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Diagnostic mutations in the cytomegalovirus UL97 kinase gene are used to assess the level of associated ganciclovir resistance and therapeutic options. The best-known mutations at codons 460, 520, or 591 to 607 individually confer 5- to 10-fold-decreased ganciclovir susceptibility, except that a 3-fold decrease occurs in the case of the amino acid substitution C592G. Less common point and in-frame deletion mutations at codons 591 to 603 remain incompletely characterized. The ganciclovir susceptibilities of 17 mutants in this codon range were evaluated by use of the same recombinant phenotyping system and extensive assay replicates in two types of cell cultures. Amino acid substitutions K599E and T601M conferred no ganciclovir resistance, while A591V conferred 3.8-fold-decreased susceptibility. In-frame deletions of three or more codons conferred at least 8-fold-increased ganciclovir resistance, while the level of resistance conferred by one- or two-codon deletions varied from 4- to 10-fold, depending on their location. Measured levels of ganciclovir resistance were closely comparable when assays were performed in either fibroblasts or modified retinal epithelial cells. The significant revision of a few previously published resistance phenotypes and the new data strengthen the interpretation of genotypic testing for cytomegalovirus drug resistance.

Quantification of cytomegalovirus (CMV) DNA in blood may aid in the identification of patients at highest risk for developing CMV disease, the evaluation of new therapeutics, and the prompt recognition of drug-resistant CMV strains. A branched-DNA (bDNA) assay was developed for the reliable quantification of CMV DNA in peripheral blood leukocytes. The bDNA assay allowed for the highly specific and reproducible quantification of CMV DNA in clinical specimens. Furthermore, the bDNA assay was at least as sensitive as culture techniques and displayed a nearly 3 log10 dynamic range in quantification. Changes in CMV DNA levels measured by the bDNA assay in a human immunodeficiency virus-positive patient undergoing therapy were consistent with CMV culture, antigen, and genotype results and correlated with disease progression and resistance markers. The bDNA assay for the quantification of CMV DNA may provide a useful tool that can be used to aid physicians in monitoring disease progression, evaluating therapeutic regimens, and recognizing viral resistance and drug failure.

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality among hematopoietic cell transplant (HCT) recipients. We describe two pediatric HCT recipients who developed persistent and severe drug-resistant CMV infections. CMV resistance to foscarnet and ganciclovir was detected after only 6 and 11 weeks of therapy, respectively. Viral pol mutations associated with drug resistance in these patients included T838A (a novel mutation) and D588N, which were shown by marker transfer to confer foscarnet and multidrug resistance, respectively. Each of these mutations significantly reduced in vitro replication of CMV, suggesting that they may decrease viral fitness. This finding was further supported by the disappearance of mutations upon withdrawal of antiviral pressure in one patient. Novel antivirals or combination therapy may be required for the treatment of drug-resistant CMV in HCT recipients and perhaps in other severely immunocompromised patients.

Immunoglobulin G to human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) in sera from solid organ recipients was measured by an enzyme-linked immunoassay (ELISA) before and after transplant. The HHV-6 ELISA was developed from glycine extracts of HHV-6-infected and uninfected HSB-2 cells. At a serum dilution of 1:500, 80 (91%) of 88 recipients were seropositive for HHV-6 before transplant, while only 14 (16%) were seropositive for CMV. Posttransplant HHV-6 serologic rises were observed in 38 (43%) recipients; rises in 25 of these recipients were associated with primary CMV infection. Titration of sera revealed much higher HHV-6 titer rises among those with primary CMV infection than among those with CMV reactivation or with no CMV infection. Elevated HHV-6 antibody titers persisted for up to 2 years after primary CMV infection. No correlation was noted between CMV and HHV-6 antibody titers in individual serum samples.

The effectiveness of three serologic assays (two enzyme-linked immunosorbent assays [ELISAs] and latex agglutination) for cytomegalovirus (CMV) serologic matching of donors and recipients was assessed over a 2-year period in a major organ transplant program. Sera with equivocal test results were investigated by repeat testing of serum samples and additional specimens from the individuals involved and monitoring of CMV infection in recipients. An in-house ELISA identified all CMV-infective donors as seropositive. Of 63 ELISA-positive donors, 5 were negative by latex agglutination; recipients from 3 of these donors developed primary CMV infection posttransplant. The in-house ELISA and a commercial ELISA (Abbott enzyme immunoassay; Abbott Laboratories, North Chicago, Ill.) had a 93% concordance of results; follow-up testing indicated that the Abbott assay was sensitive but had a false-positive rate of about 11%. One recipient with a false-positive result developed symptomatic primary CMV infection after receiving a seropositive organ. Thus, performance characteristics of currently used screening assays affect recipient outcome.

An overnight assay, based on staining cytomegalovirus-infected cells with monoclonal antibody to the 72,000-molecular-weight major immediate-early viral protein, was compared with a conventional 14-day plaque assay for quantitation of cell-free stocks of cytomegalovirus laboratory strain AD-169 and 20 other clinical strains. Viral titers were quantitatively similar when determined by either method, but centrifugation of monolayers during inoculation enhanced viral infectivity an average of 4.1-fold. When used for scoring neutralizing antibody assays, monoclonal antibody staining yielded titers within one dilution of 14-day plaque-reduction assays in 54 of 56 titrations. Of 21 cytomegalovirus strains, 2 were not recognized by the monoclonal antibody used. Assay with monoclonal antibody offers a rapid and accurate alternative to plaque assays for quantitation or neutralization of cytomegalovirus.