Detection of CMV Antiviral Resistance Mutations to Letermovir in Patients Using a Validated Clinical Sequencing Assay

Abstract

Background Antiviral resistance to human cytomegalovirus (CMV) is an important complication for immunocompromised patients on prolonged antiviral regimens, and CMV remains the most clinically significant infection following allogeneic hematopoietic-cell transplantation (HCT). Letermovir targets subunit 2 of the viral terminase complex (UL56) and is approved for CMV prophylaxis in adult HCT recipients. Resistance to letermovir is conferred by point mutations in the UL56 gene, and with the potential clinical need for antiviral resistance testing, we have developed a UL56 sequencing assay covering 24 identified resistance mutations. Here we summarize the performance characteristics of the UL56 antiviral resistance assay, along with results from de-identified samples following early reference laboratory testing. Methods This assay uses automated nucleic acid extraction followed by CMV UL56-specific polymerase chain reaction (PCR). PCR products were subjected to cycle sequencing and capillary electrophoresis, and the resulting sequences were analyzed for the presence of known resistance mutations between codons 229 and 369 of the UL56 gene. The assay's limit of detection (LOD), precision and accuracy were validated in accordance with accepted regulatory standards using multiple laboratory and clinical CMV strains. The LOD of the assay was determined to be 99 IU/mL. Results In early clinical use of the CMV letermovir resistance assay, several resistance mutations were identified. Mutations at C325 were observed at the highest frequencies, including C325F, R, Y and W, which all have reported resistance indexes > 3,000. V236M, T244K and R369S were also detected at lower frequencies, which have published resistance indexes of 32—46, 3.3 and 38—48, respectively. Polymorphisms not known to confer resistance were detected at codons 246, 247 and 335. Resistance mutations in both UL56 and UL97, conferring resistance to letermovir and ganciclovir, respectively, were also observed in CMV DNA isolated from a single patient. Conclusion The CMV letermovir resistance sequencing assay was shown to be a rapid and sensitive means of detecting mutations conferring letermovir resistance. This expands current CMV antiviral resistance testing, which includes UL54 and UL97 sequencing, and provides physicians with the ability to monitor for the emergence of antiviral resistance mutations to all current FDA-approved anti-CMV drugs. Following validation of the assay, several patient specimens have featured mutations conferring substantial levels of resistance to letermovir. The treatment status of these patients is unknown; however, these findings underscore the importance of screening and monitoring patients for the emergence of mutations in UL56 conferring resistance to letermovir.