Wastewater PCR gains a sharper way to separate avian H5N1 from seasonal influenza
Wastewater surveillance can detect influenza circulation before clinical systems fully register it, but the signal is often messy. Human seasonal influenza A, avian influenza A, and mixed environmental inputs can blur together when laboratories rely on broad matrix-gene targets alone. In a dispatch from European Commission and Italian collaborators, digital PCR assays were assembled into a dual-duplex workflow designed to distinguish highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b from seasonal human influenza A in wastewater extracts.
The practical problem is straightforward. A wastewater sample can contain RNA from humans, birds, animals, and environmental debris, all of it filtered through sewer systems that do not respect species boundaries. A generic influenza A result confirms circulation, but it can leave public health teams uncertain about whether they are seeing routine seasonal activity or an avian incursion that deserves closer follow-up. The assay strategy described here tries to reduce that ambiguity by pairing a broad influenza screen with a clade-specific readout.
A two-step assay design aimed at environmental ambiguity
Investigators combined the influenza A and B components of the CDC Flu SC2 multiplex assay with the European Commission’s Joint Research Centre JRC-HA and JRC-MP duplex assays. The JRC-HA assay targets a conserved hemagglutinin region in HPAI H5N1 clade 2.3.4.4b, while the JRC-MP assay targets the matrix gene for broader influenza A detection. In silico comparisons against publicly available primer and probe sequences suggested that the JRC-HA assay offered stronger discrimination against non-2.3.4.4b HPAI clades, and both JRC assays showed the lowest predicted cross-reactivity with low pathogenicity avian influenza H5 sequences.
That distinction matters in wastewater, where broad reactivity can create a false sense of certainty. A conserved matrix target is useful for screening, but the same conservation that makes it dependable also makes it blunt. By adding a clade-focused hemagglutinin assay, the workflow gains a second layer of specificity that can point surveillance teams toward the most consequential avian signal without abandoning the utility of a general influenza A screen.
Conservation held up in recent Italian H5N1 isolates
The next step used HPAI H5N1 clade 2.3.4.4b viruses collected from wild birds and poultry in northern Italy during October through December 2025. Sequence alignment showed high conservation across primer and probe binding regions for both JRC assays, suggesting that recent viral evolution had not materially undermined assay inclusivity. Two substitutions appeared in the JRC-HA probe-binding region in 3 samples, yet both quantitative reverse transcription PCR and RT-dPCR still detected influenza A in all 10 samples, including those with low viral concentrations.
One operational adjustment improved performance further. Lowering the JRC-HA RT-dPCR annealing temperature from 60°C to 56.4°C sharpened partition separation in three samples, and subsequent analysis found no meaningful increase in primer-dimer concerns or loss of specificity. The limit of detection remained 5 copies per reaction, consistent with previous work. The CDC influenza B assay did not detect target in any of the 10 samples, while the CDC influenza A assay detected influenza A in all 10 with performance comparable to the JRC-HA and JRC-MP assays.
Simulated wastewater co-circulation exposed the assay tradeoff
The most clinically relevant test came from simulated wastewater. Because confirmed HPAI-positive wastewater samples were unavailable, the team spiked defined amounts of avian and seasonal human influenza A RNA into influenza-negative wastewater extracts and examined three scenarios that mimic realistic surveillance conditions. Each scenario put pressure on the assays in a different way: avian virus alone, low-level avian virus against low seasonal background, and low-level avian virus amid heavy seasonal influenza circulation.
In the first scenario, only HPAI H5N1 clade 2.3.4.4b RNA was added. The JRC-HA, JRC-MP, and CDC influenza A assays produced comparable copy numbers, a pattern consistent with detection near the analytical limits of the JRC assays. In the second scenario, low concentrations of HPAI H5N1 RNA were mixed with seasonal influenza A RNA at 35 copies per microliter. Here, the JRC-HA and JRC-MP assays selectively detected the avian target, while the CDC influenza A assay detected both avian and seasonal RNA without distinction.
A third scenario reproduced the hardest surveillance setting: low-level HPAI RNA added to a high seasonal influenza A background of 3,500 copies per microliter. The JRC-HA assay still detected HPAI H5N1 RNA without cross-reactivity. The JRC-MP assay detected both avian and seasonal influenza A RNA, but it returned lower copy-number estimates than the CDC influenza A assay. In bedside terms for public health, the broad assay can say “influenza is present,” while the clade-specific assay can still call out the avian signal when seasonal circulation is drowning the sample in background noise.
Early-warning value without overclaiming the response
The proposed workflow places the CDC Flu SC2 assay first, followed by the JRC-HA and JRC-MP duplex assay. That order preserves routine communitywide influenza A and B surveillance and then adds a second pass aimed at identifying HPAI H5N1 clade 2.3.4.4b. The goal is not to replace current reporting systems but to sharpen them, especially when a wastewater signal emerges during a season of active human influenza.
This approach carries a useful epidemiologic logic. Influenza viruses evolve quickly, wastewater matrices vary, and signal strength depends on shedding, sewer inputs, and sample composition. A clade-specific result does not automatically dictate a different public health response than a broad H5-positive result, but it can reduce uncertainty, prioritize confirmatory testing, and help integrate environmental data with veterinary, wildlife, domestic animal, and human surveillance streams.
Field validation remains the next test of real-world performance
The workflow has limits that are plainly stated by the investigators. The evaluation used animal-derived viral material and spiked wastewater extracts rather than confirmed HPAI-positive wastewater specimens. Field performance in naturally contaminated wastewater, across different sewer systems and matrices, still needs to be established. Assay maintenance will also require periodic sequence review as new H5 clades emerge and primer-probe matches shift.
Even so, the main surveillance message is concrete. Broad influenza A detection remains necessary, but broad detection alone cannot resolve avian from seasonal human signals in a mixed environmental sample. In a setting where rapid recognition of HPAI H5N1 clade 2.3.4.4b could matter for veterinary, wildlife, and public health coordination, a dual-duplex digital PCR strategy offers a cleaner read on what is moving through the sewer system.