The Chemiluminescent Neuraminidase Inhibition Assay: A Functional Method for Detection of Influenza Virus Resistance to the Neuraminidase Inhibitors

Neuraminidase inhibitors (NAIs) represent a newer class of anti-influenza drugs. Widespread natural or acquired resistance to NAIs is a major public health concern as it limits pharmaceutical options available for managing seasonal and pandemic influenza virus infections. Molecular-based methods, such as pyrosequencing, sequencing, and PCR are rapid techniques for detecting known genetic markers of resistance, but they are unable to identify novel mutations that may confer resistance, or subtle differences in the susceptibility of viruses to the NAIs. This chapter describes the chemiluminescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The assay generates IC₅₀ values (drug concentration needed to reduce the NA enzymatic activity by 50%) which are determined by curve-fitting analysis. Test viruses showing elevated IC₅₀ values relative to those of NAI-sensitive reference viruses of the same antigenic type and subtype are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or new changes in the NA. The criteria for NAI resistance are currently not well defined and tend to vary by laboratory and NI assay, therefore harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve the NAI susceptibility testing and analysis.