Comparative evaluation of viral concentration and extraction protocols for wastewater-based epidemiology of respiratory viruses

Introduction:Wastewater-Based Epidemiology (WBE) enables early detection and monitoring of infectious diseases, but the lack of standardization in viral concentration and extraction methods challenges data reliability.Objectives: This study compared three protocols using magnetic bead-based viral concentration and automated nucleic acid extraction for detecting SARS-CoV-2 (SC2), Influenza A (FLUA), Influenza B (FLUB), and Respiratory Syncytial Virus (RSV) in wastewater.Methodology: Wastewater samples from Rio de Janeiro (February 2025) were divided into three aliquots, each processed with a different protocol: P1 (magnetic nanoparticles + automated extractor), P2 (same concentration method, different extractor), and P3 (integrated concentration and extraction).Synthetic wastewater spiked with target viruses validated the methods.Viral detection was performed using RT-PCR and digital PCR (dPCR -Qiacuity) with a 5plex assay developed by Bio-Manguinhos.Results: P3 had the highest detection rates and viral concentrations (SC2: 78.9%, 196.76 copies/mL; FLUA: 63.2%, 315.41 copies/mL), followed by P1 (SC2: 73.7%, 9.77 copies/mL; FLUA: 57.9%, 11.60 copies/mL) and P2 (SC2: 57.9%, 54.78 copies/mL; FLUA: 21.1%, 80.04 copies/mL).FLUB was best detected by P3 (42.1%, 302.44 copies/mL), with lower recoveries for P2 (26.3%, 76.38 copies/mL) and P1 (15.8%, 10.10 copies/mL).RSV detection was low overall, with P3 performing best (57.9%, 193.17 copies/mL), followed by P2 (42.1%, 41.27 copies/mL) and P1 (36.8%, 6.14 copies/mL).dPCR increased sensitivity by up to 20%, with high agreement for SC2 (>85%), moderate for FLUA and FLUB (~70%), and lower for RSV (~60%).Internal control recovery was consistent, confirming methodological reliability. Conclusion:P3 demonstrated superior viral recovery, making it the most effective for WBE.P1 showed intermediate performance, especially for SC2 and FLUA, while P2 had the lowest detection rates, particularly for RSV and FLUA.dPCR improved sensitivity, reinforcing its utility for detecting low viral concentrations.Standardizing methods is essential for reliable WBE applications.