Certain mutations in the viral DNA polymerase (pol) gene are known to confer drug resistance when transferred to susceptible human cytomegalovirus (CMV) strains, whereas other putative resistance mutations remain unproven. A new marker-transfer technique was used to produce recombinant CMV strains, to determine the drug susceptibility phenotypes conferred by 10 pol mutations (9 observed in clinical isolates). Various degrees of resistance to ganciclovir and cidofovir were conferred by mutations D301N, N410K, D413E, T503I, and L516R, which are located within exonuclease functional domains where D301N and D413E affect highly conserved residues. Mutations A692S, E756K, and E756D, which are not located within recognized functional domains, each conferred foscarnet resistance. This study significantly increases the number of confirmed CMV pol resistance mutations, at both conserved and nonconserved loci, with implications for molecular mechanisms and the genotypic diagnosis of antiviral resistance.

Most ganciclovir (GCV)-resistant cytomegalovirus (CMV) isolates contain UL97 gene mutations at codon 460 or 520 or between codons 590 and 607, where an increasing variety of mutations have been detected, including deletions. To determine their phenotypic effect, 9 UL97 mutations not previously studied were transferred to drug-sensitive laboratory CMV strains that contained unique restriction sites developed for this purpose. Deletion of the entire codon range 591-607 conferred a 6-fold increase in GCV resistance, with little effect on viral replication. Some mutations found in clinical isolates, including C592G and A594T, conferred only 2-3-fold decreases in GCV susceptibility. For C592G, this phenotype was confirmed by transfer to different CMV strains and by restoration of full drug susceptibility after removal of the mutation. Low drug levels resulting from oral GCV therapy may predispose the virus to the initial selection of these low-grade UL97 resistance mutations and to later accumulation of other mutations and greater resistance.

To determine the risk factors and outcomes associated with rising cytomegalovirus (CMV) antigenemia levels during preemptive therapy among stem cell allograft recipients, 119 patients with CMV antigenemia were studied. Patients were prospectively monitored for CMV antigenemia weekly; those with positive findings on antigenemia tests were treated with intravenous ganciclovir (5 mg/kg twice daily for 1 week, followed by 5 mg/kg per day for 5-6 d/wk). While on therapy, 47 of 119 (39%) patients demonstrated increases that were 2 or more times greater than their baseline values, whereas 33 of 119 (28%) patients demonstrated increases that were 5 or more times greater. Rising antigenemia was confirmed by polymerase chain reaction for CMV DNA. Multivariate analysis identified corticosteroids as the primary risk factor for increasing antigenemia: for increases greater than or equal to twice the baseline, 1 to 2 mg/kg steroids was associated with an odds ratio (OR) of 4.0. For increases greater than or equal to 2 mg/kg steroids, the OR was 10.1. CMV isolates obtained at the time of rising antigenemia were susceptible to ganciclovir, indicating that resistance was not a major factor. Overall, rising antigenemia levels were not correlated with CMV disease. All 4 patients in whom CMV disease developed during therapy, however, had rising antigenemia levels. Among the 47 patients with antigenemia increases greater than or equal to twice the baseline, 15 were re-induced with antivirals, whereas 32 continued to receive maintenance therapy. All 4 patients in whom CMV disease developed during therapy received maintenance therapy, and 3 died with CMV disease. Thus, host factors such as the receipt of corticosteroids explain increasing viral load during the early phase of preemptive therapy. Continued induction dosing or re-induction may protect against early breakthrough CMV disease and CMV-related death among patients with rising antigenemia on preemptive therapy. (Blood. 2001;97:867-874)