There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC 50 ) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC 50 values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M460V, and the GCV IC 50 results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.Human cytomegalovirus (HCMV) is a betaherpesvirus that causes serious diseases in immunocompromised hosts, particularly transplantation recipients. Drugs used currently against HCMV include ganciclovir (GCV), foscarnet, and cidofovir, and of these, GCV is the initial drug of choice (11, 13). However, the advent of HCMV resistant to GCV has made control of HCMV difficult. Mutant HCMV strains strongly resistant to GCV are unable to phosphorylate GCV owing to mutations in key regions of the UL97 gene (domains VIII, VI, and IX) (1,2,5,8,9,21,26). Mutations in UL54, the viral DNA polymerase, also confer resistance to GCV (10,15,24).There are two types of methods for assessing the susceptibility of HCMV to GCV: one is genotypic and detects the resistance-related mutant genes, and the other is phenotypic and assesses resistance directly (3, 13). The genotypic methods include DNA sequencing, detection of restriction fragment length polymorphisms (RFLP), and probe-specific and primerspecific hybridization. DNA sequencing is the reference method for detecting resistance-related GCV mutations, and RFLP has been widely used to detect GCV resistance due to mutations in the UL97 gene (4, 7). Although genotypic tests are fast, phenotypic tests are necessary to detect novel mutations and mutations in the UL54 DNA polymerase gene. Examples of phenotypic tests are the plaque reduction assay (PRA), in situ enzyme-linked immunosorbent assay, DNA reduction assay, and flow cytometry-based assay. The flow cytometric assay counts trypsinized fibroblasts expressing the HCM...

Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCRbased restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.