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Rapid emergence of HCMV drug resistance in immunocompromised paediatric patients detected using target enrichment and deep sequencing

By Charlotte Houldcroft, Josephine M Bryant, Daniel P Depledge, Ben K Margetts, Jacob Simmonds, Stephanos Nicolaou, Helena Tutill, Rachel Williams, Austen Worth, Stephen D Marks, Paul Veys, Elizabeth Whittaker, Judith Breuer, the PATHSEEK condortium

Posted 27 May 2016
bioRxiv DOI: 10.1101/055814 (published DOI: 10.3389/fmicb.2016.01317)

Background Cytomegalovirus can cause fatal disease in immunocompromised patients. With the advent of new anti-HCMV drugs there is interest in using virus sequence data to monitor resistance and identify new mutations. Methods We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed paediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analysed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Results 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 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. Conclusions Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of eleven 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.

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