Bioluminescence-Based Neuraminidase Inhibition Assay for Monitoring Influenza Virus Drug Susceptibility in Clinical Specimens

(NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveil-lance laboratory. A comprehensive panel of reference viruses (n 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50 % inhibitory concentrations (IC50s) generated were com-pared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemilumi-nescent (CL) assays. To provide proof of principle, clinical specimens (n 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyro-sequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, cer-tain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Ymarker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens. Monitoring influenza resistance to antiviral drugs is an essen-tial component of the WHO Global Influenza Surveillanc