Laboratory Methods for Monitoring Influenza Antiviral Resistance

Monitoring antiviral susceptibility is an essential component of virologic surveillance conducted by laboratories participating in the World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS). Since 2011, the WHO Expert Working Group on Surveillance of Influenza Antiviral Susceptibility (WHO-AVWG) has annually published recommendations for assessing antiviral susceptibility of circulating viruses. This approach includes (i) screening viral RNA sequences for amino acid substitutions (molecular markers) known or suspected to confer reduced susceptibility, and (ii) testing viruses for antiviral susceptibility. Recently, next-generation sequencing of the whole viral genome has been implemented in many reference laboratories and could be used to test clinical specimens. Susceptibility to neuraminidase inhibitors (NAIs) is analyzed using neuraminidase activity-based assays with small synthetic substrates, while susceptibility to baloxavir is determined using replication-based assays. Since laboratory correlates of clinically significant antiviral resistance are not yet established, interpretation of antiviral testing outcomes follows the arbitrary WHO-AVWG criteria, where 50% inhibitory/effective concentration (IC50/EC50) values are compared to a virus type-, subtype-, or lineage-specific median to determine a fold change. For NAIs, the level of the enzyme's inhibition is reported as normal (<10-fold; <5-fold), reduced (10- to 100-fold; 5- to 50-fold), or highly reduced (>100-fold; >50-fold) for types A and B, respectively. For baloxavir, reduced susceptibility is provisionally defined as >3-fold above the median EC50. Additional considerations including preclinical testing in animal models may shed further light on antiviral susceptibility related to drug exposures relevant to humans and the fitness of viruses that contain reduced drug susceptibility genotypes. This framework provides a harmonized approach for monitoring antiviral susceptibility of influenza viruses needed to inform public health responses and clinical management.