Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.Human cytomegalovirus (HCMV) is an important opportunistic pathogen for humans of all ages with immature or compromised immune functions (17). Drugs commonly used for the treatment of HCMV infections are ganciclovir (GCV), cidofovir, and foscarnet, which all target the viral DNA polymerase. However, all these compounds cause considerable adverse effects. Their prolonged application frequently results in the selection of drug-resistant HCMV mutants with eventual failure of therapy (reviewed in reference 6).The first-line drug GCV is pharmacologically a prodrug that requires phosphorylation to the monophosphate by the HCMV UL97-encoded protein kinase to gain its antiviral activity. As a consequence, over 80% of GCV-resistant isolates carry mutations in the UL97 gene (4, 8). UL97 mutations known to confer GCV resistance occur predominantly at codons 460 and 520 and at several codons between 590 and 607, particularly at codon positions 592, 594, 595, and 603 (3). During the selection process of a spontaneously occurring UL97 mutation under drug pressure, mixed virus populations exist in the patient. At present, direct dideoxy chain termination sequencing (CTSQ) of PCR products spanning this part of the UL97 gene is the method of choice for the detection of mutant viruses (16). However, the sensitivity of the CTSQ method in detecting minor mutant fractions in samples with heterogeneous mutant-wild-type HCMV mixtures is only moderate. Approximately 20% of the total virus population has to be mutant virus in order to be solidly detected (2; B. Schindele and B. Ehlers, unpublished data). However, fast and early detection of emerging resistant virus is of particular importance, since mixed HCMV UL97 populations can influence GCV susceptibility, and detection may predict the outgrowth of resistant virus variants (5). T...

Prenatally acquired human cytomegalovirus (HCMV) infection is the most frequent viral infection of newborns in developed countries. Virostatic therapy is accompanied by side effects and stepwise emergence of resistant virus variants. Different genotypic approaches show limited sensitivity in detecting on-growing minor resistant virus populations. Here, we demonstrate the superiority of pyrosequencing for the monitoring of mutant emergence. In a preterm baby born after 28 weeks of gestation and suffering from disseminated congenital HCMV infection, long-term control could not be achieved under ganciclovir/valganciclovir therapy and the infant died on the 113th day of life. Resistance-associated mutations in the HCMV UL97 gene were not detected by conventional DNA sequencing but postmortem pyrosequencing. Four different CMV variants carrying resistance-associated mutations each representing 11-17% of the total CMV population were found.