648. Baloxavir Resistance: qPCR Detection of Antiviral Resistance Markers in Influenza A Virus Open Access

Influenza (flu) infections affect a large subset of the population every year and have significant impacts on the health of patients, especially those with weak or compromised immune systems such as the elderly, children, cancer patients, and transplant recipients. Baloxavir marboxil was approved in October 2018 as a novel antiviral therapeutic for treating flu. During clinical trials, mutations were identified at the I28 codon of the polymerase acidic (PA) protein that greatly increased the resistance of a flu strain to this novel drug. In this study, a qPCR was developed and validated to identify these resistance mutations, allowing for guided therapy based on the resistance profile of the strain.

Flu A sequences (6,175) of the PA gene from the NCBI Influenza Virus Database collected over the last 5 years were compiled and aligned. Primers and probes were designed to target the I38 codon of the PA gene, and specific probes for each codon yielding a resistant amino acid mutation (I38T, -M, and -F) were designed. Locked nucleic acid (LNA) bases were used to increase the specificity of the probes. A combination of clinical flu specimens, laboratory strains, and synthetic constructs of each potential resistance mutation were used to validate the precision, sensitivity, and accuracy of the assay in nasopharyngeal swabs.

Precision of the cycle threshold (Ct) values for each detector was determined to have a standard deviation of less than 3 for inter-assay and less than 2 for intra-assay replicates. Sensitivity was determined to be 800 copies/mL in nasopharyngeal swabs. Accuracy was found to be 92.3%. A single laboratory strain from the H1N1 2009 epidemic showed cross-reactivity with both wild-type and resistant probes, but no circulating clinical H1N1 samples tested showed this response.

The precision, sensitivity, and accuracy of a qPCR for resistance mutations to baloxavir marboxil support this assay’s utility as an aid in the treatment of flu in at-risk patient groups. This assay allows for rapid detection (<24 hours) of resistance markers to aid clinicians in improving flu case outcomes.

All authors: No reported disclosures.