Figure 1

Figure 1. In silico comparative analysis of wastewater assays used to detect avian and seasonal influenza viruses in wastewater. A) Predicted detection of HA gene sequences from LPAI viruses, HPAI viruses other...

We first performed a comparative in silico analysis of the publicly available primer and probe sequences from 3 H5 assays (13–15), and the JRC-HA and JRC-MP assays (12) (Figure 1). The predicted detection of HPAI H5 clade 2.3.4.4b was broadly comparable across the assays, but the JRC-HA assay revealed greater discrimination against non-2.3.4.4b HPAI clades (Figure 1, panel A). The JRC-HA and JRC-MP assays had the lowest predicted cross-reactivity with low pathogenicity avian influenza H5 annotated sequences (Figure 1, panels A, B).

Figure 2

Figure 2. Analytical evaluation of JRC-HA and JRC-MP RT-PCRs against recent highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b isolates for differentiating avian and human influenza A viruses from wastewater samples. A) Quantification...

Then, we evaluated the JRC-HA and JRC-MP duplex assay by using HPAI H5N1 clade 2.3.4.4b viruses collected from wild birds and poultry in northern Italy during October–December 2025 (Appendix). Sequence alignment revealed high conservation across the primer and probe binding regions for both assays, indicating that recent viral evolution had not substantially affected assay inclusivity. Nevertheless, we identified 2 substitutions within the JRC-HA probe-binding region in 3 samples (Appendix Figure 1). In silico PCR predicted amplification (data not shown), and both quantitative reverse transcription PCR and RT-dPCR detected influenza A in all 10 samples, including samples with low viral concentrations (Figure 2, panels A, B). For samples H, I, and J, lowering the JRC-HA RT-dPCR annealing temperature from 60°C to 56.4°C improved partition separation (Appendix Figure 2). At the revised annealing temperature, in silico analysis indicated that stable primer-dimer formation was unlikely, and assay specificity was not affected (Figure 2, panels C–F). The limit of detection was 5 copies/reaction, consistent with previous results (12). The CDC influenza B assay failed to detect the target in any of the 10 samples analyzed, whereas the CDC influenza A assay detected the target in all 10 samples with comparable performance to that of the JRC-HA and JRC-MP assays (Figure 2, panels A, B). Such broad detection is useful for confirming the presence of influenza virus; however, it does not permit discrimination between avian and seasonal human influenza A viruses in mixed specimens such as wastewater samples collected during periods of seasonal influenza circulation.

Figure 3

Figure 3. Detection of influenza A and highly pathogenic avian influenza (HPAI) A(H5N1) in simulated wastewater samples by using reverse transcription digital PCR. A) Copy numbers of influenza A virus in wastewater...

To improve discrimination between avian and seasonal human influenza in wastewater samples, we evaluated the influenza A and B components of the CDC Flu SC2 multiplex assay together with the JRC-HA and JRC-MP duplex assay. We performed the 2 duplex assays separately, according to the conditions described previously (12) (Appendix). Because HPAI H5N1 clade 2.3.4.4b–positive wastewater samples were unavailable, we added defined amounts of avian and seasonal human influenza A RNA to RNA extracts from influenza-negative wastewater samples (Figure 3). We evaluated the 2 duplex assays under 3 simulated co-circulation scenarios.

In the first scenario, we added only HPAI H5N1 clade 2.3.4.4b RNA to the wastewater samples. The JRC-HA, JRC-MP, and CDC influenza A assays produced comparable viral RNA copy numbers, indicating detection near the analytical limits of the JRC assays (Figure 3, panel A). For the second scenario, we combined low concentrations (10 or 20 copies/µL) of HPAI H5N1 clade 2.3.4.4b RNA and seasonal influenza A RNA (35 copies/µL) to simulate periods of low seasonal influenza activity or low avian influenza prevalence. The JRC-HA and JRC-MP assays selectively detected HPAI H5N1 clade 2.3.4.4b RNA, whereas the CDC influenza A assay detected both avian and seasonal human influenza A RNA without distinction (Figure 3, panel B).

In the third scenario, we added low concentrations (10 or 20 copies/µL) of HPAI H5N1 clade 2.3.4.4b RNA to a high concentration (3,500 copies/µL) of seasonal influenza A background, representing a possible early stage of HPAI detection during peak seasonal influenza activity. The JRC-HA assay detected HPAI H5N1 RNA without cross-reactivity, whereas the JRC-MP detected both avian and seasonal human influenza A RNA, but yielded lower copy number estimates than the CDC influenza A assay (Figure 3, panel C). Together, our results suggest that JRC-HA identifies and discriminates HPAI H5N1 clade 2.3.4.4b, whereas JRC-MP provided complementary detection but might lack specificity during high seasonal influenza A circulation.

Figure 4

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...

We propose the complementary use of CDC Flu SC2 and JRC-HA and JRC-MP duplex assays in a dual-duplex approach (Figure 4). The influenza A and B components of the CDC Flu SC2 multiplex assay are useful for routine communitywide surveillance of influenza A and B, ensuring continuity of the current public health reporting system. The JRC-HA and JRC-MP duplex assay, after the CDC Flu SC2 duplex, is used to specifically identify the occurrence of HPAI H5N1 clade 2.3.4.4b virus in a population, providing an early-warning capability that is effective in the presence of seasonal influenza backgrounds.

We evaluated our dual duplex RT-dPCR by using animal-derived viral material and spiked wastewater RNA extracts, rather than confirmed HPAI-positive wastewater samples. Further field evaluation will be useful to assess performance under naturally occurring conditions and in different wastewater matrices. Because influenza viruses evolve rapidly, periodic sequence-based evaluations, followed by experimental validation and assay updates when necessary, will be required to maintain assay performance as new H5 clades emerge.

A clade-specific wastewater result would not by itself determine a different public health response from a broad H5-positive result. However, it could reduce ambiguity, prioritize confirmatory testing, and enable integration of wastewater findings with veterinary, wildlife, domestic animal, and human surveillance data.