McSharry J M, Lurain N S, Drusano G L, Landay A, Manischewitz J, Nokta M, O'Gorman M, Shapiro H M, Weinberg A, Reichelderfer P, Crumpacker C
Albany Medical College, New York 12208, USA.
J Clin Microbiol. 1998 Apr;36(4):958-64. doi: 10.1128/JCM.36.4.958-964.1998.
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 microM, respectively, and the IC50 for the ganciclovir-resistant D6/3/1 derivative of the AD169 strain was greater than 12 microM. 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 microM (+/-2.60). The plaque-reduction assay for these clinical isolates yielded an average IC50 of 2.80 microM (+/-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 microM 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.
已开发出一种流式细胞术检测方法，用于测定人巨细胞病毒（HCMV）实验室菌株和临床分离株对更昔洛韦的敏感性。该检测方法使用荧光染料标记的针对HCMV即刻早期和晚期抗原的单克隆抗体来识别HCMV感染的细胞，并利用流式细胞术检测和定量抗原阳性细胞的数量。通过该检测方法，更昔洛韦对HCMV AD169株的50%和90%抑制浓度（分别为IC50和IC90）分别为1.7和9.2微摩尔，而AD169株的更昔洛韦耐药D6/3/1衍生物的IC50大于12微摩尔。还通过流式细胞术分析更昔洛韦对HCMV感染细胞中晚期抗原合成的影响，确定了17株HCMV临床分离株对更昔洛韦的敏感性。对药物敏感的HCMV临床分离株而言，更昔洛韦的平均IC50为3.79微摩尔（±2.60）。针对这些临床分离株的蚀斑减少试验得出的平均IC50为2.80微摩尔（±1.46）。将流式细胞术检测结果与蚀斑减少试验结果进行比较，显示出可接受的偏差和精密度。即使在96微摩尔更昔洛韦的情况下，对感染更昔洛韦耐药临床分离株的细胞进行流式细胞术和蚀斑减少分析，也未显示晚期抗原阳性细胞百分比或蚀斑形成单位有减少。用于测定HCMV对更昔洛韦敏感性的流式细胞术检测是定量的、客观的，并且可能实现自动化，其结果在不同实验室之间具有可重复性。
