Abstract
Respiratory infections are a major public health threat. Significant global mortality is caused by influenza viruses, the new SARS-CoV-2 virus, and the Respiratory Syncytial Viruses (RSVs). Wastewater-based epidemiology (WBE) has recently emerged as a valuable tool for monitoring these pathogens, providing insights into their evolution, transmission patterns, and co-circulation within populations. This study aimed to track influenza viruses (A and B), the new SARS-CoV-2 virus, and the Respiratory Syncytial Viruses (RSVs) (type A and B) during the pandemic period (from October 2020 to October 2021) in a middle-size Spanish city (Valladolid) and its surrounding areas. Viral concentration was performed using an aluminum-based precipitation method, followed by RNA extraction and RT-qPCR quantification targeting the N1 and N2 regions of the SARS-CoV-2 nucleocapsid gene, the N gene for both RSV-A and RSV-B, and the M and non-structural protein genes for influenza A and B, respectively. The results demonstrated the utility of WBE in predicting increases in clinical cases of SARS-CoV-2, as evidenced by a high correlation (r > 0.5). For RSV-A, the findings aligned with previous studies. Interestingly, particularly considering the length and period of analysis, influenza A, influenza B, and RSV-B viruses were not observed during the study period. In addition, the prevalence of RSV-A decreased during the SARS-CoV-2 pandemic, likely due to the implementation of non-pharmaceutical interventions. In conclusion, this study reaffirms that WBE provides critical epidemiological insights, complements clinical surveillance, and supports public health authorities in making informed and timely decisions.