Phenotypic Evaluation of Previously Uncharacterized Cytomegalovirus DNA Polymerase Sequence Variants Detected in a Valganciclovir Treatment Trial

Background. In a large randomized trial comparing oral valganciclovir and intravenous ganciclovir for treatment of cytomegalovirus disease in solid organ transplantation, confirmed genotypic drug resistance was uncommon (<5%), but definitive interpretation was limited by the detection of 110 uncharacterized UL54 viral DNA polymerase sequence variants. Methods. Based on treatment history and genetic locus of the sequence changes, 39 of the sequence variants were prioritized for recombinant phenotyping by construction of cloned viral mutants and drug susceptibility testing in cell culture. Results. Four amino acid substitutions were newly confirmed to alter ganciclovir susceptibility: A505V and I726T conferred a borderline decrease in ganciclovir and cidofovir susceptibility, while Q578L and G841S conferred slightly decreased ganciclovir and foscarnet susceptibility. A nonviable phenotype was found for 8 mutations distributed among amino terminal, exonuclease and catalytic domains. Retesting of stored study specimens could not confirm the original detection of > 20 sequence variants, including the nonviable mutations and several resistance mutations. Conclusions. Newly phenotyped UL54 sequence variants did not significantly change the reported incidence of drug resistance in the clinical trial. Unrecognized sequence variants in diagnostic genotyping reports should be confirmed by additional testing in order to improve clinical decision making.