate and selected concentrations. Specifically, for each isolate, every possible subset consisting of two r...
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