Prevalence of UL97 gene mutations and polymorphisms in cytomegalovirus infection in the colon associated with or without ulcerative colitis

Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.

Prevalence of UL97 gene mutations in cytomegalovirus reactivation in the colon associated with or without ulcerative colitis

Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently (< 10%) in L228P, D263G, A53S, R137C, A140V, G188S, A674T, and T675A. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of mutations in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. T75A, Q126L, and D605E mutations may be used as genetic markers for CMV in East Asian countries.

Prevalence of UL97 gene mutations in cytomegalovirus reactivation in the colon associated with or without ulcerative colitis

Background: Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC) and in immunocompromised patients, and may be associated with poor prognosis. Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV colitis remains to date unknown. The present study aimed to investigate the prevalence of CMV UL97 gene mutations in Japanese patients with colonic CMV infection associated with or without UC.Methods: Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing of the PCR products was performed.Results: UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently (<10%) in L228P, D263G, A53S, R137C, A140V, G188S, A674T, and T675A. However, no known GCV resistance mutations were found. There was no significant difference between the ratio of mutations in patients with and without UC.Conclusions: We did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. In contrast, T75A, Q126L, and D605E mutations may be used as genetic markers for CMV in East Asian countries.

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

Background Drug-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. Methods This 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. Results Of 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). Conclusions UL97 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.

Mutations in the cytomegalovirus UL97 gene associated with ganciclovir resistance in recipients of allogeneic hematopoietic stem cell transplants

Objective. To identify mutations in UL97 gene associated with antiviral drug resistance in recipients of allogeneic hematopoietic stem cells transplants (allo-HSCT). Materials and Methods. A total of 9 HCMV DNA samples were studied. These samples were received from the blood of 8 allo-HSCT recipients who were infected with HCMV and undergoing treatment at NMRC of Hematology (Russia) over the period of 2016 to 2017. Sanger sequencing was used to find mutations. The sequenced part of the virus DNA was analyzed using nucleotide BLAST and Genome compiler. Mutations were identified using MRA program which compared the obtained nucleotide sequence with the reference sequence of UL97 gene from Merlin strain. Results. Rate of detection of viruses with mutations that may lead to drug resistance is relatively high – 3 of 8 patients. The following mutations were identified: C592G, C607F and C603W. The obtained data shows that the presence and characteristics of mutations affect the viral load and the time when HCMV DNA is identified in blood. Conclusions. Obtained data show that presence of mutations impacts the course infection, leading to higher viral load and longer persistence of viral DNA in blood samples. Identified mutations had different resistance factor, which also impacted on the pattern of infection.

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

Background A 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. Methods Resistance 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. Results Forty-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. Conclusion The 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.