UL97 Mutations Research Articles

Analysis of Ganciclovir-Resistant Cytomegalovirus Infection Caused by the UL97 Gene Mutation in Codons 460 and 520 in Pediatric Patients: A Case Series

BackgroundDrug-resistant cytomegalovirus (CMV) infection has been increasingly recognized. However, there are limited data in pediatric patients. In this study, the prevalence and factors associated with CMV infection with UL97 mutations in pediatric patients treated with ganciclovir but not responding to treatment were evaluated.MethodsThis retrospective study was conducted from January 2013 to December 2017. All patients who were suspected of having ganciclovir-resistant CMV infection and had never had ganciclovir prophylaxis were included. Genotypic assay for UL97 mutations in codons 460 and 520 conferring ganciclovir resistance was performed. Factors associated with the presence of UL97 mutations were analyzed.ResultsOf 34 patients included, 10 patients (29.4%) had a genotypically confirmed UL97 mutation. The median age (interquartile range [IQR]) was 3 (0.85–8.68) years. Ganciclovir resistance was tested at a median time (IQR) of 22.5 (14.3–31) days after initiation of ganciclovir. All resistant isolates harbored a UL97 mutation in codon 460. Compared with patients infected with CMV without UL97 mutation, those infected with UL97 mutation strains were younger (median age [IQR], 3.02 [0.85–8.68] vs 10.45 [2.7–16.4] years) and had a higher maximum viral load (median [IQR], 5.06 [4.74–6.05] vs 4.42 [4.03–4.87] copies/mL). Six of 10 (60%) patients were successfully treated with high-dose ganciclovir (7.5 mg/kg twice daily).ConclusionsUL97 mutation ganciclovir-resistant CMV infection was not uncommon in the pediatric population. Screening for this mutation should be considered in patients experiencing virological worsening while ganciclovir is given, even if patients have not previously received ganciclovir prophylaxis.

1746. Prevalence and Resistance Patterns of Cytomegalovirus Viremia in Immunocompromised Patients

BackgroundWe noted a recent increase in the number of patients with CMV viremia among immunocompromised children at our institution. The study was undertaken to determine the prevalence of CMV viremia and to evaluate factors associated with the development of antiviral drug resistance.MethodsA retrospective study of immunocompromised hosts 0–21 years of age who had CMV viremia (2007–2017). We collected demographic data as well as details of antiviral therapy and resistance testing.ResultsA total of 31 patients were identified including 10 (32%) during the last 2 years. The age range was 3 months to 20 years (median 12.6 years); 23 (74%) were male and 12 (39%) were African American. Among the 31 patients, 18 had hematopoietic stem cell transplantation, 5 had primary immunodeficiency (2 common variable immunodeficiency, 1 SCID, 1 Langerhans cell histiocytosis, 1 DiGeorge syndrome), 4 had malignancies receiving chemotherapy, 3 with heart transplantation and one 17 year old with newly diagnosed HIV infection who presented with CMV pneumonia and viremia. Antiviral resistance testing was performed on 7 CMV isolates: 5 due to persistent viremia (> 1 months) despite treatment, and 2 prior to starting antiviral therapy. CMV resistance was identified in 3 patients including 2 with CVID and one with Hodgkin’s disease status post bone marrow transplantation. The 2 CVID patients had other comorbidities including chronic diarrhea and malabsorption and were TPN dependent. Both were diagnosed with CMV colitis and one also had pneumonitis. One had received a prolonged oral valganciclovir course (> 1 year) prior to diagnosis of resistance and the other received long-term intermittent oral valganciclovir courses. The patient with Hodgkin’s disease received a prolonged IV ganciclovir course. All 3 tested positive for UL97 mutation and one had both UL97/UL54 gene mutations.ConclusionMost of our patients with CMV viremia were transplant patients. Antiviral drug resistance was detected among 3 of 31 (10%) of our patients during the study period. Two had malabsorption that may have resulted in sub-therapeutic blood levels. Treatment with oral valganciclovir should be avoided in patients with poor gut absorption because it may increase risk of drug resistance.Disclosures All authors: No reported disclosures.

Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro.

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.

A case of severe nephrotic syndrome caused by isolated CMV glomerulopathy with ganciclovir resistant UL 97 mutation

Introduction: While many cases of cytomegalovirus (CMV) nephropathy have been reported in the literature, isolated CMV glomerulopathy is considered a rare finding. Case Presentation: We report the case of a CMV-negative recipient of kidney allograft from a CMVpositive donor (D+/R-), the recipient subsequently developed severe nephrotic syndrome secondary to biopsy-proven isolated CMV glomerulopathy. Conclusion: The patient developed CMV viremia with ganciclovir resistant UL97 mutation. His treatment course was resistant to recommended dose of intravenous ganciclovir, so therapy was changed to foscarnet with resolution of his viremia and reduction in proteinuria.

A42 Next-generation sequencing to analyze multiple-strain infections, genotype distribution, and antiviral resistance in hematopoietic stem cell transplantation recipients with human cytomegalovirus infection

Next-generation sequencing (NGS) produces comprehensive insights across the entire genome of the human cytomegalovirus (HCMV), which is an important opportunistic pathogen following hematopoietic stem cell transplantation (HSCT). To assess the clinical impact of HCMV diversity, genotype distribution, and resistance mutations, we performed NGS directly on plasma specimens from HSCT recipients with HCMV reactivation. Twenty-nine HCMV-positive plasma samples (median viral load 1.7 × 103 IU/ml) collected from a prospective allogenic HSCT recipient cohort (n = 16) between 21 and 80 days after transplantation were sequenced on an Illumina MiSeq after preparation of target-enriched sequencing libraries. Consensus HCMV genome sequences were assembled for 24 samples. The presence of multiple-strain infections and antiviral resistance mutations in genes UL54 and UL97 was determined by variant analysis. Genotype distribution was determined by specific marker analysis of several hypervariable genes (RL5A, RL6, RL12, RL13, UL1, UL9, UL11, UL73, UL74, UL120, UL146, and UL139). Associations between genomic and clinical features (e.g. graft-versus-host disease (GvHD), donor/recipient HCMV serostatus, dynamics of HCMV antigenemia, survival) were explored. Multiple infections involving up to 3 HCMV strains were detected in seven out of sixteen patients, with one patient analyzed at > 2 time points, showing a switch of the dominant HCMV population. No known antiviral resistance mutations were detected, which may be expected due to sample collection early after HSCT from patients without antiviral prophylaxis. Multiple-strain infection was associated with an earlier peak of HCMV-antigenemia (P = 0.054), but not with duration of viremia, antigenemia peak values, donor/recipient HCMV serostatus, T-cell depletion, acute or chronic GvHD, disease relapse, or reduced survival. Genotype distribution analysis revealed a potential link of one genotype of the immunomodulatory gene UL11 with GvHD incidence after HCMV reactivation. NGS of HCMV diversity directly from plasma samples, even with low viral loads, enables the acquisition of data of potential clinical interest. To identify reliable associations between clinical features and HCMV diversity, further patient cohorts with suitable sample sizes are required.

Antiviral-resistant cytomegalovirus infections in solid organ transplantation in the Netherlands.

ObjectivesAntiviral resistance in cytomegalovirus (CMV) may result from mutations in the molecular targets of antiviral agents. The aim of this study was to investigate both the prevalence of resistance-associated mutations and the factors associated with antiviral resistance in solid organ transplant (SOT) patients with repeated high CMV loads during antiviral treatment.MethodsSOT patients were selected retrospectively, based on CMV loads of >30000 IU/mL at least twice in a period during which treatment was given. Patient samples were tested for antiviral resistance by Sanger sequencing the UL97 and UL54 genes of CMV, which code for the viral kinase and polymerase. Factors predisposing to and resulting from the development of antiviral resistance mutations were analysed.ResultsMultiple samples from 113 SOT patients were tested, showing resistance-associated mutations in 25 patients (22%). A further 20 (18%) patients showed mutations that were not known to be associated with antiviral resistance. Several factors were associated with development of resistance-associated mutations in UL97 as well as UL54, including human leucocyte antigen (HLA) mismatch, which occurred more frequently in the group of patients with resistance mutations. High-level resistance mutations were most frequently seen in UL97.ConclusionsThis study shows that by selecting patients solely on the basis of virological response to treatment, more patients with antiviral resistance mutations are identified. In this study we confirm findings by other groups that primary infections are associated with resistance development. Moreover, we show that HLA mismatch is associated with the development of antiviral resistance, which suggests a role for host immunity in the development of resistance.

Letermovir for Secondary Cytomegalovirus (CMV) Prophylaxis in a Pediatric Stem Cell Transplant Patient

Patients who undergo stem cell transplants are at a significant risk of infections. Cytomegalovirus (CMV) is an organism of particular concern in this population. Current treatments options for CMV include ganciclovir, valganciclovir, foscarnet, and cidofovir. These treatment options have adverse effects such as myelosuppression or nephrotoxicity, making them less than ideal as a treatment options. Increasing rates of drug resistance is another concern. The need for alternative treatment options is paramount. Many patients require secondary prophylaxis. Letermovir is a newly approved CMV DNA terminase complex inhibitor indicated for prophylaxis of CMV infection and disease in adults. Its role in secondary prophylaxis has not been clearly defined. We report the case of an 11-year old male who had undergone a 4/6 HLA-matched cord blood transplant for Fanconi Anemia. The patient developed CMV viremia that later progressed to CMV colitis and pneumonitis. He was initially treated with ganciclovir then was found to have UL97 mutation. He was then switched to foscarnet for induction therapy, to which the patient showed a good response. The patient was subsequently transitioned to cidofovir for maintenance therapy where CMV viremia recurred. Foscarnet was re-initiated with good response then transitioned to cidofovir maintenance therapy. Unfortunately, CMV viral load began to increase prompting a change to high-dose ganciclovir. Due to concerns for myelosuppression with using high-dose ganciclovir he was switched back to foscarnet. While on foscarnet the patient had nephrotoxicity and electrolyte disturbances and the decision was made to re-initiate high-dose ganciclovir. When the patient's CMV viral load was undetectable the patient was transitioned to letermovir 480 mg daily with successful suppression of CMV viral load. Letermovir has been well tolerated and the patient has experienced no adverse effects associated with it. The patient has not required further therapy for CMV viremia since starting letermovir. Letermovir prophylaxis was stopped after four months of therapy. The patient's CMV viral load began to rise after stopping for one month with the peak at 381 copies/mL. After restarting letermovir the CMV viral load became undetectable and has remained so. Letermovir could be useful in secondary prophylaxis in pediatric patients with ganciclovir-resistant CMV who are at risk of recurrences. Further investigation is warranted for the use of letermovir in the pediatric population.

Clinical Validation of the Definitions of Resistant and Refractory Cytomegalovirus (CMV) Infection and Disease in Hematopoietic Cell Transplant (HCT) Recipients.

Introduction Resistant or refractory CMV infections are not well defined and are associated with high morbidity and mortality in HCT recipients. Ganciclovir (GCV), Foscarnet (FOS) and valganciclovir (VGC) are available for treatment of CMV infections with successful results, however, toxicities from these drugs are common and associated resistance have been reported. Definitions for “resistant and refractory CMV infections” were published recently for clinical trials use and to provide guidance for clinical practice (Chemaly RF et al, CID, 2018). Methods We performed a single center retrospective chart review (1/2010 to 9/2018) of HCT recipients who had CMV genotype performed for suspected antiviral resistance. Based on the published definitions, we categorized patients as either having refractory CMV (defined as CMV viremia that fails to decrease after at least 2 weeks of appropriately dosed and delivered antiviral therapy; and in the absence of known genetic mutations to the available antiviral agents) or resistant CMV infection (defined as refractory infection with identification of genetic mutations in the UL97 and/or UL54 genes correlating with in vitro antiviral resistance). Primary outcomes were CMV disease and non-relapse mortality (NRM). Results CMV genotype analysis was performed in 120 patients and 29 (24%) patients had UL 97 and/or UL54 mutations. When compared to refractory CMV infection, patients with resistant CMV infection were more likely to have AML (38% vs 76%), had cord blood transplant (8% vs 24%), were diagnosed with resistant infection later after HCT (59 d vs 120 d), had greater number of prior episodes of CMV infections, had more CMV diseases (34% vs 62%), and specifically more CMV GI diseases (22% vs 55%); (all, p Conclusion Our data showed that resistant CMV infections are associated with higher morbidity including CMV disease while refractory CMV infections are associated with higher NRM. Whether this high NRM in patients with refractory CMV infections is a reflection of the host immunosuppression with persistent CMV infection needs to be determined.

Detection of CMV Antiviral Resistance Mutations to Letermovir in Patients Using a Validated Clinical Sequencing Assay

Background Antiviral resistance to human cytomegalovirus (CMV) is an important complication for immunocompromised patients on prolonged antiviral regimens, and CMV remains the most clinically significant infection following allogeneic hematopoietic-cell transplantation (HCT). Letermovir targets subunit 2 of the viral terminase complex (UL56) and is approved for CMV prophylaxis in adult HCT recipients. Resistance to letermovir is conferred by point mutations in the UL56 gene, and with the potential clinical need for antiviral resistance testing, we have developed a UL56 sequencing assay covering 24 identified resistance mutations. Here we summarize the performance characteristics of the UL56 antiviral resistance assay, along with results from de-identified samples following early reference laboratory testing. Methods This assay uses automated nucleic acid extraction followed by CMV UL56-specific polymerase chain reaction (PCR). PCR products were subjected to cycle sequencing and capillary electrophoresis, and the resulting sequences were analyzed for the presence of known resistance mutations between codons 229 and 369 of the UL56 gene. The assay's limit of detection (LOD), precision and accuracy were validated in accordance with accepted regulatory standards using multiple laboratory and clinical CMV strains. The LOD of the assay was determined to be 99 IU/mL. Results In early clinical use of the CMV letermovir resistance assay, several resistance mutations were identified. Mutations at C325 were observed at the highest frequencies, including C325F, R, Y and W, which all have reported resistance indexes > 3,000. V236M, T244K and R369S were also detected at lower frequencies, which have published resistance indexes of 32—46, 3.3 and 38—48, respectively. Polymorphisms not known to confer resistance were detected at codons 246, 247 and 335. Resistance mutations in both UL56 and UL97, conferring resistance to letermovir and ganciclovir, respectively, were also observed in CMV DNA isolated from a single patient. Conclusion The CMV letermovir resistance sequencing assay was shown to be a rapid and sensitive means of detecting mutations conferring letermovir resistance. This expands current CMV antiviral resistance testing, which includes UL54 and UL97 sequencing, and provides physicians with the ability to monitor for the emergence of antiviral resistance mutations to all current FDA-approved anti-CMV drugs. Following validation of the assay, several patient specimens have featured mutations conferring substantial levels of resistance to letermovir. The treatment status of these patients is unknown; however, these findings underscore the importance of screening and monitoring patients for the emergence of mutations in UL56 conferring resistance to letermovir.

Resistant Cytomegalovirus Infection in Solid-organ Transplantation: Single-center Experience, Literature Review of Risk Factors, and Proposed Preventive Strategies

BackgroundCytomegalovirus (CMV) infection causes morbidity and mortality in solid-organ transplant recipients. Drug-resistant CMV is an emerging problem with poor survival outcomes and limited therapeutic options. In this study we comprehensively address the issue of drug resistance in CMV when compared with standard therapies, such as ganciclovir (GCV) and foscarnet. MethodsWe conducted a retrospective review of adult patients diagnosed with CMV after solid-organ transplant at our center between 2013 and 2017, and identified 7 resistant CMV cases. To study risk factors in the published literature, we performed an extensive database search. ResultsAll patients had documented UL97 mutations, and 3 patients harbored both UL97 and UL54 mutations. For cases with increasing viral load or failure to achieve clinical improvement despite optimal therapy, genetic resistance testing was carried out. Patients received GCV and foscarnet combination therapy. As an adjunct, CMV immunoglobulin, cidofovir, and leflunomide were added. Risk factors, including donor+/recipient− serostatus, persistent high viral replication, prolonged therapeutic GCV exposure (>2.5 months), and allograft rejection, were assessed. ConclusionPatients at risk, especially those with D+/R− serostatus, should be judiciously monitored for resistance. Prolonged intravenous GCV exposure increases the risk for development of drug resistance. Therefore, precise guidelines are required for prevention of long-term GCV/VGCV exposure. Investigation regarding interferon-gamma release assay and adoptive transfer of T cells in diagnosed CMV patients is warranted to improve future prophylactic and management strategies against CMV, with a potential to reduce the requirement for available toxic antiviral drugs.

Biological relevance of Cytomegalovirus genetic variability in congenitally and postnatally infected children

BackgroundHuman cytomegalovirus (HCMV) is the leading cause of congenital infections resulting in severe morbidity and mortality among infected children. Although the virus is highly polymorphic, particularly in genes contributing to immune evasion, the mechanisms underlying its genetic variability and pathogenicity are only partially understood. ObjectivesWe aimed to characterize different HCMV clinical strains isolated from 21 congenitally- or postnatally-infected children for in vitro growth properties and genetic polymorphisms. Study designThe growth of various HCMV isolates was analyzed in different cell culture models. Genetic polymorphism was assessed by genetic and phylogenetic analysis of viral genes involved in virulence (UL144, US28, and UL18), latency (UL133-138), or drug resistance (UL54 and UL97). ResultsHere, we report a high degree of genetic and phenotypic diversity in distinct HCMV clinical isolates, as shown by their in vitro growth properties. In particular, HCMV isolates displayed the highest degree of genetic variability in the UL144 gene, where we were able to define four distinct genotypes within the cohort based on UL144 heterogeneity. Lastly, among all isolates we were able to identify 36 mutations in UL54 and 2 in UL97. ConclusionsOur findings indicate that surprisingly high levels of genetic HCMV variability correlate with a high degree of phenotypic polymorphism, which in turn might differentially influence the growth, fitness, and drug susceptibility of HCMV.

Leflunomide therapy for refractory cytomegalovirus infections in hematopoietic stem cell transplant recipients.

Currently, there are no prospective, randomized trials analyzing leflunomide for the treatment of cytomegalovirus infection or disease in allogeneic stem cell transplant patients. The primary objective of this case series was to determine the clinical and virological responses of utilizing leflunomide as therapy for refractory cytomegalovirus infections, unresponsive to first-line therapy in allogeneic stem cell transplant patients. Additionally, patient and leflunomide specific characteristics were identified and determined in this descriptive case series. This is a single-center, case series of adult allogeneic stem cell transplant patients with refractory cytomegalovirus infections receiving leflunomide between 1 January 2005 and 31 March 2015. A total of 14 patients with refractory cytomegalovirus infections received leflunomide. All patients received concurrent anti-cytomegalovirus therapy. Nine of 13 patients tested positive for phosphotransferase UL97 and/or viral DNA polymerase UL54 genotype mutations. Nine patients achieved a virological response with undetectable cytomegalovirus titers. Of the 13 patients with teriflunomide serum levels, eight patients maintained levels >40 micrograms/milliliter (mcg/mL). Common adverse effects were pancytopenia (n = 8) and elevated liver function tests (n = 4). Despite current strategies, refractory or recurrent cytomegalovirus infection and disease remain a clinical challenge to treat in the stem cell transplant patient population. Leflunomide used in combination with other concomitant therapies use for refractory cytomegalovirus infection in clinical practice may be a safe and effective option in the allogeneic stem cell transplant patient population.

Drug-resistant cytomegalovirus: clinical implications of specific mutations.

Cytomegalovirus (CMV) infection can be refractory to antiviral treatment. Although refractoriness can be due impaired host immunity, it can also be due to viral mutations that confer antiviral drug resistance. This article provides a succinct review of mutations in CMV genes that confer drug resistance, and offer guidance on clinical management. Recent advances in medical and research technology have confirmed traditional mutations and identified novel ones that confer resistance to current antiviral drugs. Resistance to ganciclovir is commonly predicted by mutations in UL97, which encode for viral kinase that catalyzes its phosphorylation. Mutations in UL54, which encode for CMV DNA polymerase, confer resistance (or cross-resistance) to ganciclovir, cidofovir and/or foscarnet. Resistance to letermovir, the new drug approved for CMV prophylaxis in allogeneic hematopoietic stem cell transplant recipients, has emerged and mapped most commonly to mutations in UL56 and less commonly UL51 and UL89, the gene complex that encode for viral terminase. Mutations in CMV genes can be selected during antiviral drug exposure, and manifests phenotypically as nonresponsive drug-resistant disease. Knowledge of specific mutations informs clinicians in selecting appropriate antivirals for managing transplant patients with CMV disease.

Cytomegalovirus resistance in CD34+ -selected hematopoietic cell transplant recipients.

Cytomegalovirus (CMV) viremia after CD34+ -selected hematopoietic stem cell transplant (HCT) often requires prolonged antiviral therapy. We report rates and outcomes of resistant CMV in a contemporary cohort of CD34+ -selected HCT recipients managed preemptively. We retrospectively reviewed 220 consecutive, CMV-seropositive recipients (R+), who received CD34+ -selected HCT at Memorial Sloan Kettering Cancer Center between June 2010 and December 2014. Patients were monitored by quantitative CMV PCR and were treated preemptively. CMV resistance was tested by a genotypic assay. One hundred and sixty-one (73%) patients developed CMV viremia and 47 (29% of viremic and 21% of total patients) had CMV resistance testing by one-year from HCT. CMV resistance was confirmed in 19 (12% of viremic and 9% of total) patients and was identified >3months from HCT in 90% of patients. Twelve patients had mutations in UL97 only; the remaining 7 patients had mutations in UL54 only or UL54 and UL97. By 1 year from HCT, 11 of 19 (58%) patients with mutations had CMV end-organ disease. CMV-related mortality in patients with resistance was 42%. Nine percent of CMV R+, CD34+ -selected HCT recipients had resistant CMV by 1 year from HCT. Of 19 patients with resistant CMV, 58% had CMV end-organ disease and 42% died of CMV. Effective strategies for CMV prevention and restoration of CMV immunity are needed for CD34+ -selected HCT.

Safety and efficacy of foscarnet for the management of ganciclovir-resistant or refractory cytomegalovirus infections: A single-center study.

Infection with cytomegalovirus (CMV) is an important cause of morbidity and mortality following solid organ transplantation. Resistance to ganciclovir can rarely develop via mutations in UL97 or UL54. There are limited published studies assessing the safety and efficacy of foscarnet for the management of ganciclovir-resistant or refractory cytomegalovirus infection and many centers are reluctant to utilize this important therapy because of concerns about toxicity. Solid organ recipients transplanted between January 1, 2006 and December 31, 2014 who received at least 1 dose of foscarnet were retrospectively reviewed to assess treatment outcomes, tolerability, and safety of foscarnet. Ten of 31 (32.3%) patients who received foscarnet during the study period died during treatment with foscarnet, whereas all 21 surviving recipients successfully cleared infection. Of these surviving patients, 3 (14.3%) developed significant renal dysfunction, defined as >25% decline in estimated glomerular filtration rate during treatment; one-third had definitive renal biopsy results consistent with foscarnet-induced toxicity. Although mortality was high in this population, foscarnet use, with proper precautions, was generally safe and significant renal dysfunction was lower than previously reported in other sources, even with extended use.

High-dose Valganciclovir Treatment for Resistant Cytomegalovirus Colitis due to UL97 and UL54 Mutations

We report the first case of a ganciclovir-resistant cytomegalovirus (CMV) involving the gastrointestinal tract that was successfully treated with high-dose valganciclovir. A kidney transplant recipient developed drug-resistant CMV colitis which was initially treated with valganciclovir, but his CMV was found to have major resistance to ganciclovir and cidofovir due to UL97 and UL54 mutations. The patient was switched to intravenous foscarnet 40 mg/kg given every twelve hours. However, foscarnet had to be discontinued after 4 days of treatment due to acute kidney injury. Patient was restarted on valganciclovir at a higher target dose of 1800 mg twice a day based on the creatinine clearance. CMV became undetectable 2 weeks after valganciclovir treatment was completed. High-dose valganciclovir along with immune suppression reduction may be a treatment option for CMV colitis with ganciclovir resistance due to dual UL97 and UL54 gene mutations.

Incidence of UL97 Resistance Mutations in Infants with Congenital Cytomegalovirus Disease Receiving 6 Months of Oral Valganciclovir Therapy

BackgroundA recently completed Phase 3 randomized, controlled, double-blind, multicenter study of infants with symptomatic congenital cytomegalovirus (CMV) disease receiving 6 months of oral valganciclovir (VGCV) therapy represents the largest such population in which to evaluate treatment-emergent antiviral resistance. The most common mechanism of CMV antiviral resistance occurs through mutations in the CMV UL97 gene that confer resistance to ganciclovir (GCV). Genotypic resistance analyses were performed on infants receiving 6 months of VGCV to assess the incidence of antiviral resistance due to UL97 sequence variants.MethodsResistance analyses were performed by conventional DNA sequencing of the UL97 gene at multiple time points. Following CMV DNA extraction from frozen whole blood specimens, the UL97 gene was amplified with a double nested polymerase chain reaction method and sequenced to identify polymorphisms and mutations that might confer GCV resistance.ResultsForty-six infants with symptomatic CMV disease who received a 6-month course of VGCV underwent resistance analysis to identify UL97 sequence variants. In addition to a range of natural polymorphisms known to have no effect on antiviral susceptibility, 2 subjects developed UL97 mutations known to confer resistance to GCV (A594V and G598S detected in one subject; E596G detected in another), yielding an incidence of 4%. Each of these resistance mutations occurred in specimens collected after at least 4 months of antiviral therapy. As evaluated in the original Phase 3 trial, neither of these infants showed an improvement in hearing outcome.ConclusionThe development of treatment-emergent UL97 resistance mutations was determined in a controlled study population of infants with congenital CMV disease receiving 6 months of VGCV. This targeted resistance analysis demonstrated an incidence approaching the total incidence of antiviral resistance for CMV disease in some immunocompromised populations, such as solid-organ transplant recipients. Further studies within this study population are warranted to elucidate the risk of emerging antiviral resistance and to assess clinical impact as well as the potential need for combination antiviral therapy.Disclosures All authors: No reported disclosures.

Human Cytomegalovirus UL54 and UL97 Mutations for Detection of Ganciclovir Resistance in Congenital Infection

Aim: Ganciclovir (GCV) is used as an antiviral drug for the treatment of human cytomegalovirus infection. The aim of this study was to demonstrate GCV-resistant human cytomegalovirus in congenitally infected neonates. Patients & methods: DNA of CMV positive newborn samples was extracted and UL97 and UL54 genes were amplified by PCR and real-time PCR. Sequencing of UL97 and UL54 genes were performed and analyzed. Results: UL97 GCV resistance mutation C603W was detected in one newborn. D605E was the most common polymorphism in UL97 observed in 7/13 (53.8%) of samples. N685S, A688V, A885T and N898D were four known common UL54 polymorphisms. Conclusion: The rate of GCV resistance in congenital CMV is low. Common polymorphisms in UL97 and UL54 genes are also reported as new mutations.

Ganciclovir-Resistant Cytomegalovirus Infection in Abdominal Solid Organ Transplant Recipients: Case Series and Review of the Literature.

Ganciclovir-resistant cytomegalovirus (GR-CMV) is emerging as a significant infection in the abdominal transplant population. GR-CMV is difficult to manage, and treatment options are limited. We report a descriptive case series of 15 patients who had documented GR-CMV at our center and review the literature on treatment of GR-CMV. The first case in this series was detected in 2012; the majority of cases occurred after January 1, 2014, with approximately 50% occurring in 2015. UL97 and UL54 viral genome mutations were present in 100% and 40% of CMV-infected patients, respectively. GR-CMV infection occurred ≤ 1 year posttransplantation in 11 patients (73%). All patients experienced dose reduction of valganciclovir (the oral prodrug of ganciclovir) before the development of GR-CMV. Initial treatment for GR-CMV included a variety of regimens, all including reduction in maintenance immunosuppression. Of the 6 patients with detectable GR-CMV by polymerase chain reaction (PCR) who were discharged without GR-CMV treatment and had a length of stay (LOS) less than 14 days, 83% were subsequently readmitted for treatment of GR-CMV within 2 months (60% in < 20 days); none received leflunomide. Of six patients with a LOS ≥ 14 days, 80% had CMV PCR below quantification on hospital discharge, and only one patient was readmitted in less than 20 days; 83% received leflunomide. Following GR-CMV, there was a 50% rejection incidence, 27% graft loss, and 20% mortality. For patients with more than three admissions for GR-CMV treatment, 100% had a major complication: 60% rejection, 20% graft loss, and 40% mortality. Common clinical characteristics of patients with GR-CMV included high-risk serostatus, lymphocyte depletion, and history of valganciclovir dose reduction. Overall, outcomes were poor. It appears that hospital readmission rate was reduced when CMV was treated to negativity with an initial treatment regimen of reduced immunosuppression, foscarnet, intravenous immunoglobulins, and leflunomide.

Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.