The misuse and overuse of antibiotics represent a critical global issue and one of the most pressing public health challenges of the 21st century. Training future healthcare professionals effectively is essential for ensuring responsible antibiotic use. This study aimed to validate a questionnaire designed to evaluate the knowledge, attitudes, and perceptions of medical students regarding the education they receive on infectious diseases, antimicrobial resistance, and antibiotic stewardship during their university studies. Methods: A self-administered questionnaire was developed and distributed to medical students at the University of Santiago de Compostela. Comprising 44 items, the questionnaire assessed eight key dimensions: “infection diagnosis”, “criteria for not prescribing antibiotics”, “initial antibiotic therapy”, “re-evaluation of therapy”, “quality of care”, “communication skills”, “antibiotic resistance”, and “teaching methodology”. Validation was carried out in two stages: Phase 1 involved content and face validity, while Phase 2 focused on reliability analysis. Results: A total of 295 students completed the questionnaire, with a mean age of 23.15 ± 1.78 years. The sample included 86 male (29.2%) and 209 female (70.8%) respondents. Content and face validity were established by a nominal group of five experts and a focus group of medicine and pharmacy students to ensure consensus on item understanding in the Spanish language. The questionnaire demonstrated high internal consistency with a Cronbach’s alpha of 0.92 and satisfactory item discrimination. Construct validity was confirmed through principal component analysis, which supported the presence of the eight predefined dimensions. Conclusions: The validated questionnaire exhibited strong reliability and validity, making it a valuable tool for assessing medical students’ training in antibiotic-related topics. Its application will enable the identification of areas for improvement in university curricula, ultimately contributing to the promotion of appropriate antibiotic use and the reduction of antimicrobial resistance.
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