Development and Dynamics of Cytomegalovirus UL97 Ganciclovir Resistance Mutations in Transplant Recipients Detected by Next Generation Sequencing

Background (Val)ganciclovir resistance mutations in CMV UL97 (UL97-GCV-R) complicate anti-CMV therapy in recipients of solid organ and hematopoietic stem-cell transplants but comprehensive data on prevalence, emergence, and outcome are scarce. Methods Using next generation sequencing (NGS) (Illumina MiSeq platform), we analysed UL97-GCV-R in patients with available plasma samples and refractory CMV replication/DNAemia (n=87) containing viral loads ≥910 IU/mL. 21 patients with CMV DNAemia resolving under antiviral therapy were analysed as controls. Detected mutations were considered induced and of potential clinical significance if they increased by ≥10% compared to the first detected frequency, or if they had a maximum frequency ≥25%. Results 19/87 (21.8%) with refractory CMV replication had >1 UL97-GCV-R detected by NGS, in comparison to 0/21 of the controls (p=0.02). One third of the recipients had 2 or more induced UL97-GCV-R mutations. The most frequently induced mutations affected codons 595 (42% (8/19)), 594 (32% (6/19)) and 603 (32% (6/19)). C592G was present in all episodes of both cases and controls at frequencies <15%, but never induced. UL97-GCV-R tended to be more frequent in donor/recipient CMV IgG mismatch or following failure to complete primary prophylaxis, and many developed invasive CMV disease. Conclusion UL97-GCV-R is common among transplant patients with refractory CMV replication. Early testing by NGS allows for identification of major mutations at codons 595, 594 and 603, and exclude a major role of C592G in ganciclovir resistance. Large prospective studies on UL97-GCV-R are warranted.

Drug Resistance Mutations and Associated Phenotypes Detected in Clinical Trials of Maribavir for Treatment of Cytomegalovirus Infection

Background In separate phase 2 trials, 120 patients received maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA <200 copies/mL) within 6 weeks. Methods Baseline and posttreatment plasma samples were tested for mutations in viral genes UL97, UL54, and/or UL27. Selected viral mutants were phenotyped for drug susceptibility. Results Baseline samples revealed UL54 mutations newly phenotyped as conferring resistance to standard DNA polymerase inhibitor(s), including K493N, P497S, K513T, L565V, V823A, A987V, and E989D. Of 29 patients (including 25 from trial 202) who cleared but later experienced recurrent CMV infection while on maribavir, 23 had available UL97 genotyping data; 17 had known resistance mutations (T409M or H411Y) and 5 additional had UL97 C480F alone. The newly phenotyped mutation C480F conferred high-grade maribavir resistance and low-grade ganciclovir resistance. Among 25 who did not respond to >14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone. Conclusions After maribavir therapy (400–1200 mg twice daily), UL97 mutations T409M, H411Y, or C480F emerge to confer maribavir resistance in patients with recurrent CMV infection while on therapy or no response to therapy. Clinical Trials Registration NCT01611974 and EudraCT 2010-024247-32.

Cytomegalovirus in Adult Allogeneic Blood and Marrow Transplant Patients Before or Around the Period of Neutrophil Recovery: A Single-Center, Retrospective, Descriptive Study

Background Few reports exist on pre-engraftment cytomegalovirus (CMV) DNAemia in allogeneic blood or marrow transplant (allo BMT) recipients. We describe this clinical entity, its management, and the potential effect of 3 different quantitative CMV deoxyribonucleic acid (DNA) tests used during the 6-year study period. Methods We performed a retrospective, single-center study of allo BMT recipients from 2010 to 2015 who developed CMV DNAemia before neutrophil recovery (absolute neutrophil count [ANC] <1000 cells/mm3, “pre-engraftment CMV”) or who became neutropenic concomitant with detectable CMV DNA (“peri-engraftment CMV”). Clinical data were collected from the electronic medical record. Results Among 1151 adult allo BMT patients, 73 developed CMV DNAemia before engraftment or while neutropenic after initial engraftment. Most patients were eventually treated (valganciclovir or ganciclovir, N = 68; foscarnet, N = 1); 4 were not treated. First CMV detection occurred at median day +12 (range, 0–48), but treatment was not started until median day +33 (range, 4–105) at median ANC of 760 cells/mm3. Six patients had peak viral loads >5000 IU/mL; none had tissue-invasive disease. One developed ganciclovir resistance. No significant differences were observed upon stratification by quantitative CMV DNA test. Conclusions Cytomegalovirus DNA was detected in 6.3% of pre- and peri-engraftment allo-HSCT patients. Ganciclovir derivatives were commonly used for treatment despite risk of neutropenia. Treatment was typically deferred until CMV DNA and ANC rose. With rare exceptions, this treatment strategy did not appear to have adverse clinical consequences with respect to acute CMV. Different CMV DNA quantification tests used performed similarly from a clinical perspective despite different analytical performance characteristics.

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.