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

The PLEX-ID platform was used to perform a pilot evaluation for the detection and subtyping or lineage characterization of human influenza virus types A and B, respectively. For this, nasopharyngeal swab specimens (NPS) collected from a network of more than 80 practitioners participating actively to the clinical surveillance of influenza cases in Switzerland and screened for influenza during the 2010-2011 season were used as follows. After viral genome extraction using the NucliSENS easyMAG (bioMérieux, Geneva, Switzerland), the following four onestep real-time RT-PCR assays were applied for the routine screening: the CDC pan-influenza A specific real-time RT-PCR assay [19] considered as a reference assay for influenza type A surveillance by the WHO, and three in-house developed assays specific for A(H1N1)pdm (Swine H1 GE), influenza A(H3N2) (A/H3), and influenza B (InfB MP) detection (supplementary Table 1 ) all validated on WHO quality controls. Each week, a batch of 22 or 46 influenzapositive specimens (according to the number of available specimens) were tested with the PLEX-ID/Flu assay in parallel to the usual real-time RT-PCR assays performed by the Swiss National Reference Centre for Influenza. NPS were selected blind of the real-time RT-PCR threshold cycle (C T ) values and the type of influenza (influenza A or B). For each assay, specific positive and negative internal controls were included systematically in each run to rule out any potential PCR inhibitors or contaminations, respectively. and Wisconsin/01/10 strains, respectively). Of note, the PLEX-ID/Flu assay's subtyping or lineage characterization for A(H3N2) and type B specimens, respectively, is less accurate than for A(H1N1)pdm specimens since HA and NA genes are not included in the analysis. Taken together, the PLEX-ID/Flu assay detected positively and gave a typing/lineage characterization result for 93% of all NPS detected positively by real-time RT-PCR. Positive results obtained by both methods were always in agreement. All non-typeable influenza A and B specimens by the PLEX-ID/Flu assay showed relatively low viral loads with C T values 33 obtained with the screening real-time RT-PCR assays.