Relative frequency of cytomegalovirus UL56 gene mutations detected in genotypic letermovir resistance testing

Genotypic testing for letermovir (LMV) resistance was performed by Sanger sequencing of cytomegalovirus terminase gene UL56 (codons 202-412) in 1165 diagnostic specimens, disclosing 36 sequence variants among 173 (14.8%) of the specimens, including one or more LMV resistance mutations in 134 specimens. Codon 325 mutations (C325Y/F/W/R) were the most common (108 specimens), followed by those at codon 369 (R369 S/G/T/K, 13 specimens) and V236M (11 specimens). Mutations V231L, N232Y, Q234R, L257F and V363I were detected in 1-3 specimens each. Combinations of codon 325 mutation and those at codons 236 or 369 were found in 6 specimens. Eleven novel sequence variants were phenotyped, validating Q234R, V363I and R369K as conferring 2- to 5-fold increased LMV 50% inhibitory concentrations (EC50). These findings indicate that UL56 codon 325 mutations conferring >3000-fold LMV EC50 are detected much more frequently in clinical practice than those conferring lower grade resistance, and suggest that a single step mutation to absolute LMV resistance is an ongoing concern in its therapeutic use.