Unraveling Cytomegalovirus Drug Resistance in Transplant Patients by Targeting Deep Sequencing

Drug-resistant cytomegalovirus (CMV) poses a major clinical challenge in transplant recipients, leading to treatment failure and increased morbidity. This study applied a next-generation sequencing (NGS) approach to identify antiviral resistance mutations (ARMs) in 71 samples from 68 CMV-positive patients who had undergone hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT) between 2018 and 2024. A custom nested-PCR protocol targeting six CMV genes (UL27, UL51, UL54, UL56, UL89, and UL97) was developed for enrichment prior to NGS. ARMs were detected in 23% of patients without clinical suspicion of resistance and in 62% of those with suspected resistance, most frequently affecting UL97. The most common UL97 mutations were A594V (24.4%), C603W (20.0%), and L595S (15.6%), while D301N (50%) predominated in UL54. Mutations associated with foscarnet and maribavir resistance were found in five and eight patients, respectively. NGS identified ARMs in 29 patients not detected by Sanger sequencing (p < 0.00001), while no additional ARMs were identified by Sanger alone. Importantly, these minority variants, revealed by NGS, are clinically relevant, as they may expand under antiviral pressure and contribute to virological failure. ARM presence was not significantly associated with viral load or mortality, though recurrent CMV reactivation showed a trend toward association (p = 0.0504). Survival was significantly lower in HSCT versus SOT recipients (p = 0.027). These findings support the routine clinical use of NGS for CMV resistance testing, particularly in complex cases and in the context of expanding antiviral options such as maribavir and letermovir.