A Standardized Plaque Reduction Assay for Determination of Drug Susceptibilities of Cytomegalovirus Clinical Isolates

Abstract

Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.Cytomegalovirus (CMV) is a major opportunistic pathogen in immunocompromised hosts. Long-term therapy with ganciclovir (GCV) and foscarnet (PFA) has been associated with development of clinical resistance and progression of disease (3,4,9,10). Standardized laboratory methods to rapidly and accurately determine the susceptibility of CMV isolates to antiviral drugs are needed. The inherent difficulties in working with CMV include the slow growth of the virus and the fact that CMV clinical isolates are strongly cell associated. Multiple passages in culture are typically required to generate sufficient extracellular virus for titration and testing. Standard susceptibility assays require the inhibition of viral replication in the presence of serial concentrations of drug. The slow growth of CMV has limited the usefulness of standard assays in patient management and in guiding clinical trials. Thus, the ultimate goal is to develop a rapid method that can be used directly on clinical samples to detect resistance mutations (1, 11). As a first step toward this goal, 12 laboratories in the CMV Resistance Working Group of the AIDS Clinical Trials Group (ACTG) have collaborated in formulating and testing a standardized plaque reduction assay to use as a "gold standard." In the first phase of this process, four characterized plaque-purified CMV strains (two laboratory strains and two clinical isolates) were distributed and tested in 12 laboratories using variations of a plaque reduction assay. In the second phase, a consensus assay was used to retest these strains, as well as six additional coded clinical isolates. The inhibition curves were calculated by computer modeling using a PROC NLIN program of SAS. The results are presented in this report. MATERIALS AND METHODS Cells and drugs.In phase 1, human diploid fibroblasts from a variety of sources, commercial and in-house, were used. In phase 2, MRHF (human foreskin) cell cultures (Biowhittaker, Walkersville, Md.) were used by all of the laboratories. GCV was provided by Syntex-Roche, and PFA was provided by Astra Pharmaceuticals. The drugs were prepared from common lots by the Viral Quality Assurance Laboratory of the ACTG at Rush-Presbyterian-St. Luke's Medical Center. GCV (4.5 mM) and PFA (20 mM) stocks were filter sterilized, shipped on dry i...

Differentiated Levels of Ganciclovir Resistance Conferred by Mutations at Codons 591 to 603 of the Cytomegalovirus UL97 Kinase Gene

“…The two cases (600del and 597del3) where the current phenotypes are more GCV resistant (Table 1) than previously reported by plaque reduction assays (5, 13) reflect the same technical considerations as those for A591V. There are well-documented difficulties in the standardization of plaque assays across studies because of limited replicates of testing (often no more than three), compounded by variable operator expertise and culture conditions leading to fluctuating baseline and mutant EC 50 values that greatly affect calculated ratios (25). A secondary objective of this study was to compare the assay performance of a cloned retinal epithelial cell line overexpressing platelet-derived growth factor receptor alpha (PDGFR␣).…”

Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection

“…12 It is thought that prolonged exposure to ganciclovir, particularly at concentrations that do not fully suppress viral replication, may lead to resistance. 7,11,13,14 Ganciclovir resistance may be assessed by phenotypic analysis by plaque reduction assay 4,15 and by genotypic analysis, which demonstrates characteristic mutations in the UL97 region or the DNA polymerase gene. 4,[16][17][18] In the past, foscarnet has been the main agent utilized for therapy for ganciclovirresistant CMV in transplant recipients, 19 but its usefulness in transplant recipients is often limited by nephrotoxicity and electrolyte disturbances.…”

Point Mutations and Antiviral Drug Resistance

“…The in vitro assay determines the antiviral's ability to reduce the number of lytic plaques, which quantifies the cytopathic effect induced by the virus. This can be determined as a 50% inhibitory concentration (IC 50 ) (Chou et al, 1999;Landry et al, 2000). This is a difficult method that requires trained personnel and standard antiviral concentrations.…”