Cytomegalovirus (CMV), a member of the Herpesviridae family, is widely distributed globally and usually causes asymptomatic infection or self-limited symptoms, followed by latency for an indeterminate time in immunocompetent individuals. However, in immunosuppressed patients, it can trigger severe clinical manifestations due to reactivation. The emergence of strains resistant to the antivirals ganciclovir, foscarnet, cidofovir, maribavir, and letermovir represents a growing challenge in treatment. In this context, early detection of mutations associated with viral resistance is essential to guide interventions and optimize therapeutic management. This study aimed to develop a molecular test based on next-generation sequencing (NGS) to identify mutations associated with resistance to the main antivirals in key CMV genes, using clinical samples.

Specific primers were designed for the target regions of the UL27, UL51, UL54, UL56, and UL97 genes, where mutations previously associated with antiviral resistance occur. Viral DNA was extracted from clinical samples previously positive for CMV and then submitted to PCR, library preparation with the Microbial Amplicon Prep kit, and sequencing on an Illumina platform. Bioinformatic analysis was conducted with an in-house pipeline, and variants were compared with the HerpesDRG DB v03112024 database.

The assay demonstrated 98% sensitivity for detecting resistance variants in samples with viral load ≥1,500 IU/mL and 80% in samples with viral load of 1,000 IU/mL. A total of 39 clinical samples were analyzed, of which 5 showed clinically relevant mutations in the UL97 gene. Mutations in the UL54 and UL56 genes were validated using synthetic DNA. The main mutations identified included C603W, M460V, L595S, and A594V (UL97), I726T, T700A, and Q578H (UL54), and C325F and T244K (UL56), associated with resistance to different antivirals. The assay performance was validated in an international reference laboratory, with advantages such as broader genomic coverage, faster turnaround time, and simultaneous analysis of multiple genes.

The developed test is robust, sensitive, and applicable to routine clinical practice for detecting CMV antiviral resistance. Its implementation contributes to personalized management of immunosuppressed patients, optimizing therapeutic choice and reducing the risk of treatment failure.