Simple Culture Assay for isolation and resistance assessment of clinical HIV-1 isolates.
Int Conf AIDS 2002 Jul 7-12; 14:(abstract no. B10189)
Mukadi BK, Delferiere N, Bodeus M, Goubau P Universite Catholique de Louvain, Brussels, Belgium
We developed a simple method giving a high isolation rate of patient HIV-1 strains and an antiviral drug susceptibility assay for clinical isolates. The two-step procedure consists in either the culture of plasma HIV-1 virus or co-cultivation of infected PBMC with PHA-stimulated CD4 from blood donors. Patients sample are treated within one hour by centrifugation of the EDTA whole blood to separate the plasma and by ultracentrifugation of a filtrated EDTA plasma. The sample residues are cultivated with fresh CD4 cells in RPMI medium. A p24 antigen ELISA (Genetic systems HIV-1 Ag EIA, Biorad, France) is used to evaluate the viral replication: about 80% of HIV-1 clinical isolates was obtained when the viral load was at leat 50,000 RNA copies/ml. In the second step, HIV-1 isolates are cultivated in the presence of antiretroviral drugs at the mean peak plasma concentrations: each well contains 1,25 to 1,6 105 CD4 cells in a total volume of 300ml and triplicate control wells without antiviral agent are added. Cultures are incubated at 37?C with 5% CO2. The supernatant is assayed for p24 antigen inhibition after suitable dilution to be readable in the controls. To validate the method, two dilutions (1:1 and 1:100) of the HTLV IIIB reference virus were evaluated in quadruplicate wells containing serial tree-fold antiretroviral dilutions in order to determine the IC50 and the influence of viral inoculum. The IC50 obtained when testing for example the ZDV-susceptible HTLV IIIb virus in the presence of ZDV concentrations ranging from 0 to 5mM are 0,0045mM and 0,0065mM respectively for the non-diluted and the diluted virus. We obtained the similar results with others antiretroviral agents (ddI, 3TC, d4T, ABC, NEV, RTV, IDV, SQV, NFV). We developed a simple method for high isolation rate of HIV-1 clinical samples and an evaluation of viral resistance at a single cut-off antiviral drug concentration without the need of prior precise virus titration.
Keywords: AEGIS, HIV-1, Viral Load, Zidovudine, Stavudine, Lamivudine, Didanosine, Acquired Immunodeficiency Syndrome, Culture, Antiviral Agents, Virus Replication, HIV-1 Reverse Transcriptase, France, Greece, Human, Ethnology, virology
