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Background: This paper describes the third large outbreak of Norovirus (NoV) gastroenteritis reported in the Southern Italy region of Puglia. Methods: A matched case control study was conducted, on 19 July 2005, for investigating risk factors, using a structured questionnaire on food consumption. A multivariate analysis was conducted to estimate the adjusted Odds Ratios. Laboratory and environmental investigation were also performed. Results: On the day of the study 41 cases were identified and 41 controls were enrolled. Controls were matched for age and gender. The mean age of the cases was 26 years old, and 58% were female. The clinical pattern of the disease was characterised by the presence of diarrhoea (95%), vomiting (70%), abdominal pain (51%) and fever (32%). Of the 41 cases included in the study, the majority (65%) were residents of Northern Italian regions. No food samples were available for testing. The matched univariate analysis revealed that cases were more likely to have consumed raw mussels, eggs or ice cubes made of tap water than controls. In the multivariate conditional logistic regression analysis, having eaten raw mussels or ice became more strongly associated with illness. All of the 20 faecal samples collected were tested for NoVs. Eighteen stools (90% of total examined) were positive by RT-PCR, and sequence analysis performed onto 3 samples confirmed the presence of a GGII NoV. No test specific for NoV was performed on water or food samples. Conclusion: The most likely hypothesis supported by the findings of the epidemiological investigation was that illness was associated with raw mussels and ice, made with tap water. These hypothesis could not be confirmed by specific microbiologic testing for NoV in food or ice. The lack of clear knowledge of NoV as a major causative agent of epidemic outbreaks of gastroenteritis in Italy is due to the absence of timely reporting of the cases to the local public health offices and the uncommon practice of saving clinical samples for virological analysis after bacteriological testing. [ABSTRACT FROM AUTHOR]