Rapid Detection of Human Cytomegalovirus UL97 and UL54 Mutations Directly from Patient Samples

Abstract

cHuman cytomegalovirus (CMV) is a significant contributor to morbidity and mortality in immunocompromised patients, particularly in the transplant setting. The availability of anti-CMV drugs has improved treatment, but drug resistance is an emerging problem. Here, we describe an improved, rapid, sequencing-based assay for the two genes in CMV where drug resistance occurs, the UL97 and UL54 genes. This assay is performed in 96-well format with a single master mix and provides clinical results within 2 days. It sequences codons 440 to 645 in the UL97 gene and codons 255 to 1028 in the UL54 gene with a limit of detection of 240 IU/ml. With this assay, we tested 43 specimens that had previously been tested for UL97 drug resistance and identified 3 with UL54 mutations. One of these patients had no concurrent UL97 mutation, pointing toward the need for an assay that facilitates dual UL97/UL54 gene testing for complete resistance profiling. Human cytomegalovirus (CMV) causes morbidity and mortality in immunocompromised patients, including transplant and HIV-infected patients (1). The first-line drug therapy for CMV infection is ganciclovir (GCV) or its prodrug valganciclovir (VGCV). GCV or VGCV must be activated by phosphorylation before they act on human CMV. This phosphorylation is carried out by the viral kinase UL97, and activated GCV subsequently inhibits the viral DNA polymerase UL54. Clinically, GCV resistance usually arises first from a UL97 mutation resulting in decreased accumulation of the activated drug. Subsequent UL54 mutations can confer high levels of GCV resistance and various degrees of cross-resistance to the second-line drugs cidofovir (CDV) and foscarnet (FOS) (2).Our group previously published a rapid PCR-and sequencingbased detection method for UL97 mutations conferring ganciclovir resistance (3). This test, currently used to detect mutations directly from clinical specimens, results in the sequence for UL97 gene codons 440 to 645. This region covers the known drug resistance mutation sites in the UL97 gene. However, because UL54 is the target of all currently marketed anti-CMV drugs, a detection method for UL54 mutations is also needed. Polymerase mutations are more likely to emerge after prolonged GCV treatment and typically add to the level of resistance conferred by UL97 mutations or introduce cross-resistance to CDV or FOS (4).Despite its predicted utility in treatment management, clinical tests for UL54 drug resistance mutations have been slower to develop for a number of reasons. First, the coding sequence of UL54 is almost twice as long as that of UL97. In addition, the baseline sequences are more variable and the number and variety of resistance mutations are greater in the UL54 gene than in the UL97 gene. Polymerase genotypic testing therefore requires more-extensive sequencing, typically covering codons 300 to 1,000. To meet the clinical need for complete CMV drug resistance profiles, we adapted our previous UL97 sequencing method (3) to develop a single assay that amplifies six region...

Drug Resistance Mutations In Transplant Recipients With Suspected Resistance

“…Surprisingly, in three patients ARM was only found in UL54; this fact may be explained by the fact that some ARM in UL97 may have reverted to wild-type after switching therapy to FOS or CDV. In this sense, previous experiments have shown that, fortunately, the most common ARM found, L595S/W, reverts after a while, provided that the selective pressure of GCV is removed (14), suggesting a certain disadvantage of this ARM compared to susceptible wild-type. Of note is the high proportion of patients with treatment failure unrelated to ARM: 72,72% (48/66) and 89,23% (58/65) regarding UL97 and UL54, respectively.…”

Antiviral Combination Therapy for Cytomegalovirus Infection in High-Risk Infants

“…Initial and peak viral load values were collected for the monotherapy group, and all available viral loads were collected for the combination therapy group. Any available CMV resistance test results for mutations at UL97 and UL54 were collected for both groups [13].…”

Cytomegalovirus and Kidney Transplantation: An Update

“…The effects of Cytomegalovirus in solid organ transplant recipients and other immunocompromised populations are significant and the clinical manifestations variable. Asymptomatic viremia, seen in up to 34% of CMV-infected transplant recipients, may be present in the absence of any overt manifestations of infection [15,45]. Symptomatic CMV infection, in addition to detectable viral replication in blood, requires the presence of clinical symptoms or illness [17,46].…”