A-Z Index × Submit A-Z Index × Submit A-Z Index Search Dropdown × Submit Facebook Twitter LinkedIn Syndicate Emerging Infectious Disease journal ISSN: 1080-6059 Disclaimer: Early release articles are not considered as final versions. Any changes will be reflected in the online version in the month the article is officially released.
Highly pathogenic avian influenza A(H5Nx) viruses, particularly clade 2.3.4.4b, pose substantial global health risks. We combined and piloted 2 duplex reverse transcription digital PCRs to discriminate between avian and seasonal influenza viruses in wastewater. This approach enabled rapid, strain-specific detection to reduce ambiguity in environmental surveillance for influenza viruses.
Highly pathogenic avian influenza (HPAI) A(H5Nx) viruses, particularly clade 2.3.4.4b, continue to circulate widely among wild and domestic birds, with recurrent spillover into mammalian species and sporadic infections in humans. The extensive geographic distribution and persistent circulation of clade 2.3.4.4b, together with its predominance among HPAI A(H5) viruses currently circulating in Europe, support its continued relevance for animal and public health surveillance ( 1 – 4 ).
Wastewater and environmental surveillance (WES) provides a population-level complement to clinical, veterinary, and wildlife surveillance and is increasingly integrated into the monitoring framework in Europe ( 5 ). WES has been widely used for SARS-CoV-2 and has also been applied to influenza A and B viruses ( 6 , 7 ).
WES typically relies on PCR-based methods to detect and quantify pathogens. Similar to other pathogens, influenza RNA concentrations in wastewater might vary according to shedding patterns, sewer inputs, matrix characteristics, and the relative contributions of human and animal sources ( 6 ). Differences in laboratory methods and reporting further complicates interpretation, underscoring the necessity of considering assay sensitivity and specificity when comparing results across studies ( 8 ).
The matrix gene is the preferred molecular target for the screening of influenza A viruses ( 9 ) because it is conserved across seasonal human and animal influenza strains, but broad reactivity limits strain discrimination in mixed environmental samples. For instance, the influenza A component of the Centers for Disease Control and Prevention (CDC) influenza SARS-CoV-2 (Flu SC2) multiplex assay, developed for clinical respiratory specimens and real-time PCR ( 10 ) and adapted for reverse transcription digital PCR (RT-dPCR) analysis of wastewater ( 11 ), can detect both seasonal human and avian influenza A signals ( 12 ).
Several wastewater assays were designed to detect H5 viruses ( 13 – 15 ). Those assays are useful for broad H5 virus screening but might require further characterization to distinguish between HPAI H5N1 clade 2.3.4.4b, another H5 lineage, or low pathogenicity avian influenza. A clade-specific assay can provide additional epidemiologic resolution and help prioritize follow-up across animal and human surveillance.
We previously developed the European Commission’s Joint Research Centre (JRC) RT-PCRs targeting the hemagglutinin (HA) and matrix protein (MP) genes. The JRC-HA assay targets a conserved HA region in HPAI H5N1 clade 2.3.4.4b and the JRC-MP assay targets the conserved matrix gene for broader detection of influenza A ( 12 ) ( Appendix ). We evaluated a dual-duplex RT-dPCR approach combining the CDC influenza A an…