Emergence and prevalence of cytomegalovirus UL97 mutations associated with ganciclovir resistance in AIDS patients

Gilbert, Clare; Handfield, Julie; Toma, Emil; Lalonde, Richard; Bergeron, Michel G.; Boivin, Guy

doi:10.1097/00002030-199802000-00001

Cited by 43 publications

(18 citation statements)

References 11 publications

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“…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.…”

Section: Genotypic Methodsmentioning

confidence: 99%

Antiviral Drug Resistance of Human Cytomegalovirus

2010

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SUMMARY The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.

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Section: Genotypic Methodsmentioning

confidence: 99%

Antiviral Drug Resistance of Human Cytomegalovirus

2010

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“…In HIV-infected patients, GCV resistance has been reported in Ͼ20% of patients before the availability of highly active antiretroviral therapy (HAART) and in 9% after and is associated with CD4 ϩ T-cell counts of Ͻ50 cells/l (18)(19)(20)(21). Continued administration of a drug to which resistance has developed can lead to an accumulation of multiple drug resistance mutations (6), increased morbidity, and, in SOT recipients, shortened graft survival (15,16).…”

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Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

et al. 2013

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Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations. H uman cytomegalovirus (CMV)is an important cause of severe systemic and tissue-invasive disease in immunocompromised patients (reviewed in reference 1), such as solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. In these patients, prophylactic or preemptive treatment with antiviral agents for CMV significantly reduces the rates of CMV disease, the risk of other viral and bacterial infections, and all-cause mortality (2-5), but these treatments place patients at risk for the development of CMV drug resistance (6). In addition to anti-CMV therapy lasting for Ն3 months, other risk factors for drug resistance are intensive immunosuppression, multiple episodes of CMV reactivation, high viral counts, and suboptimal antiviral concentrations due to poor compliance or low drug absorption (7).The number of antiviral drugs approved for CMV treatment is limited and includes ganciclovir (GCV) and its prodrug valganciclovir (VCV), foscarnet (FOS), and cidofovir (CDV). Intravenous GCV and oral VCV are used as first-line agents for both prophylaxis and treatment, largely because of their lower toxicities than those of FOS and CDV (8-13). All of the available CMV drugs ultimately target the viral DNA polymerase UL54; however, GCV and VCV also require phosphorylation by the phosphotransferase UL97 for their antiviral activity (6). Thus, mutations in the UL97 and UL54 genes can confer resistance to GCV and VCV, while CDV and FOS resistance is only associated with mutations in UL54. Large numbers of sequence variants ha...

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“…The mean viral DNA load as determined by the COBAS AMPLICOR CMV MONITOR test for those samples was 9.96 ϫ 10 3 CMV DNA copies per 4 ϫ 10 6 PMNL. The presence of various CMV UL97 mutations at codons 460, 520, 594, and 595 was assessed as previously described (12,14). Briefly, two regions (nucleotides 1088 to 1587 and 1713 to 1830) of the CMV UL97 gene were amplified using a nested-PCR protocol.…”

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“…GCV or during a second viremic episode. Previous results from our group indicate that such a genotype-based screening assay is more sensitive than phenotypic (susceptibility) analyses in evaluating GCV resistance in human immunodeficiency virus-infected individuals (12). This technique also offers the advantage of detecting minor UL97 mutant subpopulations (at least 10%) mixed with wild-type strains (7) with an absolute sensitivity of 25 copies, as determined by testing for a plasmid containing the UL97 gene.…”

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Lack of Emergence of Cytomegalovirus UL97 Mutations Conferring Ganciclovir (GCV) Resistance following Preemptive GCV Therapy in Allogeneic Stem Cell Transplant Recipients

Gilbert

Roy

Bélanger

et al. 2001

Antimicrob Agents Chemother

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Fifty allogeneic stem cell transplant recipients were enrolled in a prospective cytomegalovirus pp65 antigenemia-guided preemptive therapy trial. Among these, 10 of 34 patients who received ganciclovir exhibited sustained and/or recurrent antigenemia despite treatment. Thirteen leukocyte preparations from these 10 subjects were screened for the presence of the most frequent cytomegalovirus UL97 mutations conferring ganciclovir resistance. None of these mutations were detected after mean and median ganciclovir exposures of 31.6 and 28.0 days, respectively.Cytomegalovirus (CMV) infections represent a serious threat in patients undergoing allogeneic bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) (15). In order to reduce the morbidity and mortality associated with CMV disease, most centers have now adopted one of the two following preventive strategies. The first approach, referred to as universal prophylaxis, is based on the administration of a potent anti-CMV drug, usually ganciclovir (GCV), to all patients for the first 3 months after transplantation. Prophylaxis with GCV has been reported to be highly effective in reducing the occurrence of early CMV disease (prior to day ϩ100) but is also associated with an increased incidence of bacterial and fungal infections and with significant bone marrow toxicity (4, 13). Long-term use of GCV has raised concerns about the development of delayed CMV disease and the possibility of selecting GCV-resistant strains (11,25). The second strategy, called preemptive or early treatment, is based on early identification of patients with active CMV blood infections using sensitive detection assays followed by antiviral treatment for various lengths of time.A successful CMV pp65 antigenemia-guided preemptive strategy for allogeneic hematopoietic stem cell recipients was recently reported (5). In that study, 50 patients with hematologic malignancies received a transplant (not T cell depleted) from matched sibling donors. The subjects were monitored weekly for the presence of CMV antigens and DNA (COBAS AMPLICOR CMV MONITOR test; Roche Diagnostics, Laval, Canada) in polymorphonuclear leukocytes (PMNL) from initiation of the conditioning regimen until day ϩ98 after transplantation. Among these 50 patients, 34 (68%) were treated with GCV (14 days of intravenous [i.v.] administration [5 mg/kg of body weight twice a day] followed by 14 days of oral administration [1,000 mg three times a day]) upon a positive pp65 antigenemia assay. Eight (23.5%) of these 34 patients received a second course of therapy based on the reemergence of CMV pp65 antigens in the blood. Only one patient (2%) enrolled in the study developed CMV disease despite a negative antigenemia test. To further support the use of such a preventive approach, we now report the results of genotypic markers of CMV resistance to GCV among the same cohort of patients.Among samples from the 50 patients enrolled in our study, 13 from 10 patients were selected for genotypic analysis on the basis of...

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Emergence and prevalence of cytomegalovirus UL97 mutations associated with ganciclovir resistance in AIDS patients

Cited by 43 publications

References 11 publications

Antiviral Drug Resistance of Human Cytomegalovirus

Antiviral Drug Resistance of Human Cytomegalovirus

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

Lack of Emergence of Cytomegalovirus UL97 Mutations Conferring Ganciclovir (GCV) Resistance following Preemptive GCV Therapy in Allogeneic Stem Cell Transplant Recipients

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