Analytical Evaluation of the Microarray-Based FluChip-8G Influenza A+B Assay

Abstract Background Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8 G Influenza A + B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. Objective To evaluate key analytical performance characteristics of the FluChip-8 G Influenza A + B Assay. Study Design Analytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. Results The limit of detection (LOD) for the FluChip-8 G influenza A + B Assay ranged from 5.8 × 10 2 - 1.5 × 10 5 genome copies/mL, with most samples ˜2 × 10 3 genome copies/mL (˜160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both “non-seasonal influenza A” and A/H1N1 pdm 2009. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7 - 98.7)) regardless of operator, site, reagent lot, or testing day. Conclusion The FluChip-8 G Influenza A + B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.