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Influenza is an infectious disease with a high impact on the population in terms of morbidity and mortality, but despite International and European guidelines, vaccination coverage rates among healthcare workers (HCWs) remain very low. The aim of the present study was to evaluate influenza vaccination adherence in the three Sicilian University Hospitals of Catania, Messina, and Palermo and to understand the attitudes and perceptions of vaccinated healthcare workers and the main reasons for vaccination refusal. A cross-sectional survey th