Revealing the Unseen: Next-Generation Sequencing for Early Detection of Drug-Resistant Cytomegalovirus Variants Upon Letermovir Prophylaxis Failure

In recipients of allogeneic hematopoietic cell transplant, prophylactic management strategies are essential for preventing cytomegalovirus (CMV) reactivation and associated disease. We report on a 63-year-old male patient with a D-/R+ CMV serostatus, who showed ongoing low-level CMV replication after hematopoietic cell transplant despite receiving letermovir prophylaxis. Sanger sequencing failed to detect drug resistance mutations (DRMs) until CMV pneumonitis developed, revealing a UL56-C325R DRM linked to high-level letermovir resistance. Retrospective analysis with next-generation sequencing revealed the DRM at a low frequency of 6% two weeks prior to detection by Sanger sequencing. This study highlights the importance of advanced next-generation sequencing methods for early detection of CMV DRMs, allowing for faster adjustments in antiviral treatment strategies.