The influence of mixed HCMV UL97 wildtype and mutant strains on ganciclovir susceptibility in a cell associated plaque reduction assay.
BACKGROUND
Ganciclovir (GCV) resistance is an emerging problem following organ transplantation. A restriction fragment length polymorphism (RFLP) assay is a convenient and rapid method to discover known resistance mutations within the UL97 (phosphotransferase) gene for the determination of GCV resistance. Phenotypic resistance testing remains important for the identification of human cytomegalovirus (HCMV) strains possibly harboring novel mutations and also for the determination of foscarnet and cidofovir resistance.


OBJECTIVE
The aim of this work was to evaluate the reliability of a cell-associated plaque reduction assay with respect to an expanded UL97 RFLP assay for use on codons 460, 520, 591, 592, 594, 595 and 603. Furthermore, the influence of mixed viral populations with coexistent wildtype and mutant UL97 sequences on GCV IC(50) values was investigated.


METHODS
Twenty-eight clinical HCMV isolates were obtained from six adult patients under clinical and virological suspicion for development of GCV resistance following peripheral blood stem cell transplantation (PBSCT), and from one adult and three pediatric patients with confirmed GCV resistance following PBSCT. All isolates were tested for drug susceptibility and screened for UL97 resistance mutations.


RESULTS
The plaque reduction assay exceeded the GCV cut-off for resistance even when only a small number (5-10%) of the viral population was resistant. The proportion of UL97 mutant and wildtype strains influenced GCV IC(50) values. Genotypically detected GCV resistance always preceded phenotypically detected resistance. Long-term follow-up UL97 resistance screening revealed evidence for transient and compartment-specific UL97 mutations.


CONCLUSIONS
The stringent and longitudinal use of an expanded HCMV UL97 RFLP assay of specimens from different sites contributes to the rapid and reliable diagnosis of GCV resistance. The influence of the proportion of UL97 mutant and wildtype strains on GCV IC(50) values along with the strong correlation between phenotype and genotype suggests that the cell associated plaque reduction assay is highly reliable.
Eckle
Tobias
T
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, Elfriede-Aulhorn-Strasse 6, D-72076 Tübingen, Germany.
Jahn
Gerhard
G
Hamprecht
Klaus
K
D000293:Adolescent; D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology; D002648:Child; D003062:Codon; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000188:drug therapy / Q000821:virology; D016172:DNA Fingerprinting; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D024882:Drug Resistance, Viral / Q000235:genetics*; D015774:Ganciclovir / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D020125:Mutation, Missense; D036102:Peripheral Blood Stem Cell Transplantation; D010641:Phenotype; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D010948:Viral Plaque Assay
D000998:Antiviral Agents; D003062:Codon; D004279:DNA, Viral; D015774:Ganciclovir