Introduction 
 
Avian influenza is a highly contagious infectious disease caused by Influenza A viruses. Aquatic birds represent the natural reservoir of the virus, contributing to its spread and responsible for occasional epidemics generating significant economic losses. The highly pathogenic avian influenza viruses (HPAIV, 2.3.4.4.b clade) responsible for the 2020/21 and 2021/22 european epizootics, correspond to the H5N8 and H5N1 subtypes, both belonging to the H5 subtype, originating from the A/Goose/Guangdong/1/1996 (GSGd) lineage.
Currently, detection of HPAIV is performed by individual sampling and requires a two-step test strategy (first screening. A recent publication suggested that dust sampling would be an effective surveillance approach and would allow a faster detection (Filaire et al., 2022).
IAGE company is specialized in the development of biological diagnostic tools in the environment, based on the latest PCR generation technology (digital PCR or dPCR). We developed here an innovative method for the detection of HPAIV H5, including the specific detection of 2.3.4.4.b clade.
 
Method

IAGE designed specific probes targeting 5 markers: Galloanserae, the M gene, the H5 subtype, the H7 subtype and finally a specific probe targeting highly pathogenic influenza viruses (2.3.4.4b clade).
The sensitivity of the dPCR methods developed were evaluated on dust collected with wipes in poultry houses (Filaire et al., 2022).
Finally, the milits of detection were compared (qPCR vs. dPCR) on dust samples using the same master mix for both RT-qPCR (officially accredited kit) and RT-dPCR (QIAGEN).
IHAP performed experiments using commercial RT-qPCR kits for detection of H5 subtypes.
 
Results

First, we validated the specificity of the in-house designed probes for each target. In vitro tests were conducted by comparing the performance of the commercial kits for the detection of avian influenza viruses on animals and our dPCR methods.
Secondly, RT-qPCR vs. RT-dPCR tests showed that dPCR was particularly convenient to detect the target at very low concentration in samples, allowing early diagnosis, when RT-qPCR was unable to reveal any signal.
 
Conclusion

Thanks to this innovative assay, we could detect HPAIV and type specifically the 2.3.4.4.b clade in a single analysis within 24 hours after receiving the sample, compared to the current diagnosis which requires two steps. It also allowed a detection via environmental samples, as an alternative and complement to individual samples that may occur too late, at the onset of clinical signs.