Introduction: Healthcare workers are frequently exposed to infectious agents in their work environment; therefore, proper use of personal protective equipment and adherence to hand hygiene are critical measures in preventing healthcare-associated infections. Despite the existence of guidelines and training programs, consistent implementation of hand hygiene and both standard and isolation precautions in clinical settings remains a persistent challenge. Purpose: The purpose of this study was to assess the objective and subjective knowledge of healthcare workers regarding the use of personal protective equipment and hand hygiene, and to evaluate alignment of their knowledge with the official guidelines. Methods: A quantitative cross-sectional study was conducted using a structured questionnaire. The survey included 300 healthcare workers from various occupational profiles, employed at the Division of Surgery and the Institute of Medical Rehabilitation at the University Medical Centre Ljubljana. Data were analysed using descriptive statistics, the Kruskal-Wallis's test, and Spearman’s correlation coefficient. Results: The average objective knowledge score regarding the use of personal protective equipment and hand hygiene was 74.7 %. Nurses achieved the highest scores, followed by resident physicians, physiotherapists, and occupational therapists. Specialist physicians and healthcare assistants scored somewhat lower. More years of service were associated with higher levels of objective knowledge. However, respondents who rated the importance of correct and consistent implementation of standard and isolation precautions more highly did not demonstrate better objective knowledge. Similarly, higher self-assessed knowledge was not correlated with better objective knowledge of personal protective equipment use and hand hygiene. Discussion and Conclusion: Although the overall knowledge among participating healthcare workers was good, differences were observed between professional profiles. Regular, targeted education and training for all healthcare worker profiles, as well as strengthening a culture of safety within healthcare institutions, are essential to improving adherence to protective measures and reducing the incidence of healthcare-associated infections.
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