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The concurrent detection of hepatitis B e antigen (HBeAg) and its corresponding antibody (anti-HBe) in patients with chronic hepatitis B virus (HBV) infection is well established but the clinical features remain poorly understood. Demographic information, clinical and laboratory data were collected from 1624 consecutive inpatient records of patients with chronic hepatitis B. Viral genotype, basic core promoter and precore mutations were determined by direct sequencing. In vitro HBeAg and anti-HBe binding experiments were conducted with three pairs of HBeAg-positive and anti-HBe-positive serum samples, which were mixed at variable ratios and incubated at 37°C for 3-24h. Of the 1624 chronic patients, 169 (10.4%) had concurrent HBeAg and anti-HBe positivity, and this was associated with intermediate age and HBV-DNA load, higher alanine aminotransferase level and more pronounced liver damage compared with HBeAg-positive or anti-HBe-positive patients alone. HBeAg and anti-HBe titres (median and interquartile range, S/CO) in the concurrent positive group were 4.2 (1.8-9.6) and 0.54 (0.27-0.72), which were closer to their respective cut-off values than those of HBeAg-positive or anti-HBe-positive groups alone. For the cases successfully sequenced, 110/134 (82.1%) harboured T1762/A1764 or/and A1896 mutants. The binding experiments showed that HBeAg and anti-HBe could be concurrently observed provided an optimal ratio (HBeAg to anti-HBe) was chosen. In antiviral treatment-naive patients, concurrence of HBeAg and anti-HBe was not uncommon, and such patients had profound liver disease. An optimal ratio between HBeAg and anti-HBe led to their concurrent detection when sera were tested by sensitive assays.