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Next-generation sequencing (NGS) produces comprehensive insights across the entire genome of the human cytomegalovirus (HCMV), which is an important opportunistic pathogen following hematopoietic stem cell transplantation (HSCT). To assess the clinical impact of HCMV diversity, genotype distribution, and resistance mutations, we performed NGS directly on plasma specimens from HSCT recipients with HCMV reactivation. Twenty-nine HCMV-positive plasma samples (median viral load 1.7 × 103 IU/ml) collected from a prospective allogenic HSCT recipient cohort (n = 16) between 21 and 80 days after transplantation were sequenced on an Illumina MiSeq after preparation of target-enriched sequencing libraries. Consensus HCMV genome sequences were assembled for 24 samples. The presence of multiple-strain infections and antiviral resistance mutations in genes UL54 and UL97 was determined by variant analysis. Genotype distribution was determined by specific marker analysis of several hypervariable genes (RL5A, RL6, RL12, RL13, UL1, UL9, UL11, UL73, UL74, UL120, UL146, and UL139). Associations between genomic and clinical features (e.g. graft-versus-host disease (GvHD), donor/recipient HCMV serostatus, dynamics of HCMV antigenemia, survival) were explored. Multiple infections involving up to 3 HCMV strains were detected in seven out of sixteen patients, with one patient analyzed at &gt; 2 time points, showing a switch of the dominant HCMV population. No known antiviral resistance mutations were detected, which may be expected due to sample collection early after HSCT from patients without antiviral prophylaxis. Multiple-strain infection was associated with an earlier peak of HCMV-antigenemia (P = 0.054), but not with duration of viremia, antigenemia peak values, donor/recipient HCMV serostatus, T-cell depletion, acute or chronic GvHD, disease relapse, or reduced survival. Genotype distribution analysis revealed a potential link of one genotype of the immunomodulatory gene UL11 with GvHD incidence after HCMV reactivation. NGS of HCMV diversity directly from plasma samples, even with low viral loads, enables the acquisition of data of potential clinical interest. To identify reliable associations between clinical features and HCMV diversity, further patient cohorts with suitable sample sizes are required.

virus evolution

A42 Next-generation sequencing to analyze multiple-strain infections, genotype distribution, and antiviral resistance in hematopoietic stem cell transplantation recipients with human cytomegalovirus infection