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Volume 32, Number 11—November 2026

Dispatch

Differentiating Avian and Seasonal Influenza Viruses in Wastewater by Digital PCR

Valentina Paracchini, Mauro Petrillo, Francisco Mayo Leon, Gerhard Buttinger, Sabrina Marciano, Marika Crimaudo, Calogero Terregino, Carolina Veneri, Giuseppina La Rosa, Elisabetta Suffredini, Clementina Cocuzza, Bernd M. Gawlik, Tobias Wiesenthal, Ursula Vincent, Valentina Panzarin, and Antonio MarchiniComments to Author 
Author affiliation: European Commission, Joint Research Centre, Geel, Belgium (V. Paracchini, F.M. Leon, G. Buttinger, U. Vincent); Seidor Italy Srl., Milan, Italy (M. Petrillo); Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy (S. Marciano, M. Crimaudo, C. Terregino, V. Panzarin); Istituto Superiore di Sanità, Rome, Italy (C. Veneri, G. La Rosa, E. Suffredini); University of Milano-Bicocca, Monza, Italy (C. Cocuzza); European Commission, Joint Research Centre, Ispra, Italy (B.M. Gawlik, T. Wiesenthal, A. Marchini)

Main Article

Figure 4

Interpretation of dual duplex RT-dPCR results for differentiating avian and seasonal influenza A viruses in wastewater. Extracted RNA from wastewater samples is analyzed by using the JRC-HA and JRC-MP duplex assay for detection of HPAI A(H5Nx) clade 2.3.4.4b viruses and the influenza A and B components of the CDC influenza and SARS-CoV-2 multiplex assay for broad influenza A virus surveillance. Results from both assays are then integrated to classify wastewater detections according to the presence, absence, and relative signal intensity of assay targets. Detection patterns are interpreted as indicating no influenza A virus detected, seasonal influenza A early warning, seasonal influenza A circulation, possible LPAI or non–clade 2.3.4.4b HPAI virus, HPAI A(H5Nx) clade 2.3.4.4b circulation, or co-circulation of HPAI A(H5Nx) clade 2.3.4.4b and seasonal influenza A viruses. Minus signs (−) indicate no target detection; increasing numbers of plus signs (+ to ++++) indicate increasing relative assay signal. CDC, Centers for Disease Control and Prevention; HA, hemagglutinin; HPAI, highly pathogenic avian influenza; JRC, Joint Research Centre hemagglutinin; LPAI, low pathogenicity avian influenza; MP, matrix protein; RT-dPCR, reverse transcription digital PCR.

Figure 4. Interpretation of dual duplex RT-dPCR results for differentiating avian and seasonal influenza A viruses in wastewater. Extracted RNA from wastewater samples is analyzed by using the JRC-HA and JRC-MP duplex assay for detection of HPAI A(H5Nx) clade 2.3.4.4b viruses and the influenza A and B components of the CDC influenza and SARS-CoV-2 multiplex assay for broad influenza A virus surveillance. Results from both assays are then integrated to classify wastewater detections according to the presence, absence, and relative signal intensity of assay targets. Detection patterns are interpreted as indicating no influenza A virus detected, seasonal influenza A early warning, seasonal influenza A circulation, possible LPAI or non–clade 2.3.4.4b HPAI virus, HPAI A(H5Nx) clade 2.3.4.4b circulation, or co-circulation of HPAI A(H5Nx) clade 2.3.4.4b and seasonal influenza A viruses. Minus signs (−) indicate no target detection; increasing numbers of plus signs (+ to ++++) indicate increasing relative assay signal. CDC, Centers for Disease Control and Prevention; HA, hemagglutinin; HPAI, highly pathogenic avian influenza; JRC, Joint Research Centre hemagglutinin; LPAI, low pathogenicity avian influenza; MP, matrix protein; RT-dPCR, reverse transcription digital PCR.

Main Article

Page created: September 24, 2026
Page updated: October 15, 2026
Page reviewed: October 15, 2026
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