A New Next-Generation Sequencing Approach in Human Cytomegalovirus for the Identification of Antiviral Resistance Mutations and Genotypic Classification.

This study introduces a new procedure for antiviral resistance analysis and genetic classification of human cytomegalovirus (HCMV) using next-generation sequencing (NGS) adapted to existing methodologies, aiming for more targets due to the recent use of new antivirals. It expands the classical investigation of mutations in UL54 and UL97 genes, associated with resistance to (val)ganciclovir, foscarnet and cidofovir, to include UL27, UL56 and UL89 genes, which target newer antivirals like maribavir and letermovir. Additionally, it includes the genetic analysis of UL55 (glycoprotein B) for genotype classification. This new methodology involves multiplex-PCR for DNA enrichment, followed by NGS using Illumina MiSeq platform and data analysis through an in-house pipeline. Several validations were performed by firstly using genome sequence from wild-type sensitive reference strain (AD-169), secondly comparing to previously characterized samples by Sanger, and lastly the use of external quality controls. A new NGS technique based on amplicons approach has been developed. Validation using wild-type control material showed 100% identity with the reference genome across all replicates. Only one minority variant was detected in one replicate. Compared to Sanger sequencing, NGS revealed additional low-frequency mutations not detected by Sanger, without impacting resistance interpretation. The method also performed reliably in external quality assessment controls. Moreover, the detection limit of the technique was established at 17 894.60 IU/mL. Finally, this approach enabled the identification of HCMV genotypes. This approach improves the monitoring of antiviral resistance and viral diversity, enhancing early clinical decision-making in immunocompromised patients.