Detection of Ganciclovir Resistance Mutations and Quantitation of Cytomegalovirus (CMV) DNA in Leukocytes of Patients with Fatal Disseminated CMV Disease

Abstract

Cytomegalovirus (CMV) UL97 mutations associated with ganciclovir resistance at codons 460, 594, and 595 were detected by polymerase chain reaction (PCR) followed by restriction enzyme analysis in CMV blood isolates and directly in polymorphonuclear leukocyte (PMNL) DNA extracts of 4 subjects who died of progressive disseminated CMV disease due to ganciclovir-resistant CMV strains. The CMV DNA load was also serially determined in leukocyte fractions of these patients using a quantitative-competitive PCR assay. There was excellent concordance between specific UL97 mutations in blood culture isolates and those detected in PMNL fractions for all patients. Emergence of such UL97 mutations during ganciclovir therapy was associated with an increasing CMV DNA burden in leukocytes of the 2 patients with AIDS but not in the 2 subjects with chronic lymphocytic leukemia. Rapid molecular strategies, including detection of common CMV UL97 mutations and CMV DNA quantitation, can be used directly in leukocytes of immunocompromised subjects with CMV disease to monitor antiviral therapy.

Antiviral Drug Resistance of Human Cytomegalovirus

“…on May 10, 2018 by guest http://cmr.asm.org/ restriction sites, which are detected by running a set of restriction digests of the PCR products on an agarose gel (19,20,42,43,74,83). The pattern of fragments produced by different enzymes is indicative of the presence of specific drug resistance mutations.…”

Human Cytomegalovirus Resistance to Antiviral Drugs

“…Those assays are based on the restriction fragment length polymorphisms (RFLPs) of PCR-amplified DNA fragments and on DNA sequencing of viral genes (UL97 and UL54) that have been involved in HCMV resistance to antivirals. One of the greatest advantages of those assays is that they can be performed directly with clinical specimens (19,134), thus reducing considerably the time required to obtain results. By omitting the need to grow the virus, such methods also minimize the risks of introducing a selection bias.…”

New Strategies for Prevention and Therapy of Cytomegalovirus Infection and Disease in Solid-Organ Transplant Recipients

“…Certain mutations in the viral UL97 and UL54 genes confer ganciclovir resistance to CMV (24,42,114,131,426). Codon changes in the DNA polymerase gene UL54 are responsible for foscarnet resistance (23).…”