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Background: Hand hygiene is a cornerstone of infection prevention and control and plays a critical role in preventing healthcare-associated infections. During the COVID-19 pandemic, hand hygiene awareness and adherence increased across healthcare systems, but evidence on whether these improvements were sustained as pandemic pressures subsided remains limited. Nursing students and registered nurses are key groups for understanding long-term behavioural changes, as their knowledge, perceptions, and workplace experiences influence patient safety practices.

Objective: To compare nursing students' and registered nurses' knowledge and perceptions of hand hygiene at two time points: during the peak of the COVID-19 pandemic in 2020 and in the post-pandemic period in 2023.

Design: Comparative cross-sectional survey study conducted at two time points (2020 and 2023).

Setting: A Swedish university and its affiliated university hospital. Participants: A total of 376 individuals: first-semester nursing students, final-semester nursing students, and registered nurses surveyed in 2020 (n = 201) and 2023 (n = 175).

Methods: This study employed a two-time point, independent-sample design. Data were collected online using two validated World Health Organization instruments: the Hand Hygiene Knowledge Questionnaire for Health Care Workers and the Perception Survey for Health Care Workers. Descriptive statistics were computed for all variables. Comparisons between 2020 and 2023 were performed using the Mann-Whitney U test for ordinal variables and Fisher's exact test for categorical variables.

Results: Total knowledge scores did not differ significantly between time points. However, first-semester students had lower knowledge of transmission routes and preventive actions in 2023. Perceptions of hand hygiene differed between time points. The mean score for perceived importance of hand hygiene as a patient safety measure was significantly lower in 2023 than in 2020 for first-semester students (3.77 vs 3.30, p < 0.001), final-semester students (3.61 vs 2.89, p < 0.001), and registered nurses (3.39 vs 3.19, p = 0.041). Perceived patient expectations about hand hygiene were also lower among nursing students, and registered nurses rated perceived hand hygiene adherence among healthcare workers lower in 2023. By contrast, first-semester students reported higher ratings of educational support and tolerance of alcohol-based hand rubs in 2023.

Conclusions: The findings indicate group-level differences in hand hygiene perceptions between the pandemic-period and post-pandemic samples, while total knowledge scores did not differ significantly within any participant group. Given the independent-sample design, these findings should not be interpreted as individual-level changes over time. Lower perceived importance of hand hygiene suggests a need for continued educational reinforcement and organisational support to maintain hand hygiene practices.