Rotavirus Quantification and Genotyping in Wastewater: A Molecular Surveillance Study in Italy (2024–2025)

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Rotavirus remains a leading cause of acute gastroenteritis worldwide, particularly in young children, despite widespread vaccination efforts. This study aims to evaluate rotavirus circulation at the population level through wastewater-based epidemiology (WBE), offering a non-invasive, complementary approach to clinical surveillance. Between 2024 and 2025, a total of 172 composite 24 h samples were collected from eight urban wastewater treatment plants across Northern, Central, and Southern Italy. Viral RNA was concentrated by PEG precipitation and quantified using digital RT-PCR, while genotypes were determined via nested PCR targeting VP7 and VP4 genes. Rotavirus RNA was detected in 143 out of 172 samples (83.1%), with viral loads ranging between 4.2 × 102 to 7.3 × 105 genome copies per liter (g.c./L). Genotyping revealed G3 as the predominant VP7 type, followed by G1, G2, G6, and G9. All VP4-positive samples were classified as P8. This investigation expands current knowledge of rotavirus epidemiology in Italy using molecular surveillance of urban wastewater. By combining digital RT-PCR and genotyping, it offers a robust framework for integrating WBE into rotavirus monitoring programs, especially in settings where clinical surveillance is limited.