Rapid culture and quantitation of human immunodeficiency virus type 1 from patient cells without the use of mitogen-stimulated donor cells

Abstract

We report the development of a rapid, sensitive virus culture method for direct quantitation of human immunodeficiency virus (HIV) in peripheral blood mononuclear cells (PBMCs). This new method involves culturing 10(7) PBMCs from HIV-seropositive persons in 10 ml of medium containing phorbol 12-myristate 13-acetate and interleukin-2. Both agents stimulate cell activation and hence viral replication. Cell-associated virus and free virus are quantitated by a commercially available HIV p24 antigen capture enzyme immunoassay. Detection of cell-associated p24 antigen by flow cytometry was less sensitive than by the enzyme immunoassay. In this preliminary study, HIV was detected in 20 of 23 HIV-seropositive patients and in none of the 11 HIV seronegative low-risk individuals. One HIV-seronegative person with Guillain-Barré syndrome following high-risk activity was found to be rapid-HIV-culture positive. The overall sensitivity and specificity were 87 and 100%, respectively. By comparing the quantity of virus produced in infected cells with the amount of virus produced in chronically infected U1 monocytes and ACH-2 lymphocytes stimulated with phorbol 12-myristate 13-acetate and interleukin-2, the approximate number of infected cells per sample is calculated. In the same patient specimens, quantitation of the number of HIV infected cells by the HIV rapid-culture method correlated with the results of the 21-day cell dilution coculture assay (correlation coefficient r = 0.5; 95% confidence interval, 0.07 to 0.77). Advantages of the rapid HIV culture include no requirement for donor PBMCs or change of media, shortened culture time, and the ability to detect p24 viral antigen from cell-associated virus for quantitation of viral load.