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Background: The development of a vaccine against hepatitis B virus (HBV) has been a major achievement in terms of prevention of HBV infection. For the present study, we analysed the long-term immunogenicity and effectiveness of HBV vaccination among healthcare students with different working seniorities. Methods: A cross-sectional study of undergraduate and postgraduate students attending the Medical School of the Second University of Naples was conducted between September 2012 and December 2014. HBV serum markers were determined and multivariate logistic regression analysis was used to identify factors associated with the level of long-term immunogenicity. Results: Of the 2,932 subjects evaluated, only 33 (1.1 %) declared no history of vaccination. All vaccinated subjects were HBsAg/anti-HBc negative, 459 of which had an anti-HBs titre <10 IU/L The latter were younger, more likely to be attending a healthcare profession school (i.e., dental hygienists, nursing, paediatric nursing, radiography and midwifery) than a medical school (at either undergraduate or postgraduate level) and more likely to have been vaccinated in infancy. Conclusion: The results of this study suggest that assessment of HBV serum markers in workers potentially exposed to hospital infections is useful to identify small numbers of unvaccinated subjects or vaccinated subjects with low antibody titre, all of whom should be referred to a booster series of vaccinations. [ABSTRACT FROM AUTHOR]