Six years of clinical herpes simplex virus genotypic acyclovir resistance testing confirms common resistance mechanisms and identifies novel mutations

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations. IMPORTANCEAcyclovir-resistant herpes simplex virus infections remain an important clinical concern, especially for immunocompromised patients, but testing for acyclovir resistance has traditionally been limited to lengthy and laborious phenotypic testing. Since 2020, the University of Washington Clinical Virology Laboratory has provided genotypic acyclovir resistance testing. This testing provides faster turnaround times but relies on prior mutation characterization to identify resistance making it important to continually evaluate and report identified mutations. The clinical experience with this testing provides valuable insight into resistance mechanisms, identifies common polymorphisms, and highlights novel mutations for further characterization.