What does this research mean for the field?

A next-generation sequencing assay has been developed that can detect CMV antiviral resistance mutations with 98% sensitivity in samples with viral load ≥1,500 IU/mL. Novelty: ClaimNovelty.METHODOLOGICAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

What question did this study set out to answer?

The aim is to develop a test using next-generation sequencing to identify CMV mutations linked to antiviral resistance for better treatment management.

March 17, 2026Open Access

Next-Generation Sequencing Applied to the Identification of CMV Antiviral Resistance Mutations: Development and Validation of an Assay for Clinical Use

Key Points

The aim is to develop a test using next-generation sequencing to identify CMV mutations linked to antiviral resistance for better treatment management.
Designed specific primers for target regions in CMV genes (UL27, UL51, UL54, UL56, UL97)
Extracted viral DNA from clinical samples and performed PCR and library preparation
Sequenced samples on an Illumina platform and analyzed data with an in-house pipeline
Compared identified variants with the HerpesDRG DB database for validation
Achieved 98% sensitivity for resistance variants in viral loads ≥1,500 IU/mL
Identified 5 clinically relevant mutations in the UL97 gene from 39 samples
Validated mutations in UL54 and UL56 genes using synthetic DNA
The assay demonstrates advantages like broader genomic coverage and faster results

Abstract

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.

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