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.