Assessing normalization methods in wastewater based epidemiology: a systematic review

Abstract

Wastewater-based epidemiology (WBE) has emerged as a powerful tool for monitoring community level infectious disease trends. However, there is a lack of consistent normalization and control practices across studies that limits data interpretability and comparability. In this review, we analyzed 247 articles to assess current normalization approaches, focusing on the use of biomarkers and their associated normalization goals. We identified substantial variability in marker selection, application, and reporting, often without clear explanation. Endogenous markers such as pepper mild mottle virus (PMMoV) and CrAssphage were frequently used, primarily to adjust fecal strength, while exogenous controls such as bovine coronavirus (BCoV) and MS2 bacteriophage were commonly applied to evaluate sample recovery efficiency. However, there is no clear distinction between these functions in the literature. Additionally, normalization goals were often unspecified; when reported, they generally were for adjusting flow variability, correlating with clinical data, and accounting for population size. Mapping control types to normalization goals revealed a diverse range of applications but highlighted a lack of standardization. We emphasize the importance of standardized reporting guidelines, including clear documentation of normalization strategies and quality assurance measures, to improve reproducibility, facilitate meta-analyses, and strengthen the role of WBE in public health surveillance.