title: Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2.
abstract: BACKGROUND
  Most herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing.
  
  
  OBJECTIVE
  To enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet.
  
  
  METHODS
  The assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions.
  
  
  RESULTS
  Results agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3-37% coefficient of variation based on IC50.
  
  
  CONCLUSIONS
  Chemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States. Electronic address: keith.d.tardif@aruplab.com.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Tardif
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States.
authors.forename: Shane
authors.initials: S
authors.lastname: Jorgensen
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States.
authors.forename: Janine
authors.initials: J
authors.lastname: Langer
authors.affiliation: University of Alabama at Birmingham, AL, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Prichard
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States; University of Utah School of Medicine, Department of Pathology, Salt Lake City, UT, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Schlaberg
mesh_terms: D000212:Acyclovir / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival; D003588:Cytopathogenic Effect, Viral; D017245:Foscarnet / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000187:drug effects* / Q000302:isolation & purification*; D006801:Humans; D020128:Inhibitory Concentration 50; D008163:Luminescent Measurements / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods; D015203:Reproducibility of Results; D019562:Viral Load
chemical_list: D000998:Antiviral Agents; D017245:Foscarnet; D000212:Acyclovir
keywords: Antiviral; Assay; Chemiluminescence; HSV; Herpes; Susceptibility