Optimizing Avian Influenza Viral RNA Enrichment from Water: A Comparison of Concentration Methods for Low-Volume Samples

Feed-baited water bins may provide a complementary approach for avian influenza virus (AIV) surveillance in wild waterbird populations. In a previous study, water from these bins was sampled using swabs after exposure to visiting birds, but the low sample volume limited the detection and subsequent characterization of AIV RNA (F. I. Rondi, J. Neumann-Heise, T. Harder, V. Allendorf, K. Depner, T. Homeier-Bachmann, L. Breuer, S. Knauf, S. Dreyer, and A. Globig, One Health 23:101548, 2026, https://doi.org/10.1016/j.onehlt.2026.101548). Here, we compared five sample-processing methods for improving AIV RNA detection in small (40-mL) water samples (tap and brackish water) spiked with an AIV of the H4N6 subtype under simulated field conditions: swab sampling, polyethylene glycol precipitation, activated carbon capture, combined activated carbon capture and polyethylene glycol precipitation (AC/PEG), and electronegative membrane adsorption (EM). AC/PEG and EM showed the strongest overall performance based on RT-qPCR results, although their relative performance varied across experimental conditions. The qPCR-derived viral signal was generally higher in tap water than in brackish water, whereas wheat presence was associated with a lower signal, particularly for swab sampling. Subsequent testing was conducted to ascertain the most effective storage conditions with regard to temperature and duration. During storage, AIV RNA detection remained relatively stable at minus 20 degrees Celsius for up to four weeks, with only minor increases in mean Ct values in both water matrices. Greater increases were observed at room temperature and 4 degrees Celsius, with significantly higher Ct values at both temperatures than at minus 20 degrees Celsius in both water matrices at Weeks 2 and 4 (p < 0.05). These findings support the potential of AC/PEG and EM to facilitate the recovery, detection and subsequent characterization of AIV from low-volume water samples by improving viral RNA detection using RT-qPCR. Given its overall performance and lower cost compared with EM, AC/PEG may represent a practical option for field-based surveillance. Regarding storage, minus 20 degrees Celsius provided the best conditions for maintaining AIV RNA detection, with relatively stable results for up to four weeks.