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Despite access to efficient hepatitis B virus (HBV) vaccine and universal immunization schedules, HBV infection remains a global health concern. HBV infection has decreased by this program. Nevertheless, breakthrough infections occur due to generation of occult HBV infection (OBI) and surface gene mutants in the immunized population. We aimed to determine the presence of OBI in a population born after initiation of nationwide HBV vaccination in Tehran, Iran. A HBV mass vaccination schedule was launched in Iran in 1993. For this study, we enrolled 1120 cases younger than 24 years. ELISA was applied to evaluate the presence of HBsAg, anti-HBs and anti-HBc. HBV-DNA presence was determined in all HBsAg-negative cases using nested polymerase chain reaction. The prevalence of HBsAg, anti-HBc and anti-HBs was 0.1, 0.54 and 39.9% respectively. Out of 6 anti-HBc-positive individuals, 4 cases also had anti-HBs. One case revealed HBsAg co-existence and the other one showed isolated anti-HBc. HBV-DNA was not detected in HBsAg-negative specimens. A very low prevalence of HBsAg and isolated anti-HBc was observed and no occult HBV infection was detected. It seems that evasion mutants are not a potential threat for HBV universal immunization efficacy in the vaccinated population.