Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir

Abstract

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...

Antiviral Drug Resistance of Human Cytomegalovirus

“…They also require time to prepare the inoculum, consisting of virus-infected cells, which can be added to monolayers in 25-cm 2 flasks and incubated for 3 to 4 days in the presence of a range of drug concentrations. An alternative method of inoculation that can further shorten the assay is to accelerate transfer of virus infection by mixing infected with uninfected cells and adding them to six-well plates (121). In each case the cells are harvested, permeabilized, and treated with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody to an HCMV immediate-early (IE) antigen.…”

“…Phenotypic assays based on flow cytometry have been published (121,146). They also require time to prepare the inoculum, consisting of virus-infected cells, which can be added to monolayers in 25-cm 2 flasks and incubated for 3 to 4 days in the presence of a range of drug concentrations.…”

Rapid detection of human cytomegalovirus UL97 and UL54 mutations for antiviral resistance in clinical specimens

“…Identification from clinical specimens of the UL97 mutation alone has been reported, the 95% detection limit for obtaining usable sequence information having been <750 copies/mL for both amplicons . Rapid detection of drug resistant mutations of UL97 by novel procedures has been reported . However, using the present assay, drug‐resistant mutations in clinical specimens were successfully identified from samples with as few as 6 and 36 copies/µg of DNA.…”

Recent developments in human cytomegalovirus diagnosis

“…For phenotypic assays, the plaque-reduction assay is the standard method of antiviral susceptibility testing for HCMV. To avoid the shortcomings of plaque-reduction assays, such as their time-consuming and labor-intensive nature, other tests have been developed based on fluorescence-activated cell sorting techniques [82,83] or by simplifying the classical assay [84]. Phenotypic assays still have their justification for the detection of novel mutations or mutations affecting the resistance gene.…”