Comparative Evaluation of Crassphage, Pepper Mild Mottle Virus, and Human Mastadenovirus as Indicators of Human Fecal Contamination in Surface Water

Abstract

This study assessed the presence and concentration of crAssphage, pepper mild mottle virus (PMMoV) and human mastadenovirus (HAdV) as potential indicators of human fecal contamination in different aquatic matrices. A total of 75 stream and seawater samples were collected between 2020 and 2021 across four watersheds displaying varying urbanization degrees in Brazil. Viral particles were concentrated employing the adsorption-elution method followed by ultrafiltration and quantified by qPCR/RT-qPCR. The findings indicate that crAssphage was the most frequently detected (65.33%), followed by PMMoV (45.33%) and HAdV (44.00%). CrAssphage also exhibited the highest viral loads in stream water (median of 6.49 log₁₀ GC L⁻¹), reflecting a strong association with domestic sewage. Comparatively, PMMoV showed similar detection rates in both matrices, whereas HAdV exhibited higher detection rates in stream water. Higher crAssphage and PMMoV concentrations were observed in watersheds located in higher population density areas when compared to lower density areas. No viruses were detected upstream of the urbanized regions, confirming the specificity of the selected viral indicators. In this sense, crAssphage and HAdV are more suitable for monitoring stream water bodies affected by human activities, while PMMoV is suited for both matrices. The findings suggest that the three viruses emerged as promising indicators for tracking human fecal contamination in aquatic matrices with different characteristics.