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This study described 17 cases of children admitted to the Bambino Gesù Children's Hospital with acute hepatitis of unknown origin between mid-April and November 2022. Following the World Health Organization's working case definition of probable cases, 17 children, with a median age of 2.1 years (interquartile range: 1.0–7.1), presenting with acute hepatitis non-AE, with serum transaminase >500 IU/L, were included in the study. A pre-specified set of microbiological tests was performed on different biological specimens for all pediatric patients. All patients resulted negative for the common hepatotropic viruses. The most common pathogen detected in blood specimens was human-herpes-virus-7 (52.9%). Adenovirus was detected more frequently in stool specimens (62.5%) than in respiratory (20.0%) or blood samples (17.6%). Regarding Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, one child tested positive two days after admission, while antibodies against spike and nucleoprotein were present in 82.3% of patients. A co-pathogen detection was observed in 94.1% of children. Overall, 16 children recovered without clinical complications, while one patient required liver transplantation. In these cases of acute hepatitis of unknown origin, adenovirus was mainly detected in stool samples. A co-pathogen detection was also frequently observed, suggesting that the etiology of this acute hepatitis is most probably multifactorial. [ABSTRACT FROM AUTHOR]