AbstractBackground and AimsThe prevalence of HDV infection in HBsAg carriers is about 9.9% in Italy. However, the real prevalence is underestimated because the anti‐HDV test is not performed routinely in all HBsAg carriers. The aim of this study was to compare the prevalence and the absolute number of HDV infection identified in HBsAg‐positive subjects tested at University Hospital Federico II before and after the introduction of anti‐HDV reflex testing.MethodsFrom January to December 2022, reflex test for the detection of total HDV antibodies was performed in all HBsAg‐positive subjects tested at University Hospital Federico II. The control group consisted of all the HBsAg‐positive subjects tested at the same laboratory in 2019, before the implementation of anti‐HDV reflex testing. Sera were evaluated with ADVIA Centaur HBsAgII Qualitative, Liaison Murex HBsAg Quantitative and Liaison Murex Total Anti‐HDV Qualitative.ResultsBefore reflex testing, anti‐HDV had been tested in 16.4% (84/512) of HBsAg‐positive subjects, while after its implementation, 100% (484/484) of HBsAg‐positive patients was tested for anti‐HDV. The anti‐HDV positive prevalence was lower than before the introduction of reflex test (10.7% vs. 16.6%) but the absolute number of anti‐HDV positive patients increased (14 vs. 52 subjects). HDV‐RNA was detectable in 26 (53%) of 49 tested subjects.ConclusionsOur data showed that the implementation of anti‐HDV reflex testing increased the diagnoses of HDV infection. In this setting, due to the approval of specific anti‐HDV drugs, a reflex test for anti‐HDV should be implemented to early identify patients with HBV/HDV infection.