Flow Cytometric Determination of Ganciclovir Susceptibilities of Human Cytomegalovirus Clinical Isolates

摘要

A flow cytometric assay has been developed for the measurement of susceptibilities to ganciclovir of laboratory strains and clinical isolates of human cytomegalovirus (HCMV). The assay uses fluorochrome-labeled monoclonal antibodies to HCMV immediate-early and late antigens to identify HCMV-infected cells and flow cytometry to detect and quantitate the number of antigen-positive cells. By this assay, the 50 and 90% inhibitory concentrations (IC₅₀ and IC₉₀, respectively) of ganciclovir for the AD169 strain of HCMV were 1.7 and 9.2 μM, respectively, and the IC₅₀ for the ganciclovir-resistant D6/3/1 derivative of the AD169 strain was greater than 12 μM. The ganciclovir susceptibilities of 17 HCMV clinical isolates were also determined by flow cytometric analysis of the effect of ganciclovir on late-antigen synthesis in HCMV-infected cells. The average IC₅₀ of ganciclovir for drug-sensitive HCMV clinical isolates was 3.79 μM (±2.60). The plaque-reduction assay for these clinical isolates yielded an average IC₅₀of 2.80 μM (±1.46). Comparison of the results of the flow cytometry assays with those obtained from the plaque-reduction assays demonstrated acceptable bias and precision. Flow cytometric and plaque-reduction analysis of cells infected with ganciclovir-resistant clinical isolates failed to show a reduction in the percentage of late-antigen-positive cells or PFU, even at 96 μM ganciclovir. The flow cytometric assay for determining ganciclovir susceptibility of HCMV is quantitative, and objective, and potentially automatable, and its results are reproducible among laboratories.