FLOW CYTOMETRIC DETERMINATION OF GANCICLOVIR SUSCEPTIBILITIES OF HUMAN CYTOMEGALOVIRUS CLINICAL ISOLATES

A flow cytometric assay has been developed for the measurement of susc eptibilities to ganciclovir of laboratory strains and clinical isolate s of human cytomegalovirus (HCMV). The assay uses fluorochrome-labeled monoclonal antibodies to HCMV immediate-early and late antigens to id entify HCMV-infected cells and flow cytometry to detect and quantitate the number of antigen-positive cells. By this assay, the 50 and 90% i nhibitory concentrations (IC50 and IC90, respectively) of ganciclovir for the AD169 strain of HCMV were 1.7 and 9.2 mu M, respectively, and the IC50 for the ganciclovir-resistant D6/3/1 derivative of the AD169 strain was greater than 12 mu M. The ganciclovir susceptibilities of 1 7 HCMV clinical isolates were also dctermined by flow cytometric analy sis of the effect of ganciclovir on late-antigen synthesis in HCMV-inf ected cells. The average IC50 of ganciclovir for drug-sensitive HCMV c linical isolates was 3.79 mu M (+/-2.60). The plaque-reduction assay f or these clinical isolates yielded an average IC50 of 2.80 mu M (+/-1. 46). Comparison of the results of the flow cytometry assays with those obtained from the plaque-reduction assays demonstrated acceptable bia s and precision. Flow cytometric and plaque-reduction analysis of cell s infected with ganciclovir-resistant clinical isolates failed to show a reduction in the percentage of late-antigen-positive cells or PFU, even at 96 mu M ganciclovir. The flow cytometric assay for determining ganciclovir susceptibility of HCMV is quantitative, and objective, an d potentially automatable, and its results are reproducible among labo ratories.