The authors have declared that no competing interests exist.
Conceived and designed the experiments: MZH JSP. Performed the experiments: MZH WQH FM JCX ZL. Analyzed the data: KNF JSP. Contributed reagents/materials/analysis tools: MZH WQH FM JCX ZL. Wrote the paper: JSP.
Little is known about whether low serum HBsAg levels result from impaired HBsAg synthesis or a reduced number of hepatocytes caused by advanced liver fibrosis. Therefore, we investigated the capacity for HBsAg synthesis in a cross-sectional cohort of treatment-naïve chronic hepatitis B patients.
Chronic hepatitis B patients (n = 362) were enrolled; liver biopsies were performed and liver histology was scored, and serum HBsAg and HBV DNA levels were investigated. In the enrolled patients, 183 out of 362 have quantitative serum HBsAg levels. Tissue HBsAg was determined by immunohistochemistry.
A positive correlation between serum HBsAg and HBV DNA levels was revealed in HBeAg(+) patients (r = 0.2613,
Although serum HBsAg levels were negatively correlated with fibrosis severity in HBeAg(+) patients, aminopyrine breath test-adjusted serum HBsAg and tissue HBsAg, two indices that are unaffected by the number of residual hepatocytes, were positively correlated with fibrosis severity. Furthermore, adjusted serum HBsAg has an accurate prediction capability.
Chronic hepatitis B (CHB) remains one of the most common infectious diseases worldwide, and in Asia and Africa in particular. Interferon α (or pegylated-interferon) and nucleosides (or nucleotide analogues) are prescribed widely for the treatment of CHB. Following effective therapy, a marked decline in hepatitis B virus (HBV) DNA load and/or quantified serum hepatitis B surface antigen (HBsAg) can be observed. There is a positive correlation between HBV DNA level and liver cirrhosis and hepatocellular carcinoma (HCC)
Recently, two studies have reported on the negative correlation between serum HBsAg level and the severity of liver fibrosis
HBsAg is secreted into the circulation as tubular forms or spherical particles by HBV-infected hepatocytes. Patients with CHB usually express different levels of HBsAg when assayed by immunohistochemistry. In patients with advanced fibrosis, residual hepatocytes diminish dramatically, which has a profound effect on serum HBsAg quantification, but not on intrahepatic HBsAg (tissue HBsAg) quantification. Tissue HBsAg levels are determined by the establishing the percentage of HBsAg-positive hepatocytes and the grade of staining reflected by immunohistochemistry. Therefore, the number of hepatocytes has limited effects on tissue HBsAg levels.
Research by Herold
Treatment-naïve CHB patients reporting to the 174th Hospital of the PLA in Fujian, China, between 2010 and 2013 were enrolled. Patients were investigated retrospectively and informed consent was obtained from all the patients. The present study was approved by the Institutional Ethics Board of Chenggong Hospital (the 174th Hospital of the PLA) and Zhongshan Hospital, Xiamen University. All patients provided written consent prior to liver biopsy and study entry. Entire study was conducted according to the principles of the Declaration of Helsinki. Inclusion criteria for patients were: HBsAg-positive, known HBeAg status, and scheduled liver biopsy. Patients were excluded in the following situations: Hepatitis C virus, Hepatitis D virus, or Human Immunodeficiency Virus co-infection, significant steatosis, alcoholic fatty liver, and decompensated cirrhosis. A total of 362 treatment-naïve CHB patients were included in the present study.
Liver biopsies were performed on the same day that serum samples were collected, or in less than 2 days thereafter.
HBsAg levels were assayed using the HBsAg assay kit (Wantai Biological Co., Beijing, China). HBV DNA levels were determined by quantitative fluorescence PCR on an ABI 7000 (Applied Biosystems, Carlsbad, USA), with a lower limit of detection of 500 IU/ml. HBsAg and HBV DNA were expressed as log IU/ml. Serum ALT and AST levels were expressed as IU/ml. Liver histology was assessed using the Scheuer scoring system
Quantitative testing of liver functions in a large sample of fibrosis patients related to chronic hepatitis have been reported by Herold
Statistical analyses were performed using R platform (version 3.0.1,
Among the 362 enrolled patients, 210 (58.0%) were HBeAg(+) and 152 (42.0%) were HBeAg(−) (
(A) grade of inflammatory activity; (B) stage of fibrosis; (C) immunohistochemistry grade of tissue HBsAg; (D) HBV DNA (log10 IU/ml); (E) serum HBsAg (log10 IU/ml).
Patient group | All(N = 362) | HBeAg(+) (n = 210) | HBeAg(−) (n = 152) | |
Age, yr (Mean ± SD) | 35.5±9.9 | 32.7±8.7 | 40.2±9.6 | <0.0001 |
Male, n (%) | 285 (78.7) | 160 (76.2) | 125 (82.2) | 0.1935 |
ALT, IU/L (Mean ± SD) | 144.7±207.0 | 176.6±236.9 | 100.2±145.2 | <0.0001 |
Normal ALT, n (%) | 62 (17.1) | 19 (9.0) | 43 (28.3) | <0.0001 |
AST, IU/L (Mean ± SD) | 77.0±111.9 | 92.6±133.2 | 55.5±67.6 | <0.0001 |
Normal AST, n (%) | 163 (45.0) | 73 (34.8) | 90 (59.2) | <0.0001 |
Activity, n (%) | ||||
G0 | 19 (5.2) | 6 (2.9) | 13 (8.6) | 0.0290 |
G1 | 145 (40.1) | 75 (35.7) | 70 (46.0) | 0.0511 |
G2 | 132 (36.5) | 86 (40.9) | 46 (30.3) | 0.0463 |
G3 | 62 (17.1) | 39 (18.6) | 23 (15.1) | 0.4800 |
G4 | 4 (1.1) | 4 (1.9) | 0 (0) | 0.1423 |
S0 | 53 (14.6) | 21 (10.0) | 32 (21.1) | 0.0041 |
S1 | 152 (42.0) | 99 (47.1) | 53 (34.9) | 0.0234 |
S2 | 84 (23.2) | 51 (24.3) | 33 (21.7) | 0.6148 |
S3 | 54 (15.0) | 33 (15.7) | 21 (13.8) | 0.0083 |
S4 | 19 (5.2) | 6 (2.9) | 13 (8.5) | 0.0290 |
0 | 14 (3.9) | 7 (3.3) | 7 (4.6) | 0.5875 |
1 | 83 (22.9) | 55 (26.2) | 28 (18.4) | 0.0905 |
2 | 134 (37.0) | 73 (34.8) | 61 (40.1) | 0.3217 |
3 | 111 (30.7) | 61 (29.0) | 50 (32.9) | 0.4886 |
4 | 20 (5.5) | 14 (6.7) | 6 (4.0) | 0.3526 |
HBV DNA, log10 IU/ml (Mean ± SD) | 6.01±1.98 | 7.07±1.49 | 4.55±1.63 | <0.0001 |
HBeAg(+) vs. HBeAg(−);
According to the Scheuer scoring system.
The study cohort was analyzed both as a whole and stratified according to HBeAg status.
All patients underwent an HBV DNA assay. However, due to cost, quantitative HBsAg measurement was performed in only 183 of the 362 enrolled patients. Detailed informations of the 183 patients were listed in
Patient group | All (N = 183) | HBeAg(+) (n = 113) | HBeAg(−) (n = 69) | |
Age, yr (Mean ± SD) | 34.7±10.2 | 31.0±8.1 | 40.9±10.3 | <0.0001 |
Male, n (%) | 147 (80.3) | 86 (75.4) | 61 (88.4) | 0.0358 |
ALT, IU/L (Mean ± SD) | 143.2±199.6 | 168.3±220.0 | 101.1±152.0 | <0.0001 |
Normal ALT, n (%) | 30 (16.4) | 10 (8.8) | 20 (29.0) | 0.0007 |
AST, IU/L (Mean ± SD) | 72.8±92.8 | 83.8±104.9 | 54.3±64.4 | <0.0001 |
Normal AST, n (%) | 87 (47.5) | 46 (40.7) | 41 (59.4) | 0.0152 |
Activity, n (%) | ||||
G0 | 10 (5.5) | 4 (3.5) | 6 (8.7) | 0.1824 |
G1 | 75 (41.0) | 45 (39.5) | 30 (43.5) | 0.6444 |
G2 | 67 (36.5) | 47 (41.2) | 20 (29.0) | 0.1130 |
G3 | 27 (14.8) | 14 (12.3) | 13 (18.8) | 0.2837 |
G4 | 4 (2.2) | 4 (3.5) | 0 (0) | 0.2990 |
Fibrosis, n (%) | ||||
S0 | 25 (13.6) | 14 (12.3) | 11 (15.9) | 0.5127 |
S1 | 81 (44.3) | 55 (48.2) | 26 (37.7) | 0.1681 |
S2 | 38 (20.8) | 26 (22.8) | 12 (17.4) | 0.4531 |
S3 | 26 (14.2) | 14 (12.3) | 12 (17.4) | 0.3864 |
S4 | 13 (7.1) | 5 (4.4) | 8 (11.6) | 0.0807 |
0 | 6 (3.3) | 3 (3.3) | 3 (4.6) | 0.6748 |
1 | 40 (21.9) | 27 (26.2) | 13 (18.4) | 0.4654 |
2 | 64 (35.0) | 36 (34.8) | 28 (40.1) | 0.2641 |
3 | 59 (32.2) | 37 (29.0) | 22 (32.9) | 1.0000 |
4 | 14 (7.6) | 11 (6.7) | 3 (4.0) | 0.2553 |
HBsAg, log10 IU/ml (n, Mean ± SD) | 183, 3.80±0.58 | 114, 3.87±0.64 | 69, 3.68±0.44 | 0.2600 |
HBV DNA, log10 IU/ml (Mean ± SD) | 6.05±2.08 | 7.17±1.45 | 4.20±1.58 | <0.0001 |
According to the Scheuer scoring system.
The study cohort was analyzed both as a whole and stratified according to HBeAg status.
In the 183 patients in whom serum HBsAg levels were quantified, there was a positive correlation between serum HBsAg and HBV DNA levels (r = 0.3053,
(A–C) correlation between serum HBsAg (log10 IU/ml) and HBV DNA (log10 IU/ml) in 183 patients with quantified serum HBsAg values, HBeAg(+) patients, and HBeAg(−) patients, respectively. (D–F) correlation between serum HBsAg (log10 IU/ml) and stage of fibrosis in 183 patients with quantified serum HBsAg values, HBeAg(+) patients, and HBeAg(−) patients, respectively.
Severity of liver fibrosis was scored using the Scheuer system
S0 |
S1 | S2 | S3 | S4 | |
G0 | 0.55 | 0.40 | 0.42 | 0.24 | 0.21 |
G1 | 0.52 | 0.38 | 0.40 | 0.22 | 0.19 |
G2 | 0.47 | 0.34 | 0.36 | 0.20 | 0.17 |
G3 | 0.48 | 0.35 | 0.37 | 0.21 | 0.18 |
G4 | 0.43 | 0.31 | 0.33 | 0.19 | 0.16 |
Herold
In all 183 patients, or when stratifying this cohort into HBeAg(+) and HBeAg(−) patients, increasing ABT-adjusted serum HBsAg values were associated with increasing severity of fibrosis (
(A–C) correlation between ABT-adjusted serum HBsAg (log10 IU/ml) and fibrosis severity in 183 patients with quantified serum HBsAg values, HBeAg(+) patients and HBeAg(−) patients, respectively. Stage of fibrosis was determined according to the Scheuer scoring system. (D–F) diagnostic performance (AUC) of aminopyrine breath test -adjusted serum HBsAg in recognizing moderate to severe fibrosis (S3 and S4) in all of 183 patients, HBeAg(+) patients, and HBeAg(−) patients, respectively.
Stages S0–S1 fibrosis were defined as ‘no to mild’, while stages S3–S4 were defined as ‘moderate to severe’ fibrosis. In order to evaluate the diagnostic performance of ABT-adjusted serum HBsAg levels in differentiating patients with S0–S1 fibrosis from patients with S3–S4 fibrosis, ROC curves were generated. In the 183 patients with quantified serum HBsAg levels, the ABT-adjusted serum HBsAg values had a nearly perfect area under the curve (AUC) for differentiating stages S0–S2 from stages S3–S4 (AUC: 0.998, 95% confidence interval [CI]: 0.994–1.000;
When stratifying the enrolled 362 patients according to the severity of fibrosis, there was a trend toward increasing tissue HBsAg levels with increasing fibrosis severity in HBeAg(+) patients (
(A–C) grade of tissue HBsAg in CHB patients stratified according to the stage of fibrosis in all enrolled patients, HBeAg(+) patients, and HBeAg(−) patients, respectively. Stage of fibrosis was determined according to the Scheuer scoring system.
In brief, we preformed this cross-sectional study in a group of 362 unselected CHB patients and attempted to elucidate the relationship between serum HBsAg or tissue HBsAg and severity of fibrosis. In China, due to medical insurance cover, some of the patients accepted qualitative measurement of HBsAg. Thus, quantitative HBsAg levels could be determined in only 183 patients. An investigation performed by Zeng
We observed that HBeAg(−) patients tended to have a higher percentage of normal ALT or AST levels, lower HBV DNA load, with mild necroinflammation, as compared to HBeAg(+) patients. Loss of HBeAg may indicate that the hosts had attained a certain level of immune-control over HBV and these hosts may become ‘inactive carriers’
In the past several years, increasing numbers of studies have been performed to establish the role of serum HBsAg quantification in the prediction of therapy response
In treatment-naïve CHB patients, pre-core promoter mutations may lead to impaired secretion of HBeAg and HBV virions
Similar to previous reports
As for the prediction of severity of fibrosis, ABT-adjusted serum HBsAg values demonstrated a nearly perfect AUC in both HBeAg(+) and HBeAg(−) CHB patients (
We also found that there was a positive correlation between tissue HBsAg and the severity of fibrosis for all of the enrolled patients, and more particularly for HBeAg(+) patients. Using immunohistochemistry, tissue HBsAg was determined from the percentage and the strength of staining in hepatocytes. The number of viable hepatocytes has no effect on the expression of tissue HBsAg; thus, the positive correlation of tissue HBsAg with the severity of fibrosis further confirmed the relationship that we observed between ABT-adjusted serum HBsAg levels and the stage of fibrosis. Therefore, it seemed reasonable to deduce that the capacity for synthesis of HBsAg by individual hepatocyte is enhanced in cases with moderate to severe fibrosis.
Compared with recent studies
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We are much indebted to Prof. Yu-Qing Zheng and Prof. Tian-Hai Ji for reviewing the liver histology. We thank Dr. Rong-Hua Fan, Wei-Bin Wu and Li Zhang for collecting the clinical information.