Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens

Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

Among these 37 specimens, the PLEX-ID/Flu assay was able to subtype 9/19 influenza A (47.5%) and characterize the lineage for 14/18 influenza B (77.8%). The Sanger-based sequencing method was able to subtype 13/19 influenza A (68.4%) and identify the lineage for 9/18 influenza B (50%). Therefore, for these selected specimens, the PLEX-ID/Flu assay demonstrates a sensitivity for influenza B virus lineage characterization that is at least similar to the Sanger-based method, which is directly dependent on conditions used for viral genome amplification by classical PCR. However, the latter appears more sensitive for influenza A subtyping under these conditions. This observation can be explained by the PLEX-ID/Flu assay algorithm that requires the analysis of nucleotide base composition signatures of a minimum of three positive PCRs out of eight independent primer pairs present in the assay, with the mandatory coupling of detection and subtyping processes, for influenza A specimen analysis. Therefore, the typing constraint of the PLEX-ID/Flu assay explains partially the higher influenza A subtyping performance of the Sanger-based method for low viral load specimens. Indeed, 9/10 influenza A NPS detected positive by real-time RT-PCR could not be subtyped with the PLEX-ID/Flu assay, although one or two positive PCR signals were observed. Although the minimal requirement of three independent PCR signals for subtyping was not obtained, specific influenza A PCR amplifications were detected, suggesting a sensitivity threshold close to the realtime RT-PCR.