Viral concentration method biases in the detection of viral profiles in wastewater

Viral detection methodologies used for wastewater-based epidemiology (WBE) studies have a broad range of efficacies. The complex matrix and low viral particle load in wastewater emphasize the importance of the concentration method. This study focused on comparing three commonly used virus concentration methods: polyethylene glycol precipitation (PEG), immuno-magnetic nanoparticles (IMNP), and electronegative membrane filtration (EMF). Influent and effluent wastewater samples were processed by the methods and analyzed by DNA/RNA quantification and sequencing for the detection of human viruses. SARS-COV-2, Astrovirus, and Hepatitis C virus were detected by all the methods in both sample types. PEG precipitation resulted in the detection of 20 types of viruses in influent and 16 types in effluent samples. The corresponding number of virus types detected was 21 and 11 for IMNP, and 16 and 8 for EMF. Certain viruses were unique to only one concentration method. For example, PEG detected three types of viruses in influent and six types in effluent compared to IMNP, which detected seven types in influent and one type in effluent samples. However, the EMF method appeared to be the least effective, detecting three types in influent and none in effluent samples. Rotavirus was detected in influent sample using IMNP method, whereas EMF and PEG methods failed to yield a similar outcome. Consequently, the potential false negative results pose a risk to the credibility of WBE applications. Therefore, implementation of a proper concentration technique is critical to minimize method biases and ensure accurate viral profiling in WBE studies.