UL97 Mutations Research Articles

Incidence of Ganciclovir Resistance in CMV-positive Renal Transplant Recipients and its Association with UL97 Gene Mutations.

Human cytomegalovirus (CMV) remains the most common infection affecting organ transplant recipients. Despite advances in the prophylaxis and acute treatment of CMV, it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant recipient. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. Common mechanisms of CMV resistance to ganciclovir have been described chiefly with the UL97 mutations. Here we evaluate Incidence of ganciclovir resistance in 144 CMV-positive renal transplant recipients and its association with UL97 gene mutations. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 gene sequencing. Six mutations in six patients were detected. Three patients (2.6%) of 112 patients with history of ganciclovir (GCV) treatment had clinical resistance with single UL97 mutations at loci known to be related to resistance (including mutations at codon 594, codon 460, and codon 520). three patients who were anti-CMV drug naïve had single UL97 mutations (D605E) without clinical resistance. Our results confirm and extend our earlier findings on the specific mutations in the UL97 phosphotransferase gene in loci that have established role in ganciclovir resistance and also indicate that clinical ganciclovir resistance due to UL97 gene mutations is an issue in subjects with history of with ganciclovir treatment. D605E mutations remains a controversial issue that needs further investigations.

Epidemiology and Outcome of Ganciclovir-Resistant Cytomegalovirus Infection After Solid Organ Transplantation: A Single Transplant Center Experience in Thailand

BackgroundData on drug-resistant cytomegalovirus (CMV) infection in solid organ transplantation (SOT) are not often reported from resource-limited settings. We aimed to investigate the epidemiology and outcomes of this infection in SOT recipients at our institution. MethodsThis was a retrospective study conducted from January 2012 to May 2015. We included all SOT recipients who were suspected for drug-resistant CMV infection. Genotypic assay for UL97 gene mutation was analyzed by real-time polymerase chain reaction. Patients were reviewed for demographic data, clinical presentation, virologic data, treatment, and outcomes. ResultsThe population consisted of 18 (12 kidney, 6 liver) SOT recipients with a median age of 20 years (interquartile range [IQR], 1–49); 44% were male. Anti-CMV resistance testing was analyzed at a median time of 23 days (IQR, 14–33) after initiation of anti-CMV therapy with a median CMV load of log 3.79 copies/mL (IQR, 3.37–4.58). During a median period of 2 years (IQR, 1–3), 6 SOT recipients were identified with UL97 gene mutation in codon 460, conferring ganciclovir (GCV) resistance. Patients with UL97 gene mutation had a longer mean duration of CMV DNAemia compared with those without mutation (263 vs 107 days; P = .04). All patients received high-dose GCV. Two patients received foscarnet and cidofovir. Two patients died (non–CMV-related), and 4 patients. ConclusionsGCV-resistant CMV infection in SOT recipients is an emerging clinical problem in resource-limited country. Those with UL97 mutation CMV infection have prolonged duration of CMV DNAemia. Clinicians should be aware of this condition when caring for SOT recipients.

Genotypic resistance of cytomegalovirus to antivirals in hematopoietic stem cell transplant recipients from Portugal: A retrospective study

The aim of this study was to characterize Human Cytomegalovirus (HCMV) drug resistance mutations in UL97 and UL54 genes in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients in Portugal. We have performed a retrospective study with 22 patients from a cohort of patients with different haematological malignancies submitted to allo-HSCT between 2010 and 2014. Patients were selected according to clinical and laboratory data of HCMV infection and management. HCMV resistance mutations were characterized by sequencing of UL97 and UL54 genes. Sequence data were compared with: 1) HCMV genome reference strain AD169; and also 2) UL97 from Merlin strain (GenBank: AY446894.2), and UL54 from TB40/E strain (GenBank: ABV71585.1). Resistance mutations were identified in seven patients (32%): five harboured resistance mutations in UL97: A594V (n = 2), C592G (n = 1), L595W (n = 1), and C603W (n = 1); and two harboured resistance mutations in UL54: P522S and L957F, one in each patient. Several natural polymorphisms and unknown mutations were found in both UL97 and UL54, with the majority of the patients harbouring more than one unknown mutation in UL97 but only one in UL54. No simultaneous mutations were found. This is the first study in Portugal to characterize HCMV UL97 and UL54 sequences and to identify HCMV drug-resistance mutations in allo-HSCT patients. The UL97 resistance mutations found were amongst the most frequent resistant mutations, while UL54 L957F mutation was here reported for the first time in a clinical specimen. This information provides important information regarding HCMV strains and antiviral resistance in our population.

Molecular Analysis of Ganciclovir-Resistant Cytomegalovirus in Renal Transplant Recipients with High Viral Load.

Gancyclovir-resistant (GanR) cytomegalovirus (CMV) remains an issue, especially in solid organ transplant (SOT) recipients. Some mutations in UL54 and UL97 confer this resistance. Long-lasting high-dose drug exposure, high viral load, together with lack of sufficient compliance with treatment may account for these mutations. The aim of this study was to detect UL97 and UL54 putative mutations conferring ganciclovir-resistance in renal organ transplant recipients with high CMV load. In this cross-sectional study, 58 serum samples were collected from renal transplant recipients who had referred to three hospitals in Tehran from January 2014 to June 2015. Specific criteria such as CMV syndrome, presence of CMV in blood and organ dysfunction were considered. Then, they were tested for viral load in their early fourth month of intravenous ganciclovir treatment. Fifty cases revealing more than 200 copies/mL were analyzed for mutations. Two fragments of UL54 and Ul97 genes were amplified and sequenced bidirectionally. Sequence alignment and statistical analysis were performed by Mutation Surveyor software and t-test respectively. A significant difference was observed in viral load between seronegative and seropositive recipients (P = 0.036). The most frequent mutation was related to D605E in UL97 gene with the rate of 25%. Regardless of viral load, neither putative mutation nor simultaneous mutation was detected in either UL97 and UL54 regions. In spite of high viral load and persistence of symptoms, our population study did not reveal putative mutations. Hence, the direct relationship between the presence of high quantity of CMV and the occurrence of putative mutation cannot be considered. Non-putative gancyclovir resistant mutations and prolonged drug exposure may have a role in these manifestations.

Brincidofovir (CMX-001) for refractory and resistant CMV and HSV infections in immunocompromised cancer patients: A single-center experience

BackgroundThe emergence of resistance to available antiviral drugs may cause higher Cytomegalovirus (CMV)-associated morbidity and mortality in hematopoietic cell transplant (HCT) recipients. Few novel antiviral drugs are being developed with potential effect against refractory or resistant CMV infections. Brincidofovir, an oral nucleotide analog and prodrug of cidofovir, has shown in vitro activity against CMV. MethodsWe reviewed data of 4 cancer patients with resistant CMV or herpes simplex virus (HSV) infections and were treated with brincidofovir under emergency IND application. ResultsThree out of the 4 patients with resistant CMV or HSV infections responded to brincidofovir. Brincidofovir achieved virological response in 2 patients with confirmed UL97 mutation (ganciclovir-resistant CMV infection), but failed to control CMV when a specific UL54 mutation was present (conferring resistance to foscarnet and cidofovir), as seen with patient 3. Furthermore, brincidofovir was effective for treatment of acyclovir resistant HSV infection, as described in patient 4 which is in alignment with the high in vitro potency of brincidofovir (about 100-fold more than acyclovir) for inhibition of HSV replication. Only one patient had brincidofovir-related diarrhea without any evidence of concurrent gastrointestinal GVHD. ConclusionWithout the nephrotoxicity of cidofovir, brincidofovir could be an effective alternative for CMV-resistant and HSV-resistant infections. Combination therapy of brincidofovir with other antiviral agent, at a conventional or a lower dose, could be considered to potentiate efficacy and minimize side effects of the approved antiviral agents.

Identification of resistance-associated HCMV UL97- and UL54-mutations and a UL97-polymporphism with impact on phenotypic drug-resistance

BackgroundHuman cytomegalovirus (HCMV) drug-resistance remains of high clinical importance. While UL97-mutations can confer ganciclovir-resistance, UL54-mutations can be associated with resistance to ganciclovir, foscarnet and/or cidofovir. ObjectiveThree UL97-mutations (A619V, P468Q, del597-599), three UL54-mutations (V715A, A492D, L516W) and two UL97/UL54-mutation combinations (A594TUL97+V715MUL54; A591VUL97+D515EUL54, L516MUL54, I521TUL54) were characterised phenotypically. All mutations were introduced into the bacterial artificial chromosome (BAC) TB40-BACKL7-UL32EGFP. A revertant of HCMV-TB40-BACKL7-UL32EGFP/A591VUL97+D515EUL54, L516MUL54, I521TUL54 was generated. ResultsThe UL97-mutation del597-599 showed GCV-resistance while A619V and P468Q were drug-sensitive. The UL54-mutation V715A was FOS-resistant/CDV-hypersensitive and L516W was GCV/CDV cross-resistant. Mutation A594TUL97+V715MUL54 showed GCV/FOS cross-resistance. HCMV-BACKL7-UL32EGFP/A591VUL97+D515EUL54,L516MUL54, I521TUL54 was GCV/CDV cross-resistant with a remarkably increased GCV-ratio compared to a strain where only the UL54-mutations D515E+L516M+I521T were present. Since the revertant was drug-sensitive again, the increased drug-ratio is supposed to be due to the UL97-polymorphism A591V. ConclusionPhenotypic characterisation of newly detected mutations in UL97 and UL54 remain of high importance. Only mutations with a confirmed phenotype allow reliable interpretation of genotypic methods. Here, we provide the first description of a UL97-polymorphism that contributes to the overall drug-resistance when combined with resistance-associated UL54-mutations. The finding shows the high importance to look at mutations in the context of their genetic background.

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

Approach to drug-resistant cytomegalovirus in transplant recipients.

The purpose of this study is to provide updated information on diagnosis of cytomegalovirus (CMV) drug resistance, treatments for drug-resistant infection and potential uses of experimental antiviral compounds. For established CMV antivirals, uncommon viral UL97 kinase and UL54 DNA polymerase drug resistance mutations are sporadically described that expand an extensive existing database. Some novel mutations reported from treated patients have no drug-resistant phenotype and may be genotyping artefacts. Next-generation sequencing technology may enable earlier detection of emerging resistance mutations in treated patients. Management options for drug-resistant infection include optimization of host defenses, antiviral dose escalation, substitutions or combinations of standard or experimental antivirals. Maribavir and letermovir have antiviral targets distinct from the classic DNA polymerase. UL97 mutations elicited by ganciclovir and maribavir are different, although a single p-loop mutation can confer significant cross-resistance. High-grade resistance mutations in the UL56 terminase gene are readily selected in vitro under letermovir and await clinical correlation. Technical advancements can enhance the accurate and timely genotypic detection of drug resistance. Antivirals undergoing clinical trial offer the prospect of new viral targets and drug combinations, but unresolved issues exist with regard to their therapeutic potential for drug-resistant CMV and their genetic barriers to resistance.

Detection of Two Drug-Resistance Mutants of the Cytomegalovirus by High-Resolution Melting Analysis.

Human cytomegalovirus (CMV) is an opportunistic pathogen that can be treated with ganciclovir. Mutations in the UL97 gene of CMV render the virus ganciclovir resistance. These include H520Q and C603W mutations, against which we developed a novel genotyping assay for their identification. PCR reactions were performed to amplify fragments of the UL97 gene containing H520Q or C603W mutations. High resolution melting analysis (HRMA) coupled with unlabeled DNA probes was employed to identify the shift in melting temperature of the probe-template complex, which reflexes the presence of point mutations. Melting point analysis performed on the dimeric DNA of PCR products of UL97 gene could not identify mutations in the gene. When coupled to unlabeled probes, point mutations in UL97 can be identified by analyzing the melting curve of probe-template complex. When WT and mutant UL97 DNAs were mixed together to mimic heterogeneous viral population in clinical samples, the genotyping assay is sensitive enough to detect H520Q and C603W mutants that constitute 10% of total DNA input. Probe-based HRMA is effective in detecting H520Q and C603W mutations in the UL97 gene of CMV.

Human Cytomegalovirus Infection in Solid-Organ Transplantation

Human cytomegalovirus (CMV) continues to be a major threat against solid-organ transplant recipients despite significant advancements in its prophylaxis and therapy. Primary CMV infection or reactivation of latent CMV in the transplant recipients may cause CMV diseases such as flu-like viral syndrome and tissue-invasive CMV disease. In addition, CMV infection in the recipients is associated with graft rejection and higher risk of other opportunistic infections, which are collectively known as the indirect effects of CMV infection. Prevention strategies with antiviral drugs including ganciclovir remarkably decreased CMV disease and the indirect effects. Two commonly employed strategies are universal prophylaxis and preemptive therapy. However, gangciclovir-resistant CMV has emerged due to mutations in CMV UL97 and UL54 genes, now requiring alternative therapeutic options to be developed. This review provides an overview of CMV infection and disease, indirect effects on hosts, prevention strategies, and drug resistance in solid-organ transplant recipients.

The impact of drug-resistant cytomegalovirus in pediatric allogeneic hematopoietic cell transplant recipients: a prospective monitoring of UL97 and UL54 gene mutations.

Cytomegalovirus (CMV) infection is a major cause of morbidity in allogeneic hematopoietic cell transplant (alloHCT) recipients. Little is known about the epidemiology of antiviral resistance in the pediatric population. We performed the prospective study to assess the impact of drug-resistant CMV infections in pediatric alloHCT recipients. Pediatric alloHCT recipients who developed CMV infection were consecutively enrolled from May 2009 to April 2012. CMV polymerase chain reaction amplification and sequencing analysis for UL97 and UL54 genes were performed at enrollment and during follow-up. In total, 208 sequence data from viruses in 49 recipients were eligible for the final analysis. Resistant CMV infection caused by UL97 and UL54 mutations occurred in 4.1% (2/49) and 2.0% (1/49), respectively. Known UL97 mutations, M460V and C592G, were observed in each of 2 patients. One patient with the M460V UL97 mutation had an additional T700A UL54 mutation. Drug-resistant CMV attributable mortality was 2.0% (1/49). One or more known sequence variants (drug-sensitive) were observed in all 49 patients. Thirty-one (63.3%) and 28 patients (60.9%) already had known UL97 and UL54 sequence variants before antiviral therapy, respectively. This study provides comprehensive information on the epidemiology of both UL97 and UL54 variants and mutations in alloHCT recipients.

Detecting human cytomegalovirus drug resistant mutations and monitoring the emergence of resistant strains using real-time PCR

BackgroundAntiviral resistance development is a serious complication of human cytomegalovirus virostatic therapy caused by mutations in UL 97 and/or UL54 genes. ObjectivesTo determinate the presence of sensitive and resistant strains in patients developing antiviral resistance. Study designWe used three different molecular biological methods for mutation analysis–restriction fragment length polymorphism, sequencing and real-time PCR approach. ResultsWe describe three allogeneic hematopoietic stem cell transplant patients developing the GCV resistant HCMV strains manifested by virostatic treatment failure. In these patients we identified UL97 mutations L595S, A594V and A594T and monitored the dynamics of coexisted sensitive/resistant strains. We confirmed the presence of mixed HCMV populations and in two patients a phenomenon of sensitive strain repopulation which occurred after 6.5 months and 1 month after removing GCV pressure. ConclusionsOur results show changes in proportions of sensitive/resistant subpopulations over time but other studies would be required to demonstrate the beneficial impact of their monitoring on clinical outcome.

Cytomegalovirus antiviral resistance: characterization of results from clinical specimens.

Cytomegalovirus (CMV) infections are a major cause of disease among immunocompromised patients. Prolonged antiviral therapy is often necessary to prevent or treat CMV disease, and may lead to development of antiviral resistance (AVR). Timely identification of viral mutations conferring resistance is essential for effective patient management. Amplification by polymerase chain reaction followed by bi-directional nucleotide sequencing was performed for relevant regions of the CMV UL97 and UL54 genes. Results from 570 samples submitted to a commercial reference laboratory for testing were reviewed and characterized with respect to the frequency of mutations detected, association with viral load (VL), and consistency of results in a subset of patients with multiple samples. Only AVR mutations confirmed by marker transfer experiments were included in the analysis. AVR mutations were identified in 176 (30.9%) of the 570 samples evaluated. A total of 17 different UL97 mutations and 29 different UL54 mutations were detected. A single mutation per sample was most commonly observed, although 61 samples (10.7%) had >1 mutation, with 40 samples (7.0%) having mutations in both UL97 and UL54 genes. The VL of samples with AVR mutations ranged from 2.03-7.15 log10 copies/mL, and the VL did not differ significantly from samples without AVR mutations. A subset of patients (N = 85) had >1 sample tested, and 48.2% of these patients returned the same result for each sample analyzed, while the remainder had a different results. Genomic mutations conferring resistance in CMV to antiviral drugs were commonly identified in samples submitted from clinical patients to a reference laboratory for AVR testing. Mutations were identified over the full range of VLs and no correlation was identified between VL and the presence of AVR mutations. In patients with multiple samples submitted for analysis, approximately half of the patients had samples with variable results when the initial result was compared to subsequent results.

UL54 foscarnet mutation in an hematopoietic stem cell transplant recipient with cytomegalovirus disease

We present a case of foscarnet (FOS) resistance arising from a UL54 mutation after a short duration of FOS exposure, which has not been previously described in a stem cell transplant recipient, to our knowledge. We discuss the use of FOS to treat other viral infections and the implications this may have for the development of resistance mutations and treatment of cytomegalovirus disease.

Resolution of Mild Ganciclovir-Resistant Cytomegalovirus Disease with Reduced-Dose Cidofovir and CMV-Hyperimmune Globulin.

Ganciclovir-resistant cytomegalovirus (CMV) is associated with significant morbidity in solid organ transplant recipients. Management of ganciclovir-resistant CMV may be complicated by nephrotoxicity which is commonly observed with recommended therapies and/or rejection induced by “indirect” viral effects or reduction of immunosuppression. Herein, we report a series of four high serologic risk (donor CMV positive/recipient CMV negative) kidney transplant patients diagnosed with ganciclovir-resistant CMV disease. All patients initially developed “breakthrough” viremia while still receiving valganciclovir prophylaxis after transplant and were later confirmed to exhibit UL97 mutations after failing to eradicate virus on adequate dosages of valganciclovir. The patients were subsequently and successfully treated with reduced-dose (1-2 mg/kg) cidofovir and CMV-hyperimmune globulin, given in 2-week intervals. In addition, all patients exhibited stable renal function after completion of therapy, and none experienced acute rejection. The combination of reduced-dose cidofovir and CMV-hyperimmune globulin appeared to be a safe and effective regimen in patients with mild disease due to ganciclovir-resistant CMV.

Panresistant Cytomegalovirus in a Kidney Transplant Recipient

Cytomegalovirus (CMV) is an important pathogen often encountered after solid organ transplantation and is associated with increased morbidity and mortality. Resistance of CMV to antiviral agents is becoming more common but with few treatment strategies. Two specific mutations in the CMV genome--the UL97 and UL54 genes--correlate with antiviral drug resistance. We describe a 49-year-old, CMV-seronegative woman who received a CMV-seropositive donor kidney transplant and appropriate CMV prophylaxis. Approximately 1 month after transplantation, the patient developed CMV viremia that responded to valganciclovir. She was later diagnosed with recurrent CMV infection, CMV resistance, and both the UL97 and UL54 gene mutations. The patient responded to foscarnet and significant reduction of immunosuppression; she was negative for CMV viremia for the next 12 months. This case illustrates the importance of having heightened awareness for the possibility of panresistant CMV early and decreasing immunosuppression as the cornerstone of treatment.

Ganciclovir-Resistant Cytomegalovirus Infections among Lung Transplant Recipients Are Associated with Poor Outcomes despite Treatment with Foscarnet-Containing Regimens

Ganciclovir-resistant cytomegalovirus (CMV) infections are reported infrequently among lung transplant recipients receiving extended valganciclovir prophylaxis. We performed a single-center, retrospective review of ganciclovir-resistant CMV infections in a program that employed valganciclovir prophylaxis for ≥6 months after lung transplant. CMV infections were diagnosed in 28% (170/607) of patients. UL97 mutations were detected in 9.4% (16/170) of CMV-infected patients at a median of 8.5 months posttransplant (range, 5 to 21) and despite prophylaxis for a median of 7 months (range, 4 to 21). UL97 mutations were canonical; 25% (4/16) of strains carried concurrent UL54 mutations. Ganciclovir-resistant CMV was more likely with breakthrough infections (75% [12/16] versus 19% [30/154]; P = 0.00001) and donor positive/recipient negative (D+/R-) serostatus (75% versus 45% [69/154]; P = 0.03). The median whole-blood CMV load was 4.13 log10 copies/cm(3) (range, 2.54 to 5.53), and 93% (14/15) of patients had low-moderate immune responses (Cylex Immunoknow). Antiviral therapy was successful, failed, or eradicated viremia followed by relapse in 12% (2/16), 31% (5/16), and 56% (9/16) of patients, respectively. Eighty-seven percent (14/16) of patients were treated with foscarnet-containing regimens; toxicity developed in 78% (11/14) of these. Median viral load half-life and time to viremia eradication among foscarnet-treated patients were 2.6 and 23 days, respectively, and did not correlate with protection from relapse. Sixty-nine percent (11/16) of patients developed CMV pneumonitis, and 25% (4/16) died of it. Serum viral load was independently associated with death among foscarnet-treated patients (P = 0.04). In conclusion, ganciclovir-resistant CMV infections remained a major cause of morbidity and mortality following lung transplantation. Foscarnet-based regimens often eradicated viremia rapidly but were ineffective in the long term and limited by toxicity.

Human Cytomegalovirus UL97 Kinase Is Involved in the Mechanism of Action of Methylenecyclopropane Analogs with 6-Ether and -Thioether Substitutions

Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC50) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

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.

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

Human 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.