A rapid pyrosequencing assay for the molecular detection of influenza viruses with reduced baloxavir susceptibility due to PA/I38X amino acid substitutions

Baloxavir marboxil is a novel endonuclease inhibitor licensed for treatment of otherwise healthy or high‐risk individuals infected with influenza. Viruses with reduced baloxavir susceptibility due to amino acid substitutions at residue 38 of the PA have been detected in some individuals following treatment. Here, we describe a genotypic pyrosequencing method that can be used to rapidly screen circulating influenza A and B viruses for substitutions in the PA/I38 codon and to quantify mixed viral populations. This method is suitable for surveillance of baloxavir susceptibility and to analyse samples from hospitalised patients undergoing baloxavir treatment to aid in clinical decision making.