Sensitivity, throughput, and cost analysis of concentration methods for multi-target pathogen wastewater monitoring

Wastewater-based epidemiology is an efficient method for monitoring the transmission of diverse pathogens in communities. Standard wastewater surveillance workflows typically involve wastewater concentration, nucleic acid extraction, and pathogen quantification. While various concentration methods are used, most comparisons of concentration methods have focused primarily on SARS-CoV-2, highlighting the need for further research to guide method selection for monitoring a suite of diverse pathogens. In this study, a head-to-head comparison of six different concentration methods was performed, including direct extraction (with and without bead beating), electronegative (HA) filtration, solids concentration, and magnetic bead-based concentration (using Nanotrap® particles; with and without bead beating). Methods were assessed for sensitivity, inhibitor removal, and recovery rates of fourteen microorganisms, including viruses, bacteria, and fungal pathogens. The cost of each method was also estimated. Results showed that the concentration method selection significantly impacts the sensitivity and economic costs of the wastewater monitoring workflow. Based on the results, a concentration approach that combines HA filtration and solids concentration is recommended to optimize detection across various pathogens. This study provides data-driven insights to enhance the reliability and cost-effectiveness of wastewater surveillance systems that can support public health responses for a broad range of diseases.