Abstract
Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1–27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus with significant disease-causing potential in immunocompromised patients, including children with congenital immune deficiencies or immune suppression following solid organ or bone marrow transplantation
Three drugs are currently licensed for HCMV prophylaxis and treatment, including ganciclovir (GCV), foscarnet (FOS), cidofovir (CDV); brincidofovir, and letermovir are in phase III clinical trials; maribavir (MBV) is available on a compassionate use basis
To capture the genes currently implicated in antiviral resistance simultaneously, we made use of novel target enrichment (Depledge et al, 2011) and deep sequencing to analyse the UL27, 54, and 97 genes in serial samples from patients with prolonged HCMV viraemia despite anti-HCMV therapy
Summary
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus with significant disease-causing potential in immunocompromised patients, including children with congenital immune deficiencies or immune suppression following solid organ or bone marrow transplantation. Deep sequencing of PCR amplicons has enabled detection of minority resistance variants at frequencies as low as 1% (Görzer et al, 2010) which could lead to earlier detection of HCMV resistance and better treatment. PCR and nested PCR are known to generate mutations which could make the identification of low level resistance mutations more difficult (Depledge et al, 2011) To minimize this problem, and to capture the genes currently implicated in antiviral resistance simultaneously, we made use of novel target enrichment (Depledge et al, 2011) and deep sequencing to analyse the UL27, 54, and 97 genes in serial samples from patients with prolonged HCMV viraemia despite anti-HCMV therapy. We include 11 retrospectively identified patients from Great Ormond Street Hospital for Children who had high HCMV loads for 2 weeks or longer, with clinician suspicion of anti-viral drug resistance
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