| BACKGROUND:Chronic infection with hepatitis B virus (HBV) is a significant health problem worldwide and a major cause of morbidity and mortality. China is a high-endemic area, where the majority of hepatitis B virus infections are acquired perinatally or during early childhood. The natural history of perinatal HBV infection is generally divided into three phases according to liver damage and the status of hepatitis B e antigen (HBeAg) and serum HBV-DNA loads:immune tolerant phase, immune clearance phase, and inactive residual phase; hepatitis B re-activation can occur in some patients with inactive disease, which is defined as the fourth phase (re-activation phase). After progression to chronic HBV infection, HBeAg is initially positive in most cases during the immune tolerance phase. Transition to the immune active phase with activation of disease can be abrupt, usually followed by the loss of HBeAg and the development of antibody to hepatitis B e antigen (anti-HBe) (HBeAg seroconversion), sooner or later. In fact, anti-HBe antibody was produced for years and was present in the form of HBeAg-containing immune complexes.The HBeAg status distinguishes two categories of chronic HBV infection. HBeAg positive CHB is accompanied by high-level HBV replication; however, patients with HBeAg-negative chronic HBV infection tend to have progressive liver injury, fluctuating alanine aminotransferase (ALT) activity, and lower levels of HBV DNA.The precore/core gene contains two regions, the precore and core, each containing an initiation (AUG) codon. This gene encodes the core nucleocapsid protein (HBcAg, which is important in viral packaging) and hepatitis B e antigen (HBeAg). The synthesis of HBeAg is doubly controlled at transcription and translation levels. At the transcriptional level, mutations in the basal core promoter (BCP) at nt1762 and nt1764 (resulting in diminished production of HBeAg to 1/5～1/3) were well defined. At the translational level, the mutation at nt1896 caused a stop codon in the precore Open Reading Frame and resulted in the cession of HBeAg expression. High rate of mutations in nt1762/1764 and/or nt1896 were found in HBeAg negative CHB patients, however, these mutations could also be found in HBeAg positive CHB patients and were correlated with serum HBeAg titers.The HBV serum markers include the surface antigen (HBsAg), the e antigen, the surface antibody (anti-HBs), the e antibody and the core antibody. The mode of these markers' combination is associated with different clinical status, which is helpful for the diagnosis of CHB and antiviral treatment evaluation. HBsAg, HBeAg and anti-HBc positive mode or HBsAg, anti-HBe and anti-HBc positive mode is common in clinical practice. However, a special serological pattern of chronic hepatitis B with concurrent HBeAg and anti-HBe was sometimes seen in clinical practices. A few concurrent patients had been observed in several studies previously, but not been studied in detail to date. Little is known about the clinical and virological features and the mechanisms elucidating this unique serological pattern of HBV e system.AIMS:The aims of this study were to determine the prevalence, analyze the clinical and virological characteristics of patients with and explore the mechanism for the concurrent detection of HBeAg and anti-HBe. METHODS:1, Ranged from January 2002 to July 2009, a Chinese cohort of consecutive inpatients from the Hepatology Unit, Nanfang Hospital was studied. All patients fulfilled the following criteria:1) Diagnosed with chronic HBV infection related liver disease, compensated (Child-Pugh class A) or decompensated cirrhosis (Child-Pugh class B or C); 2) Aged not less than 14 years old; 3) Antiviral treatment-naive prior to admission, and 4) Persistent or intermittent elevation of alanine aminotransferase (ALT) serum levels within 12 months before entry. The exclusion criteria were: coinfection with other hepatitis viruses, human immunodeficiency virus or other causes of hepatitis (autoimmune hepatitis, alcoholic liver disease, etc.). Patients with hepatocellular carcinoma or HBV reactivation by chemotherapy or identified immune suppression therapy were also excluded.2, The patients included were grouped according to serum HBeAg and anti-HBe status. The difference of demography, clinical features, pathology, serology and virology among groups were analyzed.3, The HBeAg or anti-HBe mono-positive patients were stratified into three subgroups according to the HBeAg or anti-HBe titers and features of the three subgroups were analyzed, respectively.4, For the recovered 135 sera samples from concurrent patients, viral genotype, basic core promoter and precore mutations were determined by direct sequencing.5, To explore the possible mechanisms for concurrence of HBeAg and anti-HBe, in vitro simulation of concurrent HBeAg/anti-HBe was performed with three pairs of HBeAg-positive and anti-HBe-positive serum samples, which were randomly selected and paired off. Before analyzing using the Abbott i2000 instrument, sera were mixed at variable ratios between paired samples, vortexed for 10 seconds and incubated at 37℃for 3 hours. The first parameter examined was the optimal ratio between each pair of sera, at which HBeAg and anti-HBe could be concurrently observed. Briefly, sera in each pair were mixed with equal volume to determine whether antigen or antibody was in excess. Then a second mixture was made by decreasing the volume of the serum with the excess marker. More mixed samples were prepared according to the results already obtained until HBeAg and anti-HBe were concurrently observed. The second issue was to test whether concurrence was correlated with the initial concentration of HBeAg and anti-HBe. Mixtures with the optimal ratio were prepared without dilution or with a dilution factor from 10 to 500 before incubation. The third issue was to detrmine the effect of extending the incubation time on concurrent detection. The mixtures with the optimal ratio for each pair were incubated for12 or 24 hours at 37℃.RESULTS:1, A total of 1624 chronic hepatitis B patients fulfilled the study criteria, of which 12 HBeAg/anti-HBe dual negative patients were not further analyzed. The prevalence of patients with concurrent detection of both markers was 10.4%.2, For the age and HBV DNA level, there were significant difference among the three groups (age:F=90.180, P<0.001; HBV DNA:F=217.282, P<0.001). The mean age of the concurrent patients (n=169,35.2±11.0 y) fell in between the HBeAg-positive (n=887,32.0±11.3 y) and anti-HBe-positive (n=556,41.0±13.0 y) groups. The mean HBV-DNA levels (5.8±1.5 log10 copies/mL) of the concurrent group also fell in between those in the HBeAg-positive (6.6±1.2 log) and anti-HBe-positive (5.2±1.2 log) groups. Concurrent patients were associated with the highest ALT levels [166 (65-460) IU/L] compared with HBeAg-positive [144 (65-314) IU/L] and anti-HBe-positive [104 (51-299) IU/L] group (χ2= 12.656, P= 0.002). Percentages of patients with total bilirubin levels over 34.2μmol/L, which is taken as a sign of liver dysfunction, were significantly higher in the concurrent group (47.4%) and anti-HBe-positive group (46.5%) than that in HBeAg-positive patients (25.5%). Thirty five (20.7%) concurrent patients were diagnosed as decompensated cirrhosis, which were comparable to anti-HBe-positive group and were higher than that of HBeAg-positive group. Liver histology evaluations showed that inflammation grades and fibrosis stages were higher in concurrent than in HBeAg-positive patients, but both were comparable to anti-HBe-positive group.3, The median HBeAg titer in the concurrent group was lower than that of the HBeAg-positive group (Z=-33.934, P<0.001). Similarly, the anti-HBe concentration was lower than that of the anti-HBe-positive group (Z=-32.039, P< 0.001). HBeAg and anti-HBe titers (median and interquartile range) in concurrent group were 4.2 (1.8-9.6) S/CO and 0.54 (0.27-0.72) S/CO, most of the HBeAg and anti-HBe titers in the concurrent group were much close to their respective cutoff values than those of the HBeAg-positive or anti-HBe-positive alone patients, respectively.4, The HBeAg mono-positive patients were stratified into three subgroups according to the HBeAg titers (The low titer subgroup,1.0 S/CO< HBeAg≤130.9 S/CO; The medium titer subgroup,130.9< HBeAg≤293.7 S/CO; the high titer subgroup, HBeAg> 293.7 S/CO). The low titer subgroup were associated with the oldest age (F=18.604, P< 0.001), the lowest serum HBV DNA level (F=64.725, P<0.001) and the most profound liver damage. The serum ALT level was the lowest among the three subgroups(χ2=9.962, P=0.007).5, The serum ALT level of concurrent patients was higher than the low HBeAg titer subgroup (Z=-2.663, P=0.008). However, the other features (such as age, HBV DNA, etal.) were comparable between the two groups.6, The anti-HBe mono-positive patients were also stratified into three subgroups according to the anti-HBe titers (The low titer subgroup,0.105 S/CO< anti-HBe< 1.00 S/CO; The medium titer subgroup,0.064 S/CO< anti-HBe≤0.105 S/CO; the high titer subgroup, anti-HBe≤0.064 S/CO). The serum TBil level was highest in low anti-HBe titer subgroup and was lowest in high anti-HBe titer subgroup (χ2= 8.037, P=0.018). However, there was no other difference of demography, the clinical features, the pathology among the three anti-HBe subgroups.7, The HBV core promoter and precore region was successfully amplified and sequenced in 134 cases. For the cases successfully sequenced,110/134 (82.1%) harbored T1762/A1764 or/and A1896 mutants. Of the 134 individuals,95 (70.9%) carried genotype B and 39 (29.1%)C.8, The in vitro simulation showed that HBeAg and anti-HBe could be concurrently observed provided an optimal ratio (HBeAg to anti-HBe) was chosen. Once an optimal ratio was chosen, diluted (dilution factors were 10-500) samples were prepared and concurrent detection was observed at a certain concentration of HBeAg and anti-HBe, as well as dual negative status with excessive dilution. When incubated at 37℃for 12 or 24 hours, HBeAg and anti-HBe could still be concurrently observed.CONCLUSIONS:In antiviral treatment-naive patients, concurrence of HBeAg and anti-HBe was not uncommon, and such patients had profound liver disease. HBeAg mono-positive chronic hepatitis B patients were of heterogeneous patients with different HBeAg titer, there were clinical difference among the three subgroups. The HBeAg/anti-HBe double positive group and the low titer HBeAg-positive subgroup may be in the similar stage during the infection history. An optimal ratio between HBeAg and anti-HBe led to their concurrent detection when sera were tested by sensitive assays.
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