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doi:10.1111/jgh.12502
H E PAT O L O G Y
Hepatitis B surface antigen level complements viral load in predicting viral reactivation in spontaneous HBeAg seroconverters Tai-Chung Tseng,*,†† Chun-Jen Liu,†,‡ Wan-Ting Yang,§ Chi-Ling Chen,‡ Hung-Chih Yang,†,** Tung-Hung Su,†,‡ Chia-Chi Wang,*,†† Stephanie Fang-Tzu Kuo,‡‡‡ Chen-Hua Liu,†,‡ Pei-Jer Chen,†,‡ Ding-Shinn Chen†,‡ and Jia-Horng Kao†,‡,§,¶ *Division of Gastroenterology, Department of Internal Medicine, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, †Division of Gastroenterology, Department of Internal Medicine, ‡Graduate Institute of Clinical Medicine, §Hepatitis Research Center, ¶Department of Medical Research and **Microbiology, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, and ††School of Medicine, Tzu Chi University, Hualien, Taiwan; and ‡‡‡St Vincent’s Hospital, Melbourne, Victoria, Australia
Key words chronic hepatitis B, HbeAg, HbsAg, liver disease, SEARCH-B, viral replication. Accepted for publication 22 December 2013. Correspondence Professor Jia-Horng Kao, Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, 1 Chang-Te St., Taipei 10002, Taiwan. Email: [email protected] Grant/funding support: This work was supported by grants from the Buddhist Tzu-Chi General Hospital Taipei Branch (TCRD-TPE-101-12 and TCRD-TPE-NSC102-02), the National Taiwan University Hospital (NTUH100-S1534), the Department of Heath (DOH99-DC-1001 and DOH100DC-1019), and the National Science Council, Executive Yuan, Taiwan (NSC100-2314-B303-012, and NSC101-2314-B-303-006).
Abstract Background and Aims: The level of hepatitis B surface antigen (HBsAg) has been shown to complement hepatitis B virus (HBV)-DNA level in predicting disease progression in hepatitis B e antigen (HBeAg)-negative patients, especially those with low viral loads. Whether this finding could be seen in spontaneous HBeAg seroconverters remains unclear. Methods: A retrospective cohort of 390 Taiwanese spontaneous HBeAg seroconverters with a mean follow-up period of 7.4 years was enrolled. The relationships between HBV-DNA/HBsAg levels and HBeAg-negative hepatitis/active viral replication (HBVDNA level ≥ 2000 IU/mL) were investigated. Results: In the overall cohort, serum HBV-DNA level served as a better predictor for HBeAg-negative hepatitis compared with HBsAg level. However, in those with HBVDNA level < 2000 IU/mL, a higher HBsAg level was associated with a higher risk of HBeAg-negative hepatitis (P = 0.015). Multivariate analysis showed the hazard ratio of HBsAg level ≥ 1000 IU/mL versus < 1000 IU/mL was 4.1 (95% confidence interval: 1.3–13.6). When using the end-point of active viral replication, HBsAg ≥ 1000 IU/mL remained as an independent risk factor, with a hazard ratio of 2.5 (95% confidence interval: 1.1–5.9). Conclusions: In spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/ mL, HBsAg level ≥ 1000 IU/mL is associated with increased risks of HBeAg-negative hepatitis and active viral replication. Combining HBV-DNA < 2000 IU/mL and HBsAg level < 1000 IU/mL may be used to define minimal viral activity.
Introduction Hepatitis B virus (HBV) infection is a global health threat, resulting in over one million deaths per year. Patients with chronic HBV infection are at increased risk of developing liver cirrhosis, hepatic decompensation, and hepatocellular carcinoma (HCC).1–3 It is estimated that 25–40% of HBV carriers who are infected with the virus early in life will eventually develop these disastrous complications.3 It is known that hepatitis B e antigen (HBeAg) seroconversion is an important event in the natural history of chronic HBV infection as it usually indicates a remission of liver disease and confers a favorable clinical outcome.3,4 Nevertheless, 20–40% of HBeAgnegative patients do not achieve sustained viral suppression and 1242
develop HBeAg-negative hepatitis, the precursor event of cirrhosis and HCC.5–9 Currently, HBV-DNA and hepatitis B surface antigen (HBsAg) levels are two serum biomarkers able to be quantified to predict HCC development.10 The elevation of HBV-DNA level has been shown to be associated with increased HCC risk in adult HBV carriers, and the risk starts to increase when HBV-DNA level is higher than 2000 IU/mL.11 The recent cohort study, ERADICATE-B (Elucidation of Risk fActors for DIsease Control or Advancement in Taiwanese hEpatitis B carriers) study, further indicated that HBV-DNA level is a better predictor than HBsAg level for 10-year and 15-year HCC development.12 However, when focusing on the HBeAg-negative patients with low viral loads (HBV-DNA level < 2000 IU/mL), HBsAg level ≥ 1000 IU/mL, Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249
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but not HBV-DNA level, is associated with a higher risk of HCC.12 A subsequent study showed that HBsAg level ≥ 1000 IU/mL is also associated with increased risks of HBeAg-negative hepatitis, hepatitis flare, and cirrhosis.13 Whether these reported findings could be reproduced in other independent cohorts, such as spontaneous HBeAg seroconverters, remains unclear. In this study, we investigated the SEARCH-B (Study of E Antigen seRoClearance of Hepatitis B) cohort, which is a hospital-based cohort that enrolled 390 spontaneous HBeAg seroconverters,9,14 to address this interesting and important issue. Although HCC is a solid and ideal end-point to evaluate the adverse outcome of HBV carriers, it is not suitable for a small cohort because of its low incidence rate. We, thus, used HBeAgnegative hepatitis as our end-point and addressed the following issues specifically. First, although HBV-DNA and HBsAg levels at 1-year post-HBeAg seroconversion have been shown to be associated with HBeAg-negative hepatitis,9,14 it is still unclear which biomarker is the better predictor. Second, HBsAg level has been shown to be associated with disease progression in HBeAgnegative patients with HBV-DNA level < 2000 IU/mL, but most of them were in the middle or late stage of HBeAg-negative phase.13 In this study, we intended to investigate whether such relationship still holds true in an independent cohort with a different clinical setting, of which patients are in the very early stage of HBeAgnegative phase. Following the second aim, we analyzed whether there is a consistent relationship between HBsAg level and active viral replication, which is regarded as a fundamental event of all HBV-related complications.
Methods Patient cohort. Figure 1 shows the inclusion and exclusion criteria of patients in the SEARCH-B cohort study. In brief, a total of 1278 HBeAg-positive chronic hepatitis B patients above 16 years of age who received regular follow-up for more than 3 years at the National Taiwan University Hospital were consecutively enrolled from 1985 to 2004. Among them, 490 patients experienced spontaneous HBeAg seroconversion. Since all the viral factors were determined at 1-year post-HBeAg seroconversion, we excluded patients who developed HBeAg-negative hepatitis before that time point. We also excluded those who developed liver cirrhosis or HCC before HBeAg seroconversion because antiviral therapy was warranted for them.15–17 Thus, a total of 390 sustained spontaneous HBeAg seroconverters were enrolled, 239 of them had HBV-DNA levels < 2000 IU/mL at 1-year postHBeAg seroconversion and were included for the subgroup analysis. The participants of this study did not overlap with another large cohort study, ERADICATE-B,12,13 because most of them at study entry were younger than 28 years, which is different from the enrollment criteria for the ERADICATE-B study. Informed consents were obtained from all participants as per the ethical committee.
Data collection. During the follow-up period, patients were tested for serological markers (HBsAg, HBeAg, anti-HBe, antibodies against hepatitis C virus [anti-HCV], and antibodies against hepatitis D virus [anti-HDV]) at baseline. Liver function tests and
Figure 1 Flow of SEARCH-B study. *Patients received treatment within 1 year before HBeAg seroconversion, including lamivudine, adefovir dipivoxil, standard interferon, and peginterferon alpha-2a. HBV, hepatitis B virus; HCC, hepatocellular carcinoma.
alpha-fetoprotein levels were assayed every 6 months if the alanine aminotransferase (ALT) levels were within normal limits, and at least every 3 months if ALT levels became elevated. HBeAg and anti-HBe were assayed at least every 6 months until the first year after HBeAg seroconversion, and then upon the development of hepatitis. Serum samples collected at each visit were stored at −20°C until tested. High-resolution abdominal ultrasonography was performed regularly every 3–6 months from enrollment for the surveillance of liver cirrhosis and HCC.
Definitions of HBeAg-negative hepatitis and active viral replication. HBeAg seroconversion was defined as persistent HBeAg negativity and anti-HBe positivity for at least 1 year. We defined HBeAg-negative hepatitis and active viral replication to be the first and second end-points, respectively. HBeAg-negative hepatitis was defined as ALT elevation more than twice the upper limit of normal, with a concomitant serum HBVDNA level ≥ 2000 IU/mL.9,13–15 The upper limit of normal for serum ALT level is 40 U/L. Apart from the aforementioned criteria, exclusion of drugs or alcohol usage, and the absence of serological evidence suggestive of other viral hepatitis infection or autoimmune liver disease, was essential to ensure correct diagnosis. Active viral replication was defined as an upsurge of
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HBV-DNA level from < 2000 IU/mL to ≥ 2000 IU/mL.15 To investigate this end-point, we retrospectively quantified HBV-DNA levels from the stored serum samples annually to determine the first time when HBV-DNA level ≥ 2000 IU/mL, which was defined as the time point of active viral replication.
Table 1 Baseline characteristics of the overall cohort and the subcohort with low viral loads
Serological assays. Serum HBsAg, HBeAg, anti-HBe, antiHCV, and anti-HDV were tested using commercially available kits (Abbott Laboratories, Abbott Park, IL, USA).
Gender Female Male Age at HBeAg seroconversion (years) < 30 30–39 ≥ 40 Serum ALT (U/L) < 40 40–79 Serum HBV-DNA level (IU/mL) < 200 200–1999 2000–19 999 20 000–199 999 ≥ 200 000 HBV genotype B C B+C Serum HBsAg level (IU/mL) < 100 100–999 ≥ 1000
Extraction of HBV-DNA. Viral DNA in the serum was extracted using commercial kits (QIAamp DNA Blood and Tissue Mini Kit; QIAGEN Inc., Valencia, CA, USA). The extracted DNA was used for HBV-DNA quantification and HBV genotype determination. Quantification of serum HBV-DNA and HBV genotyping. Serum HBV-DNA levels and HBV genotype were determined using real-time polymerase chain reaction (PCR)-based single-tube assay as previously described.18 In short, this method consists of two consecutive steps. The first step uses real-time PCR to quantify HBV-DNA, and the second step uses melting curve analysis to genotype HBV. The dynamic range of HBV-DNA levels under this method is from 20 to 2 × 1010 IU/mL (100 to 1011 copies/mL). Since HBV-DNA level quantification with real-time PCR was not available until 2004, most HBV-DNA levels were determined retrospectively using stored sera. In addition, for patients who had undetectable HBV-DNA levels at HBeAg-negative phase, we determined HBV genotype either by IMMUNIS HBV Genotype EIA kit (Institute of Immunology Co. Ltd, Tokyo, Japan), which is an ELISA assay and detects genotype-specific epitopes in preS2,19,20 or using serum samples during their HBeAg-positive stage. Quantification of HBsAg levels. HBsAg levels were quantified using the Architect HBsAg QT (Abbott Laboratories) according to the manufacturer’s instructions.21,22 The detection range of Architect assay is from 0.05 to 250 IU/mL. If the HBsAg level was found to be higher than 250 IU/mL, the samples were diluted to 1:100 to 1:1000 to obtain a reading within the calibration curve range. Statistical analysis. Mean and standard deviation (SD) were calculated for continuous variables, and percentages were used for categorical variables. We calculated person-years from 1-year post-HBeAg seroconversion because that is when the viral factors were determined. The person-years were censored on the date of diagnosing the aforementioned end-points, the date of initiating antiviral therapy, the last date of follow-up, or June 30, 2012, whichever came first. For patients with multiple episodes of HBeAg-negative hepatitis or active viral replication, each endpoint was counted once. The cumulative incidence of the designated end-points was derived using the Kaplan–Meier method, and the log–rank test was used for statistical significance. To compare the predicting ability of HBV-DNA level and HBsAg level for HBeAg-negative hepatitis in the overall cohort,
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No. (%) of patients Overall Subjects with cohort HBV-DNA < 2000 IU/mL 126 (32.3) 264 (67.7)
82 (34.3) 157 (65.7)
243 (62.3) 95 (24.4) 52 (13.3)
162 (67.8) 52 (21.8) 25 (10.5)
331 (84.9) 59 (15.1)
222 (92.9) 17 (7.1)
139 (35.6) 100 (25.6) 63 (16.2) 30 (7.7) 58 (14.9)
139 (58.2) 100 (41.8)
316 (81.0) 73 (18.7) 1 (0.3)
203 (84.9) 36 (15.1)
18 (4.6) 34 (8.7) 338 (86.7)
16 (6.7) 24 (10.0) 199 (83.3)
ALT, alanine aminotransferase; HBV, hepatitis B virus.
we restricted the study population to patients who were followed up for at least 3 years and 6 years. Receiver operating characteristic (ROC) curve analysis was used to calculate the area under ROC curve (AUROC), which was used to compare the predictive performance of HBV-DNA level and HBsAg level for 3-year and 6-year risk of HBeAg-negative hepatitis. Cox proportional hazards regression model was used to calculate the crude and multivariate-adjusted hazard ratios (HR) of each end-point. Age, sex, HBV genotype, and serum levels of HBsAg and ALT were included as adjusting variables in analyzing the prognosis of patients with low viral load (≤ 2000 IU/mL).12–14,23 HBsAg levels were categorized into < 1000 IU/mL and ≥ 1000 IU/mL according to previous report.12,13 Statistical significance of all tests was defined as P < 0.05 by two-tailed tests. All analyses were performed using the Stata statistical software (version 9.2; Stata Corp, College Station, TX, USA).
Results Baseline characteristics. Table 1 shows the demographic data of 390 spontaneous HBeAg seroconverters and 239 spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL. Most patients were males with genotype B Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249
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infection. In the subcohort with low viral loads, 40 patients (16.7%) had HBsAg levels < 1000 IU/mL at 1-year post-HBeAg seroconversion. Follow-up results by different end-points. The follow-up results of the overall cohort and the subcohort with low viral loads are summarized in Table S1. In the overall cohort, 142 patients developed HBeAg-negative hepatitis within 2901.2 person-years of follow-up, with an average annual rate of 4.9%. In the subcohort with low viral loads, 58 patients developed HBeAgnegative hepatitis within 1977.9 person-years of follow-up, with an average annual rate of 2.9%, and 76 developed active viral replication in the 1794.9 person-years of follow-up, with an average annual rate of 4.2%.
HBsAg levels predict viral reactivation
we investigated whether an HBsAg level ≥ 1000 IU/mL was associated with a higher likelihood of subsequent active viral replication in patients with low viral loads at study entry. The Kaplan– Meier curve analysis showed that HBsAg level ≥ 1000 IU/mL was also associated with higher cumulative incidence of active viral replication (P = 0.032, Fig. 2b). Univariate analysis indicated that male sex, elevated ALT level, genotype C infection, and HBsAg level ≥ 1000 IU/mL were associated with higher risks of active viral replication (Table 3). Multivariate analysis found that, in addition to male sex, elevated ALT level, and genotype C infection, HBsAg ≥ 1000 IU/mL was an independent risk factor for active viral replication, with an HR of 2.5 (95% CI: 1.1–5.9) (Table 3).
Discussion The value of HBV-DNA and HBsAg in predicting 3-year and 6-year risk of HBeAg-negative hepatitis. Both HBV-DNA level (Fig. S1a, P for trend < 0.001) and HBsAg level (Fig. S1b, P for trend = 0.003) were positively associated with HBeAg-negative hepatitis in a dose response manner, which has been shown in our previous reports.9,14 The risk of HBeAg-negative hepatitis started to increase when HBV-DNA level ≥ 2000 IU/mL. We, thus, compared the predictive values of HBV-DNA level and HBsAg level for 3-year and 6-year risk of HBeAg-negative hepatitis using the ROC curve analysis. The results showed that HBV-DNA level, as compared with HBsAg level, served as a better predictor for both 3-year risk of HBeAgnegative hepatitis (AUROC [95% CI]: 0.77 [0.70–0.83] vs 0.61 [0.54–0.69], P ≤ 0.001) and 6-year risk of HBeAg-negative hepatitis (AUROC [95% CI]: 0.74 [0.67–0.80] vs 0.58 [0.54–0.68], P = 0.005) (The P-value here refers to the comparison of AUROC between HBV-DNA and HBsAg levels). Factors associated with HBeAg-negative hepatitis in patients with low viral loads. We explored whether HBsAg could serve as a predictor in patients with HBV-DNA level < 2000 IU/mL since the risk of HBeAg-negative hepatitis starts to increase when viral load ≥ 2000 IU/mL. The patients with low viral loads were categorized by HBsAg of 1000 IU/mL. The Kaplan–Meier curve analysis showed a higher cumulative incidence of HBeAg-negative hepatitis in patients with HBsAg ≥ 1000 IU/mL when compared with those with HBsAg < 1000 IU/mL (P = 0.015, Fig. 2a). Univariate analysis also showed male gender, older age at HBeAg seroconversion, HBV genotype C infection, and higher levels of ALT and HBsAg were associated with higher risks of HBeAg-negative hepatitis (Table 2). When compared with HBsAg < 1000 IU/mL, HBsAg level ≥ 1000 IU/mL was associated with a higher risk of HBeAgnegative hepatitis, with an HR of 3.8 (95% CI: 1.2–12.2). Using multivariate analysis, in addition to male gender, older age, HBV genotype C infection, and higher levels of ALT, HBsAg level ≥ 1000 IU/mL was an independent risk factor for HBeAgnegative hepatitis, with an HR of 4.1 (95% CI: 1.3–13.6) (Table 2). HBsAg level and active viral replication in patients with HBV-DNA < 2000 IU/mL. Since HBeAgnegative hepatitis is usually preceded by active viral replication,
Serum HBV-DNA and HBsAg levels have been shown to be two important biomarkers to predict the HBV carriers’ outcomes.10,24 The recent ERADICATE-B study has shown that HBV-DNA level, compared with HBsAg level, serves as a better predictor for HCC when evaluating the overall cohort, while HBsAg level turned out to be a better predictor for HCC when focusing on patients with low viral loads.12 In this study, we confirmed the complimentary role of HBsAg level in predicting HBeAgnegative hepatitis, using a cohort of 390 spontaneous HBeAg seroconverters. In addition to male sex, older age at HBeAg seroconversion, higher level of ALT, and genotype C infection, HBsAg level ≥ 1000 IU/mL was shown to be associated with higher risks of developing HBeAg-negative hepatitis and active viral replication in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL. The first longitudinal studies investigating HBsAg level have suggested that a lower HBsAg level serves as a marker for better viral suppression because it is strongly associated with higher chances of HBsAg loss and being inactive status.14,23,25 If the conclusion is correct, it is reasonable to see that a higher HBsAg level is associated with unstable viral control, thus more HBVrelated complications. According to the ERADICATE-B cohort study, an HBsAg level ≥ 1000 IU/mL has been shown to be associated with higher risks of hepatitis activity, cirrhosis, and HCC development in patients with HBV-DNA level < 2000 IU/mL.12,13 In this independent cohort, we not only confirmed the positive relationship between HBsAg ≥ 1000 IU/mL and risk of HBeAgnegative hepatitis, but also identified a consistent relationship between HBsAg ≥ 1000 IU/mL and a higher risk of active viral replication. All these lines of evidence first suggest that HBsAg level can serve as a marker for immune control of the virus. Furthermore, HBsAg level < 1000 IU/mL plus HBV-DNA level < 2000 IU/mL can be used to define “minimal viral activity,” which is consistent with Brunetto et al.’s finding that combining these two criteria can define 3-year inactive state.25 If the patient’s liver fibrosis stage is mild, we believe this “minimal viral activity” can be translated into “minimal cirrhosis risk” and “minimal HCC risk,” which are the conclusions of the ERADICATE-B cohort study.12,13 Importantly, it is recommended to define sustained response as HBeAg seroconversion plus HBV-DNA level < 2000 IU/mL at 6 months off therapy in HBeAg-positive patients receiving all kinds of antiviral therapy.17 However, more and more data have shown that there exists a high relapse rate
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(a)
(b)
Figure 2 In spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/ mL, HBsAg level ≧ 1000 IU/mL was associated with higher risks of (a) HBeAg-negative hepatitis and (b) active viral replication. HBV, hepatitis B virus. Serum HBsAg level (IU/mL): , < 1000; , ≥ 1000.
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Table 2 Univariate and multivariate analysis of factors associated with HBeAg-negative hepatitis in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL
Gender Female Male Age at HBeAg SC (years) < 30 30–39 ≥ 40 Serum ALT level (U/L) < 40 40–79 Serum HBsAg level (IU/mL) < 1000 ≥ 1000 HBV genotype B C
Case number
P-years of follow-up
Annual incidence rate
Crude HR (95% CI)
P-value
Adjusted HR (95% CI)
P-value
8 50
707.6 1509.2
1.13 2.51
1.0 3.0 (1.4–6.4)
0.004
1.0 3.1 (1.5–6.8)
0.003
34 16 8
1647.0 380.0 190.0
2.1 4.2 4.2
1.0 2.1 (1.1–3.8) 2.2 (1.0–4.7)
0.016
1.0 1.8 (1.0–3.4) 3.0 (1.3–7.2)
0.057
48 10
2111.0 105.8
2.3 9.4
1.0 4.5 (2.2–8.9)
3 55
383.9 1832.9
0.8 3.0
1.0 3.8 (1.2–12.2)
0.024
1.0 4.1 (1.3–13.6)
0.019
42 16
1900.7 316.2
2.2 5.1
1.0 2.3 (1.3–4.1)
0.005
1.0 2.1 (1.1–3.8)
0.021
0.050 < 0.001
1.0 4.4 (2.1–9.0)
0.010 < 0.001
ALT, alanine aminotransferase; CI, confidence interval; HBeAg SC, hepatitis B e antigen seroconversion; HBV, hepatitis B virus; HR, hazard ratio; P-years, person-years.
Table 3 Univariate and multivariate analysis of factors associated with active viral replication in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL
Gender Female Male Age at HBeAg SC (years) < 30 30–39 ≥ 40 Serum ALT level (U/L) < 40 40–79 Serum HBsAg level (IU/Ml) < 1000 ≥ 1000 HBV genotype B C
Case number
P-years of follow-up
Annual incidence rate
14 62
646.7 1387.2
2.2 4.5
49 19 8
1491.6 360.0 182.3
65 11
Crude HR (95% CI)
P-value
Adjusted HR (95% CI)
P-value
1.0 2.1 (1.2–3.7)
0.014
1.0 2.1 (1.2–3.8)
0.016
3.3 5.3 4.4
1.0 1.7 (1.0–2.9) 1.4 (0.7–3.0)
0.057
1.0 1.6 (0.9–2.7) 1.9 (0.9–4.2)
0.092
1931.8 102.1
3.4 10.8
1.0 3.5 (1.8–6.7)
< 0.001
1.0 3.8 (1.9–7.4)
< 0.001
6 70
354.2 1680.0
1.7 4.2
1.0 2.4 (1.1–5.6)
0.038
1.0 2.0 (1.2–3.5)
0.014
58 18
1738.5 295.4
3.3 6.1
1.0 1.9 (1.1–3.2)
0.021
1.0 2.5 (1.1–5.9)
0.033
0.360
0.118
ALT, alanine aminotransferase; CI, confidence interval; HBeAg SC, hepatitis B e antigen seroconversion; HBV, hepatitis B virus; HR, hazard ratio; P-years, person-years.
of hepatitis activity even if this treatment end-point is achieved.20,26 Based on this study, we believe that including HBsAg level < 1000 IU/mL to define sustained response can confer to a better long-term suppression of viral replication and a remission of hepatitis activity. This study has several unique features. First, prior HBV viral kinetic studies rarely focus on HBV carriers with low viral loads.27 This study has shown that active viral replication and HBeAg-
negative hepatitis do occur in HBV carriers with low viral loads. Second, determining HBV genotype using PCR-based methods is difficult in patients with low viral loads; thus, our study adopted the ELISA assay as an alternative, and successfully determined the HBV genotype. Third, prior studies investigating HBeAgnegative hepatitis generally enrolled patients already in their HBeAg-negative phase without specifying the time point of HBeAg seroconversion.7,13 Consequently, it would be difficult to
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accurately record the rate of HBeAg-negative hepatitis from the beginning of the HBeAg-negative phase. On the other hand, our results were derived from the early HBeAg-negative disease, hence may assist in determining the natural history of HBV. We recognized the main limitation that most HBeAg-negative hepatitis events were diagnosed serologically, not by histology in this study. However, in our daily practice, it is impractical to perform invasive liver biopsies routinely. In order to compensate for this, we adopted active viral replication as another end-point, which is supposedly a leading event of HBV-related hepatitis. We consistently found HBsAg level ≥ 1000 IU/mL to be an independent predictor of active viral replication. Both data support HBsAg level as a biomarker for viral suppression and disease activity in HBeAg-negative patients with low viral loads. The other limitation was the small number of patients with low viral load, which made it impossible to address the events with low incidence rate, such as cirrhosis and HCC. We, thus, used HBeAg-negative hepatitis as the end-point, of which the number of incidence could offer adequate statistical power. In summary, in Asian spontaneous HBeAg seroconverters with genotype B or C infection, HBV-DNA level at 1-year post-HBeAg seroconversion, compared with HBsAg level, serves as a better predictor for HBeAg-negative hepatitis. However, in patients with HBV-DNA level < 2000 IU/mL, a higher HBsAg level was associated with subsequent active viral replication and HBeAgnegative hepatitis. This fact is consistent with the relationship between HBsAg level and HCC. Taken together, HBsAg level < 1000 IU/mL, in addition to HBV-DNA level < 2000 IU/mL, is useful for defining minimal viral activity in Asian spontaneous HBeAg seroconverters.
Acknowledgments
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We thank Abbott Company for providing the quantitative HBsAg kits and colleagues at National Taiwan University Hospital, Taipei, Taiwan, who enrolled and followed the patients. We also thank all the research assistants who assisted in laboratory analyses and collection of clinical information.
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References
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carriers in relation to changes of alanine aminotransferase levels over time. Hepatology 2009; 49: 1859–67. Chu CM, Hung SJ, Lin J, Tai DI, Liaw YF. Natural history of hepatitis B e antigen to antibody seroconversion in patients with normal serum aminotransferase levels. Am. J. Med. 2004; 116: 829–34. Tseng TC, Liu CJ, Chen CL et al. Serum hepatitis B virus-DNA levels correlate with long-term adverse outcomes in spontaneous hepatitis B e antigen seroconverters. J. Infect. Dis. 2012; 205: 54–63. Tseng TC, Kao JH. Clinical utility of quantitative HBsAg in natural history and nucleos(t)ide analogue treatment of chronic hepatitis B: new trick of old dog. J. Gastroenterol. 2013; 48: 13–21. Chen CJ, Yang HI, Su J et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006; 295: 65–73. Tseng TC, Liu CJ, Yang HC et al. High levels of hepatitis B surface antigen increase risk of hepatocellular carcinoma in patients with low HBV load. Gastroenterology 2012; 142: 1140–9 e3; quiz e13–4. Tseng TC, Liu CJ, Yang HC et al. Serum hepatitis B surface antigen levels help predict disease progression in patients with low hepatitis B virus loads. Hepatology 2013; 57: 441–50. Tseng TC, Liu CJ, Su TH et al. Serum hepatitis B surface antigen levels predict surface antigen loss in hepatitis B e antigen seroconverters. Gastroenterology 2011; 141: 517–25 e2. Liaw YF, Kao JH, Piratvisuth T et al. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2012 update. Hepatol. Int. 2012; 6: 531–61. European Association for the Study of the L. EASL Clinical Practice Guidelines: management of chronic hepatitis B virus infection. J. Hepatol. 2012; 57: 167–85. Lok AS, McMahon BJ. Chronic hepatitis B: update 2009. Hepatology 2009; 50: 661–2. Yeh SH, Tsai CY, Kao JH et al. Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis. J. Hepatol. 2004; 41: 659–66. Liu SF, Hsieh MH, Hou NJ et al. Hepatitis B virus genotyping by enzyme-linked immunosorbent assay in Taiwan. Hepatol. Int. 2010; 4: 601–7. Tseng TC, Liu CJ, Su TH et al. Young chronic hepatitis B patients with nucleos(t)ide analogue-induced hepatitis B e antigen seroconversion have a higher risk of HBV reactivation. J. Infect. Dis. 2012; 206: 1521–31. Su TH, Hsu CS, Chen CL et al. Serum hepatitis B surface antigen concentration correlates with HBV DNA level in patients with chronic hepatitis B. Antivir. Ther. 2010; 15: 1133–9. Su TH, Liu CJ, Tseng TC et al. Longitudinal change of HBsAg in HBeAg-negative patients with genotype B or C infection. PLoS ONE 2013; 8: e55916. Tseng TC, Liu CJ, Yang HC et al. Determinants of spontaneous surface antigen loss in hepatitis B e antigen-negative patients with a low viral load. Hepatology 2012; 55: 68–76. Tseng TC, Liu CJ, Chen CL et al. Risk stratification of hepatocellular carcinoma in hepatitis B virus e antigen-negative carriers by combining viral biomarkers. J. Infect. Dis. 2013; 208: 584–93. Brunetto MR, Oliveri F, Colombatto P et al. Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers. Gastroenterology 2010; 139: 483–90. Reijnders JG, Perquin MJ, Zhang N, Hansen BE, Janssen HL. Nucleos(t)ide analogues only induce temporary hepatitis B e antigen seroconversion in most patients with chronic hepatitis B. Gastroenterology 2010; 139: 491–8.
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27 Chen CF, Lee WC, Yang HI et al. Changes in serum levels of HBV DNA and alanine aminotransferase determine risk for hepatocellular carcinoma. Gastroenterology 2011; 141: 1240–8, 8 e1–2.
Supporting information
HBsAg levels predict viral reactivation
Figure S1 Cumulative incidence of HBeAg-negative hepatitis was positively associated with serum (a) HBV-DNA levels and (b) HBsAg levels in 390 spontaneous HBeAg seroconverters. Table S1 Results of long-term follow-up by different end-points.
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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doi:10.1111/jgh.12502
H E PAT O L O G Y
Hepatitis B surface antigen level complements viral load in predicting viral reactivation in spontaneous HBeAg seroconverters Tai-Chung Tseng,*,†† Chun-Jen Liu,†,‡ Wan-Ting Yang,§ Chi-Ling Chen,‡ Hung-Chih Yang,†,** Tung-Hung Su,†,‡ Chia-Chi Wang,*,†† Stephanie Fang-Tzu Kuo,‡‡‡ Chen-Hua Liu,†,‡ Pei-Jer Chen,†,‡ Ding-Shinn Chen†,‡ and Jia-Horng Kao†,‡,§,¶ *Division of Gastroenterology, Department of Internal Medicine, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, †Division of Gastroenterology, Department of Internal Medicine, ‡Graduate Institute of Clinical Medicine, §Hepatitis Research Center, ¶Department of Medical Research and **Microbiology, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, and ††School of Medicine, Tzu Chi University, Hualien, Taiwan; and ‡‡‡St Vincent’s Hospital, Melbourne, Victoria, Australia
Key words chronic hepatitis B, HbeAg, HbsAg, liver disease, SEARCH-B, viral replication. Accepted for publication 22 December 2013. Correspondence Professor Jia-Horng Kao, Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, 1 Chang-Te St., Taipei 10002, Taiwan. Email: [email protected] Grant/funding support: This work was supported by grants from the Buddhist Tzu-Chi General Hospital Taipei Branch (TCRD-TPE-101-12 and TCRD-TPE-NSC102-02), the National Taiwan University Hospital (NTUH100-S1534), the Department of Heath (DOH99-DC-1001 and DOH100DC-1019), and the National Science Council, Executive Yuan, Taiwan (NSC100-2314-B303-012, and NSC101-2314-B-303-006).
Abstract Background and Aims: The level of hepatitis B surface antigen (HBsAg) has been shown to complement hepatitis B virus (HBV)-DNA level in predicting disease progression in hepatitis B e antigen (HBeAg)-negative patients, especially those with low viral loads. Whether this finding could be seen in spontaneous HBeAg seroconverters remains unclear. Methods: A retrospective cohort of 390 Taiwanese spontaneous HBeAg seroconverters with a mean follow-up period of 7.4 years was enrolled. The relationships between HBV-DNA/HBsAg levels and HBeAg-negative hepatitis/active viral replication (HBVDNA level ≥ 2000 IU/mL) were investigated. Results: In the overall cohort, serum HBV-DNA level served as a better predictor for HBeAg-negative hepatitis compared with HBsAg level. However, in those with HBVDNA level < 2000 IU/mL, a higher HBsAg level was associated with a higher risk of HBeAg-negative hepatitis (P = 0.015). Multivariate analysis showed the hazard ratio of HBsAg level ≥ 1000 IU/mL versus < 1000 IU/mL was 4.1 (95% confidence interval: 1.3–13.6). When using the end-point of active viral replication, HBsAg ≥ 1000 IU/mL remained as an independent risk factor, with a hazard ratio of 2.5 (95% confidence interval: 1.1–5.9). Conclusions: In spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/ mL, HBsAg level ≥ 1000 IU/mL is associated with increased risks of HBeAg-negative hepatitis and active viral replication. Combining HBV-DNA < 2000 IU/mL and HBsAg level < 1000 IU/mL may be used to define minimal viral activity.
Introduction Hepatitis B virus (HBV) infection is a global health threat, resulting in over one million deaths per year. Patients with chronic HBV infection are at increased risk of developing liver cirrhosis, hepatic decompensation, and hepatocellular carcinoma (HCC).1–3 It is estimated that 25–40% of HBV carriers who are infected with the virus early in life will eventually develop these disastrous complications.3 It is known that hepatitis B e antigen (HBeAg) seroconversion is an important event in the natural history of chronic HBV infection as it usually indicates a remission of liver disease and confers a favorable clinical outcome.3,4 Nevertheless, 20–40% of HBeAgnegative patients do not achieve sustained viral suppression and 1242
develop HBeAg-negative hepatitis, the precursor event of cirrhosis and HCC.5–9 Currently, HBV-DNA and hepatitis B surface antigen (HBsAg) levels are two serum biomarkers able to be quantified to predict HCC development.10 The elevation of HBV-DNA level has been shown to be associated with increased HCC risk in adult HBV carriers, and the risk starts to increase when HBV-DNA level is higher than 2000 IU/mL.11 The recent cohort study, ERADICATE-B (Elucidation of Risk fActors for DIsease Control or Advancement in Taiwanese hEpatitis B carriers) study, further indicated that HBV-DNA level is a better predictor than HBsAg level for 10-year and 15-year HCC development.12 However, when focusing on the HBeAg-negative patients with low viral loads (HBV-DNA level < 2000 IU/mL), HBsAg level ≥ 1000 IU/mL, Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249
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but not HBV-DNA level, is associated with a higher risk of HCC.12 A subsequent study showed that HBsAg level ≥ 1000 IU/mL is also associated with increased risks of HBeAg-negative hepatitis, hepatitis flare, and cirrhosis.13 Whether these reported findings could be reproduced in other independent cohorts, such as spontaneous HBeAg seroconverters, remains unclear. In this study, we investigated the SEARCH-B (Study of E Antigen seRoClearance of Hepatitis B) cohort, which is a hospital-based cohort that enrolled 390 spontaneous HBeAg seroconverters,9,14 to address this interesting and important issue. Although HCC is a solid and ideal end-point to evaluate the adverse outcome of HBV carriers, it is not suitable for a small cohort because of its low incidence rate. We, thus, used HBeAgnegative hepatitis as our end-point and addressed the following issues specifically. First, although HBV-DNA and HBsAg levels at 1-year post-HBeAg seroconversion have been shown to be associated with HBeAg-negative hepatitis,9,14 it is still unclear which biomarker is the better predictor. Second, HBsAg level has been shown to be associated with disease progression in HBeAgnegative patients with HBV-DNA level < 2000 IU/mL, but most of them were in the middle or late stage of HBeAg-negative phase.13 In this study, we intended to investigate whether such relationship still holds true in an independent cohort with a different clinical setting, of which patients are in the very early stage of HBeAgnegative phase. Following the second aim, we analyzed whether there is a consistent relationship between HBsAg level and active viral replication, which is regarded as a fundamental event of all HBV-related complications.
Methods Patient cohort. Figure 1 shows the inclusion and exclusion criteria of patients in the SEARCH-B cohort study. In brief, a total of 1278 HBeAg-positive chronic hepatitis B patients above 16 years of age who received regular follow-up for more than 3 years at the National Taiwan University Hospital were consecutively enrolled from 1985 to 2004. Among them, 490 patients experienced spontaneous HBeAg seroconversion. Since all the viral factors were determined at 1-year post-HBeAg seroconversion, we excluded patients who developed HBeAg-negative hepatitis before that time point. We also excluded those who developed liver cirrhosis or HCC before HBeAg seroconversion because antiviral therapy was warranted for them.15–17 Thus, a total of 390 sustained spontaneous HBeAg seroconverters were enrolled, 239 of them had HBV-DNA levels < 2000 IU/mL at 1-year postHBeAg seroconversion and were included for the subgroup analysis. The participants of this study did not overlap with another large cohort study, ERADICATE-B,12,13 because most of them at study entry were younger than 28 years, which is different from the enrollment criteria for the ERADICATE-B study. Informed consents were obtained from all participants as per the ethical committee.
Data collection. During the follow-up period, patients were tested for serological markers (HBsAg, HBeAg, anti-HBe, antibodies against hepatitis C virus [anti-HCV], and antibodies against hepatitis D virus [anti-HDV]) at baseline. Liver function tests and
Figure 1 Flow of SEARCH-B study. *Patients received treatment within 1 year before HBeAg seroconversion, including lamivudine, adefovir dipivoxil, standard interferon, and peginterferon alpha-2a. HBV, hepatitis B virus; HCC, hepatocellular carcinoma.
alpha-fetoprotein levels were assayed every 6 months if the alanine aminotransferase (ALT) levels were within normal limits, and at least every 3 months if ALT levels became elevated. HBeAg and anti-HBe were assayed at least every 6 months until the first year after HBeAg seroconversion, and then upon the development of hepatitis. Serum samples collected at each visit were stored at −20°C until tested. High-resolution abdominal ultrasonography was performed regularly every 3–6 months from enrollment for the surveillance of liver cirrhosis and HCC.
Definitions of HBeAg-negative hepatitis and active viral replication. HBeAg seroconversion was defined as persistent HBeAg negativity and anti-HBe positivity for at least 1 year. We defined HBeAg-negative hepatitis and active viral replication to be the first and second end-points, respectively. HBeAg-negative hepatitis was defined as ALT elevation more than twice the upper limit of normal, with a concomitant serum HBVDNA level ≥ 2000 IU/mL.9,13–15 The upper limit of normal for serum ALT level is 40 U/L. Apart from the aforementioned criteria, exclusion of drugs or alcohol usage, and the absence of serological evidence suggestive of other viral hepatitis infection or autoimmune liver disease, was essential to ensure correct diagnosis. Active viral replication was defined as an upsurge of
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HBV-DNA level from < 2000 IU/mL to ≥ 2000 IU/mL.15 To investigate this end-point, we retrospectively quantified HBV-DNA levels from the stored serum samples annually to determine the first time when HBV-DNA level ≥ 2000 IU/mL, which was defined as the time point of active viral replication.
Table 1 Baseline characteristics of the overall cohort and the subcohort with low viral loads
Serological assays. Serum HBsAg, HBeAg, anti-HBe, antiHCV, and anti-HDV were tested using commercially available kits (Abbott Laboratories, Abbott Park, IL, USA).
Gender Female Male Age at HBeAg seroconversion (years) < 30 30–39 ≥ 40 Serum ALT (U/L) < 40 40–79 Serum HBV-DNA level (IU/mL) < 200 200–1999 2000–19 999 20 000–199 999 ≥ 200 000 HBV genotype B C B+C Serum HBsAg level (IU/mL) < 100 100–999 ≥ 1000
Extraction of HBV-DNA. Viral DNA in the serum was extracted using commercial kits (QIAamp DNA Blood and Tissue Mini Kit; QIAGEN Inc., Valencia, CA, USA). The extracted DNA was used for HBV-DNA quantification and HBV genotype determination. Quantification of serum HBV-DNA and HBV genotyping. Serum HBV-DNA levels and HBV genotype were determined using real-time polymerase chain reaction (PCR)-based single-tube assay as previously described.18 In short, this method consists of two consecutive steps. The first step uses real-time PCR to quantify HBV-DNA, and the second step uses melting curve analysis to genotype HBV. The dynamic range of HBV-DNA levels under this method is from 20 to 2 × 1010 IU/mL (100 to 1011 copies/mL). Since HBV-DNA level quantification with real-time PCR was not available until 2004, most HBV-DNA levels were determined retrospectively using stored sera. In addition, for patients who had undetectable HBV-DNA levels at HBeAg-negative phase, we determined HBV genotype either by IMMUNIS HBV Genotype EIA kit (Institute of Immunology Co. Ltd, Tokyo, Japan), which is an ELISA assay and detects genotype-specific epitopes in preS2,19,20 or using serum samples during their HBeAg-positive stage. Quantification of HBsAg levels. HBsAg levels were quantified using the Architect HBsAg QT (Abbott Laboratories) according to the manufacturer’s instructions.21,22 The detection range of Architect assay is from 0.05 to 250 IU/mL. If the HBsAg level was found to be higher than 250 IU/mL, the samples were diluted to 1:100 to 1:1000 to obtain a reading within the calibration curve range. Statistical analysis. Mean and standard deviation (SD) were calculated for continuous variables, and percentages were used for categorical variables. We calculated person-years from 1-year post-HBeAg seroconversion because that is when the viral factors were determined. The person-years were censored on the date of diagnosing the aforementioned end-points, the date of initiating antiviral therapy, the last date of follow-up, or June 30, 2012, whichever came first. For patients with multiple episodes of HBeAg-negative hepatitis or active viral replication, each endpoint was counted once. The cumulative incidence of the designated end-points was derived using the Kaplan–Meier method, and the log–rank test was used for statistical significance. To compare the predicting ability of HBV-DNA level and HBsAg level for HBeAg-negative hepatitis in the overall cohort,
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No. (%) of patients Overall Subjects with cohort HBV-DNA < 2000 IU/mL 126 (32.3) 264 (67.7)
82 (34.3) 157 (65.7)
243 (62.3) 95 (24.4) 52 (13.3)
162 (67.8) 52 (21.8) 25 (10.5)
331 (84.9) 59 (15.1)
222 (92.9) 17 (7.1)
139 (35.6) 100 (25.6) 63 (16.2) 30 (7.7) 58 (14.9)
139 (58.2) 100 (41.8)
316 (81.0) 73 (18.7) 1 (0.3)
203 (84.9) 36 (15.1)
18 (4.6) 34 (8.7) 338 (86.7)
16 (6.7) 24 (10.0) 199 (83.3)
ALT, alanine aminotransferase; HBV, hepatitis B virus.
we restricted the study population to patients who were followed up for at least 3 years and 6 years. Receiver operating characteristic (ROC) curve analysis was used to calculate the area under ROC curve (AUROC), which was used to compare the predictive performance of HBV-DNA level and HBsAg level for 3-year and 6-year risk of HBeAg-negative hepatitis. Cox proportional hazards regression model was used to calculate the crude and multivariate-adjusted hazard ratios (HR) of each end-point. Age, sex, HBV genotype, and serum levels of HBsAg and ALT were included as adjusting variables in analyzing the prognosis of patients with low viral load (≤ 2000 IU/mL).12–14,23 HBsAg levels were categorized into < 1000 IU/mL and ≥ 1000 IU/mL according to previous report.12,13 Statistical significance of all tests was defined as P < 0.05 by two-tailed tests. All analyses were performed using the Stata statistical software (version 9.2; Stata Corp, College Station, TX, USA).
Results Baseline characteristics. Table 1 shows the demographic data of 390 spontaneous HBeAg seroconverters and 239 spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL. Most patients were males with genotype B Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249
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infection. In the subcohort with low viral loads, 40 patients (16.7%) had HBsAg levels < 1000 IU/mL at 1-year post-HBeAg seroconversion. Follow-up results by different end-points. The follow-up results of the overall cohort and the subcohort with low viral loads are summarized in Table S1. In the overall cohort, 142 patients developed HBeAg-negative hepatitis within 2901.2 person-years of follow-up, with an average annual rate of 4.9%. In the subcohort with low viral loads, 58 patients developed HBeAgnegative hepatitis within 1977.9 person-years of follow-up, with an average annual rate of 2.9%, and 76 developed active viral replication in the 1794.9 person-years of follow-up, with an average annual rate of 4.2%.
HBsAg levels predict viral reactivation
we investigated whether an HBsAg level ≥ 1000 IU/mL was associated with a higher likelihood of subsequent active viral replication in patients with low viral loads at study entry. The Kaplan– Meier curve analysis showed that HBsAg level ≥ 1000 IU/mL was also associated with higher cumulative incidence of active viral replication (P = 0.032, Fig. 2b). Univariate analysis indicated that male sex, elevated ALT level, genotype C infection, and HBsAg level ≥ 1000 IU/mL were associated with higher risks of active viral replication (Table 3). Multivariate analysis found that, in addition to male sex, elevated ALT level, and genotype C infection, HBsAg ≥ 1000 IU/mL was an independent risk factor for active viral replication, with an HR of 2.5 (95% CI: 1.1–5.9) (Table 3).
Discussion The value of HBV-DNA and HBsAg in predicting 3-year and 6-year risk of HBeAg-negative hepatitis. Both HBV-DNA level (Fig. S1a, P for trend < 0.001) and HBsAg level (Fig. S1b, P for trend = 0.003) were positively associated with HBeAg-negative hepatitis in a dose response manner, which has been shown in our previous reports.9,14 The risk of HBeAg-negative hepatitis started to increase when HBV-DNA level ≥ 2000 IU/mL. We, thus, compared the predictive values of HBV-DNA level and HBsAg level for 3-year and 6-year risk of HBeAg-negative hepatitis using the ROC curve analysis. The results showed that HBV-DNA level, as compared with HBsAg level, served as a better predictor for both 3-year risk of HBeAgnegative hepatitis (AUROC [95% CI]: 0.77 [0.70–0.83] vs 0.61 [0.54–0.69], P ≤ 0.001) and 6-year risk of HBeAg-negative hepatitis (AUROC [95% CI]: 0.74 [0.67–0.80] vs 0.58 [0.54–0.68], P = 0.005) (The P-value here refers to the comparison of AUROC between HBV-DNA and HBsAg levels). Factors associated with HBeAg-negative hepatitis in patients with low viral loads. We explored whether HBsAg could serve as a predictor in patients with HBV-DNA level < 2000 IU/mL since the risk of HBeAg-negative hepatitis starts to increase when viral load ≥ 2000 IU/mL. The patients with low viral loads were categorized by HBsAg of 1000 IU/mL. The Kaplan–Meier curve analysis showed a higher cumulative incidence of HBeAg-negative hepatitis in patients with HBsAg ≥ 1000 IU/mL when compared with those with HBsAg < 1000 IU/mL (P = 0.015, Fig. 2a). Univariate analysis also showed male gender, older age at HBeAg seroconversion, HBV genotype C infection, and higher levels of ALT and HBsAg were associated with higher risks of HBeAg-negative hepatitis (Table 2). When compared with HBsAg < 1000 IU/mL, HBsAg level ≥ 1000 IU/mL was associated with a higher risk of HBeAgnegative hepatitis, with an HR of 3.8 (95% CI: 1.2–12.2). Using multivariate analysis, in addition to male gender, older age, HBV genotype C infection, and higher levels of ALT, HBsAg level ≥ 1000 IU/mL was an independent risk factor for HBeAgnegative hepatitis, with an HR of 4.1 (95% CI: 1.3–13.6) (Table 2). HBsAg level and active viral replication in patients with HBV-DNA < 2000 IU/mL. Since HBeAgnegative hepatitis is usually preceded by active viral replication,
Serum HBV-DNA and HBsAg levels have been shown to be two important biomarkers to predict the HBV carriers’ outcomes.10,24 The recent ERADICATE-B study has shown that HBV-DNA level, compared with HBsAg level, serves as a better predictor for HCC when evaluating the overall cohort, while HBsAg level turned out to be a better predictor for HCC when focusing on patients with low viral loads.12 In this study, we confirmed the complimentary role of HBsAg level in predicting HBeAgnegative hepatitis, using a cohort of 390 spontaneous HBeAg seroconverters. In addition to male sex, older age at HBeAg seroconversion, higher level of ALT, and genotype C infection, HBsAg level ≥ 1000 IU/mL was shown to be associated with higher risks of developing HBeAg-negative hepatitis and active viral replication in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL. The first longitudinal studies investigating HBsAg level have suggested that a lower HBsAg level serves as a marker for better viral suppression because it is strongly associated with higher chances of HBsAg loss and being inactive status.14,23,25 If the conclusion is correct, it is reasonable to see that a higher HBsAg level is associated with unstable viral control, thus more HBVrelated complications. According to the ERADICATE-B cohort study, an HBsAg level ≥ 1000 IU/mL has been shown to be associated with higher risks of hepatitis activity, cirrhosis, and HCC development in patients with HBV-DNA level < 2000 IU/mL.12,13 In this independent cohort, we not only confirmed the positive relationship between HBsAg ≥ 1000 IU/mL and risk of HBeAgnegative hepatitis, but also identified a consistent relationship between HBsAg ≥ 1000 IU/mL and a higher risk of active viral replication. All these lines of evidence first suggest that HBsAg level can serve as a marker for immune control of the virus. Furthermore, HBsAg level < 1000 IU/mL plus HBV-DNA level < 2000 IU/mL can be used to define “minimal viral activity,” which is consistent with Brunetto et al.’s finding that combining these two criteria can define 3-year inactive state.25 If the patient’s liver fibrosis stage is mild, we believe this “minimal viral activity” can be translated into “minimal cirrhosis risk” and “minimal HCC risk,” which are the conclusions of the ERADICATE-B cohort study.12,13 Importantly, it is recommended to define sustained response as HBeAg seroconversion plus HBV-DNA level < 2000 IU/mL at 6 months off therapy in HBeAg-positive patients receiving all kinds of antiviral therapy.17 However, more and more data have shown that there exists a high relapse rate
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(a)
(b)
Figure 2 In spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/ mL, HBsAg level ≧ 1000 IU/mL was associated with higher risks of (a) HBeAg-negative hepatitis and (b) active viral replication. HBV, hepatitis B virus. Serum HBsAg level (IU/mL): , < 1000; , ≥ 1000.
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Table 2 Univariate and multivariate analysis of factors associated with HBeAg-negative hepatitis in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL
Gender Female Male Age at HBeAg SC (years) < 30 30–39 ≥ 40 Serum ALT level (U/L) < 40 40–79 Serum HBsAg level (IU/mL) < 1000 ≥ 1000 HBV genotype B C
Case number
P-years of follow-up
Annual incidence rate
Crude HR (95% CI)
P-value
Adjusted HR (95% CI)
P-value
8 50
707.6 1509.2
1.13 2.51
1.0 3.0 (1.4–6.4)
0.004
1.0 3.1 (1.5–6.8)
0.003
34 16 8
1647.0 380.0 190.0
2.1 4.2 4.2
1.0 2.1 (1.1–3.8) 2.2 (1.0–4.7)
0.016
1.0 1.8 (1.0–3.4) 3.0 (1.3–7.2)
0.057
48 10
2111.0 105.8
2.3 9.4
1.0 4.5 (2.2–8.9)
3 55
383.9 1832.9
0.8 3.0
1.0 3.8 (1.2–12.2)
0.024
1.0 4.1 (1.3–13.6)
0.019
42 16
1900.7 316.2
2.2 5.1
1.0 2.3 (1.3–4.1)
0.005
1.0 2.1 (1.1–3.8)
0.021
0.050 < 0.001
1.0 4.4 (2.1–9.0)
0.010 < 0.001
ALT, alanine aminotransferase; CI, confidence interval; HBeAg SC, hepatitis B e antigen seroconversion; HBV, hepatitis B virus; HR, hazard ratio; P-years, person-years.
Table 3 Univariate and multivariate analysis of factors associated with active viral replication in spontaneous HBeAg seroconverters with HBV-DNA level < 2000 IU/mL
Gender Female Male Age at HBeAg SC (years) < 30 30–39 ≥ 40 Serum ALT level (U/L) < 40 40–79 Serum HBsAg level (IU/Ml) < 1000 ≥ 1000 HBV genotype B C
Case number
P-years of follow-up
Annual incidence rate
14 62
646.7 1387.2
2.2 4.5
49 19 8
1491.6 360.0 182.3
65 11
Crude HR (95% CI)
P-value
Adjusted HR (95% CI)
P-value
1.0 2.1 (1.2–3.7)
0.014
1.0 2.1 (1.2–3.8)
0.016
3.3 5.3 4.4
1.0 1.7 (1.0–2.9) 1.4 (0.7–3.0)
0.057
1.0 1.6 (0.9–2.7) 1.9 (0.9–4.2)
0.092
1931.8 102.1
3.4 10.8
1.0 3.5 (1.8–6.7)
< 0.001
1.0 3.8 (1.9–7.4)
< 0.001
6 70
354.2 1680.0
1.7 4.2
1.0 2.4 (1.1–5.6)
0.038
1.0 2.0 (1.2–3.5)
0.014
58 18
1738.5 295.4
3.3 6.1
1.0 1.9 (1.1–3.2)
0.021
1.0 2.5 (1.1–5.9)
0.033
0.360
0.118
ALT, alanine aminotransferase; CI, confidence interval; HBeAg SC, hepatitis B e antigen seroconversion; HBV, hepatitis B virus; HR, hazard ratio; P-years, person-years.
of hepatitis activity even if this treatment end-point is achieved.20,26 Based on this study, we believe that including HBsAg level < 1000 IU/mL to define sustained response can confer to a better long-term suppression of viral replication and a remission of hepatitis activity. This study has several unique features. First, prior HBV viral kinetic studies rarely focus on HBV carriers with low viral loads.27 This study has shown that active viral replication and HBeAg-
negative hepatitis do occur in HBV carriers with low viral loads. Second, determining HBV genotype using PCR-based methods is difficult in patients with low viral loads; thus, our study adopted the ELISA assay as an alternative, and successfully determined the HBV genotype. Third, prior studies investigating HBeAgnegative hepatitis generally enrolled patients already in their HBeAg-negative phase without specifying the time point of HBeAg seroconversion.7,13 Consequently, it would be difficult to
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accurately record the rate of HBeAg-negative hepatitis from the beginning of the HBeAg-negative phase. On the other hand, our results were derived from the early HBeAg-negative disease, hence may assist in determining the natural history of HBV. We recognized the main limitation that most HBeAg-negative hepatitis events were diagnosed serologically, not by histology in this study. However, in our daily practice, it is impractical to perform invasive liver biopsies routinely. In order to compensate for this, we adopted active viral replication as another end-point, which is supposedly a leading event of HBV-related hepatitis. We consistently found HBsAg level ≥ 1000 IU/mL to be an independent predictor of active viral replication. Both data support HBsAg level as a biomarker for viral suppression and disease activity in HBeAg-negative patients with low viral loads. The other limitation was the small number of patients with low viral load, which made it impossible to address the events with low incidence rate, such as cirrhosis and HCC. We, thus, used HBeAg-negative hepatitis as the end-point, of which the number of incidence could offer adequate statistical power. In summary, in Asian spontaneous HBeAg seroconverters with genotype B or C infection, HBV-DNA level at 1-year post-HBeAg seroconversion, compared with HBsAg level, serves as a better predictor for HBeAg-negative hepatitis. However, in patients with HBV-DNA level < 2000 IU/mL, a higher HBsAg level was associated with subsequent active viral replication and HBeAgnegative hepatitis. This fact is consistent with the relationship between HBsAg level and HCC. Taken together, HBsAg level < 1000 IU/mL, in addition to HBV-DNA level < 2000 IU/mL, is useful for defining minimal viral activity in Asian spontaneous HBeAg seroconverters.
Acknowledgments
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We thank Abbott Company for providing the quantitative HBsAg kits and colleagues at National Taiwan University Hospital, Taipei, Taiwan, who enrolled and followed the patients. We also thank all the research assistants who assisted in laboratory analyses and collection of clinical information.
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Supporting information
HBsAg levels predict viral reactivation
Figure S1 Cumulative incidence of HBeAg-negative hepatitis was positively associated with serum (a) HBV-DNA levels and (b) HBsAg levels in 390 spontaneous HBeAg seroconverters. Table S1 Results of long-term follow-up by different end-points.
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Journal of Gastroenterology and Hepatology 29 (2014) 1242–1249 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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