HBsAg seroclearance in chronic hepatitis B in the Chinese: Virological, histological, and clinical aspects
Few studies have examined Chinese patients with chronic hepatitis B who exhibit hepatitis B surface antigen (HBsAg) seroclearance. We comprehensively studied the biochemical, virological, histological, and clinical aspects of 92 patients with HBsAg seroclearance (median follow-up, 126 months). Ninety-two HBsAg-positive controls matched for age, sex, and duration of follow-up were also recruited. Liver biochemistry, serum hepatitis B virus (HBV) DNA levels, and development of clinical complications were monitored. Intrahepatic total and covalently closed circular (ccc) HBV DNA were measured quantitatively in 16 patients. HBV genotype was determined in 30 patients. The mean age at HBsAg seroclearance was 48.8 (+ 13.81) years. There was a significant improvement in serum alanine aminotransferase levels after HBsAg seroclearance (p<0.0001). Patients with genotype B had a higher chance of HBsAg seroclearance than those with genotype C (P = .014). Ninety-eight percent of patients had undetectable serum HBV DNA. Thirty-seven percent of patients had low titer of intrahepatic HBV DNA, mainly in the form of cccDNA (71%-100%). All 14 patients with liver biopsies had near normal histology. There was no difference in the risk of development of hepatocellular carcinoma (HCC) between patients with and without HBsAg seroclearance. However, the mean age of HBsAg seroclearance was significantly older in patients with HCC than in patients without HCC (P = .016). In conclusion, patients with HBsAg seroclearance had favorable biochemical, virological, and histological parameters. Intrahepatic HBV DNA level was low and predominantly in the form of cccDNA. However, HCC could still develop, particularly in patients with cirrhosis who had HBsAg seroclearance at an older age. (HEPATOLOGY 2004;39:1694–1701.)
Hepatitis B surface antigen (HBsAg) seroclearance is a rare event in Chinese patients with chronic hepatitis B virus (HBV) infection who acquire the disease early in life. The estimated annual incidence of HBsAg seroclearance is 0.1% to 0.8%.1, 2 Undetectable HBsAg in the serum is usually due to a decrease in viremia rather than the emergence of HBsAg mutants.3 Some studies demonstrate a favorable outcome in terms of histological features and development of hepatocellular carcinoma (HCC).2, 4 However, these findings are not confirmed by other studies.5, 6
The presence of HBV DNA within the liver in patients with HBsAg seroclearance has been demonstrated in some studies,7–9 but it is not known whether the intrahepatic HBV DNA is replicative or nonreplicative—that is, in the form of covalently closed circular (ccc) DNA. Also, it is uncertain how much residual virus is in extrahepatic reservoirs like peripheral blood mononuclear cells (PBMC).
Therefore, we sought to determine the factors associated with and the significance of HBsAg seroclearance in Chinese HBV patients by examining the following parameters: (1) liver biochemistry, (2) HBV DNA in the serum and PBMC, (3) HBV genotypes, (4) intrahepatic total and cccDNA, (5) liver histology, and (6) the development of cirrhosis-related complications and HCC.
Patients and Methods
Between August 1975 and October 2001, a total of 3,843 chronic HBV patients had been followed up every 3 to 6 months in the Hepatitis Clinic of the University of Hong Kong Queen Mary Hospital in Hong Kong. Liver biochemistry and HBV serology, including HBsAg, antibody to HBsAg (anti-HBs; for patients who became HBsAg-negative), hepatitis Be antigen (HBeAg), and antibody to HBeAg (anti-HBe) were checked (Abbott Laboratories, Chicago, IL). Ninety-two patients (HBeAg/anti-HBe, 21:71 on presentation) had HBsAg seroclearance. Six patients had prior interferon-alfa treatment. HBsAg seroclearance was defined as loss of serum HBsAg on repeated testing for a period of at least 6 months and during subsequent follow-up until the time of analysis. None of the patients had concomitant hepatitis C and hepatitis D infection. Patients with cirrhosis-related complications or HCC on presentation were excluded.
Ninety-two consecutive HBV patients presenting during the same period of time and matched for sex, age, and HBeAg/anti-HBe status on presentation and for the duration of follow-up were recruited as controls.
Measurement of HBV DNA Levels in Serum, PBMC, and Liver
Serum obtained at the last follow-up was tested for HBV DNA using the Cobas Amplicor HBV Monitor Test (Roche Diagnostics, Branchburg, NJ; lower limit of detection, 200 copies/mL). PBMC were isolated by standard Ficoll-Hypaque density gradient centrifugation from heparinized peripheral blood. The cell pellets were washed 3 times in phosphate-buffered saline (PBS) and resuspended in 200 μL of PBS. Total cellular DNA was isolated from the PBMC by the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany), with a final elution volume of 200 μL. HBV DNA isolated from PBMC was measured by the Cobas Amplicor HBV Monitor Test. A TaqMan PCR Reagent Kit (Applied Biosystems, Foster City, CA), which contained specific primers and probe for detection of β-actin gene was used to measure the amount of human genomic DNA. The final concentrations of the primers and probe in each reaction were 300 μmol and 200 μmol, respectively. Real-time polymerase chain reaction (PCR) amplification and detection was performed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems).
Liver biopsies were performed in 16 patients with HBsAg seroclearance (2 were subsequently found inadequate for histological assessment), 13 control patients, and 9 non-HBV subjects. Liver samples (0.5-1.5 cm in length) were homogenized in PBS with a pellet pestle-microtube set (Kontes Glass Co., Vineland, NJ), followed by DNA extraction with a QIAamp DNA Blood Mini Kit. The extracted DNA was eluted in 400 μL of the elution buffer.
The amount of total HBV DNA, cccDNA, and human genomic DNA were quantified using the Invader HBV DNA Assay (Third Wave Technologies, Inc., Madison, WI). The general principle of the Invader assay has been described.10–12 Briefly, 2 oligonucleotides, the primary probe and Invader oligonucleotide, hybridized to a target DNA sequence to form a partially overlapping structure. A Cleavase enzyme recognized this structure and cut off a short nucleotide fragment (called a 5′-flap) from the primary probe. At an isothermal reaction temperature close to the melting temperature of the primary probe and lower than the melting temperature of the Invader oligonucleotide, the primary probe cycles on the target DNA, and the released 5′-flaps were amplified in proportion to the concentration of the target DNA. Fluorescence resonance energy transfer (FRET) cassettes were used to react with the released flaps and generate a fluorescence signal, which was measurable by real-time PCR machines.
Three sets of Invader assays were used; the sequences of the oligonucleotides for each of the Invader assay sets are shown in Table 1. Set MS1, designed for the detection of the minus-strand sequences of the HBV genome, was used for quantitation of all replicative forms of HBV DNA. Set PS2, designed for the detection of plus-strand sequences with the cleavage site located at the 5′ base of the direct repeat (DR)-2 element, was used to determine cccDNA. A third set of Invader assay was designed for quantitation of the human insulin growth factor I (hIGF1) gene. Since the Cleavase acting site for set PS2 is at the 5′ base of the DR2 element, cleavage is specific for cccDNA, which contains a covalent linkage of the 3′ end of the plus strand to the 5′ base of the DR2 element.
Table 1. Oligonucleotide Sequences for Each Invader* Assay Set
|Set MS1||Invader* oligonucleotide||5′-ctcatctgccggwccgtgtgcacttcgt-3′||1565–1591|
| ||Primary probe||5′-cgcgccgaggcttcacctctgcacgt-NH3-3′||1592–1606|
| ||FRET cassette||5′-F-tctZagccggttttccggctgagacctcggcgcg-3′|| |
|Set PS2||Invader* oligonucleotide||5′-gtckccatgcracgtgcagaggtgaat-3′||1618–1593|
| ||Primary probe||5′-aggccacggacggcgaagtgcacacg-NH3-3′||1592–1579|
| ||FRET cassette||5′-F-actZagccggttttccggctgagtcgtccgtggcct-3′|| |
|hgDNA||Invader* oligonucleotide||5′-ccagcctccttagatcacagctccggaagt-3′|| |
|hIGF gene||Primary probe||5′-atgacgtggcagaccagcactcatccacga-3′|| |
| ||FRET cassette||5′-F-tctZagccggttttccggctgagagtctgccacgtcat-3′|| |
Fifteen microlitres of heat-denatured DNA extracted from the liver biopsy samples was added to 5 μL of a reaction master mix containing 40 mmol/L 3-(N-Morpholino) propane sulfonic (pH 7.5), 50 mmol/L MgCl2, 2 μmol/L primary probe, 2 μmol/L Invaderoligonucleotide, 1 μmol/L FRET cassette and 50 ng Cleavase. The reaction mixtures were then incubated in the Rotor-Gene 2000 Real-time Cycler (Corbett Research, Mortlake, Australia) with a temperature setting of 80°C for 2 minutes, followed by a single temperature incubation of 64°C for 240 minutes, with fluorescence signal reading at 1-minute intervals.
Signal generation of the Invader assay follows quadratic kinetics, and there is a linear relationship between the target copy concentration and the quadratic coefficient.10 The amount of total HBV DNA and cccDNA in a sample was calculated by extrapolation from a standard curve of the quadratic coefficients generated by reactions using the probe sets MS1 and PS2 on external plasmid standards of known concentrations. Intrahepatic total HBV DNA and cccDNA levels were then standardized with respect to the amount (in nanograms) of human genomic DNA present in the samples, as determined by hIGFI detection by the Invader assay. Cell number was calculated based on the estimate of 6.667 pg of human genomic DNA per cell. Experiment with serial diluted plasmid standards shows that the Invader assay has a lower limit of detection of 0.002 HBV DNA copies/cell for both the relaxed circular and ccc forms of HBV DNA and a dynamic range of 5 orders of magnitude. Intrahepatic total HBV DNA and cccDNA levels were then standardized with respect to the amount (in nanograms) of human genomic DNA present in the samples, as determined by hIGFI detection by the Invader assay. The intrahepatic total HBV DNA measurement was verified by using Cobas Amplicor HBV Monitor Test and β-actin gene measurement mentioned above.
Serum from 30 patients with HBsAg seroclearance (serum at the time of presentation before HBsAg seroclearance) and 50 controls were tested for HBV genotypes. Briefly, HBV DNA was extracted as described in Stuyver et al.13 The HBsAg region spanning aa107-205 was amplified and analyzed by INNO-LiPA HBV Genotyping (Innogenetics NV, Ghent, Belgium). The INNO-LiPA HBV Genotyping assay contains oligonucleotide probes specific for HBV genotypes A to G applied as 14 different lines on a membrane strip. Multiple probes are present for each HBV genotype. The correct HBV genotype was determined by consulting an interpretation chart showing probe reactivity patterns for each genotype.
Histological activity index scores were assessed according to the criteria of Knodell et al.14 Immunoperoxidase staining was performed using monoclonal antibodies against HBsAg and HBcAg (Signet, Dedham, MA; dilution, 1:2).
All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 10.0 for Windows; SPSS Inc., Chicago, IL). All continuous variables were tested for normality by Kolmogorov-Smirnov test. Comparison between continuous variables with normal distribution was tested by Student t test. Continuous skewed variables were tested by Mann-Whitney U test and Kruskal-Wallis test for 2- and 3-group comparison, respectively. Two related skewed variables were tested by Wilcoxon signed rank test. Correlation between 2 variables was tested by Spearman rank correlation. Categorical variables were tested by chi-square test or Fisher exact test.
Demographic Data and Liver Biochemistry on Presentation
Demographic data for the 92 patients with HBsAg seroclearance and 92 control patients are listed in Table 2. There were no significant differences in any of the parameters between the 2 groups.
Table 2. Demographic Data and Liver Biochemistry on Presentation for 92 Patients With HBsAg Seroclearance and 92 Control Patients
|Demographic data|| || |
| Mean age (±SD)||42.55 (14.37)||41.90 (15.09)|
| Median duration of follow-up, mo (range)||126.28 (32.3–282)||125.50 (42–315)|
| Median duration of follow-up after HBsAg seroclearance, mo (range)||51.12 (18.72–199.56)||—|
|Liver biochemistry|| || |
| Median albumin, U/L (range)||47 (26–56)||47 (24–58)|
| Median bilirubin, μmol/L (range)||11 (3–553)||10 (2–76)|
| Median ALT, U/L (range)||27 (6–3390)||31 (8–1020)|
| Median AST, U/L (range)||24 (10–614)||30.5 (13–476)|
HBeAg Seroconversion and HBsAg Seroclearance.
The 21 HBeAg-positive patients with subsequent HBsAg seroclearance had HBeAg seroconversion at a mean (± SD) age of 38.09 (+ 16.65) years. The median alanine aminotransferase (ALT) decreased from 99.5 U/L (range, 13-3990 U/L) to 22 U/L (range, 9-1806 U/L; P < .0001) after HBeAg seroconversion. Only 14 of the 21 HBeAg-positive patients in the control group had HBeAg seroconversion.
The mean age of HBsAg seroclearance for the 92 patients was 48.8 (+ 13.81) years. Fifty-four patients (58.7%) had detectable anti-HBs with the median level of 60.8 mIU/mL (range, 4.4-1000 mIU/mL). With HBsAg seroclearance, the median ALT of the 92 patients improved from 22 U/L (range, 6-1806 U/L) to 19 U/L (range, 6-106 U/L; P < .0001).
There was no significant difference in liver biochemistry on presentation between patients with HBsAg seroclearance and controls (Table 2). The control patients had a lower incidence of HBeAg seroconversion though they were of similar age and had similar duration of follow-up (p = .009). For the control who had HBeAg seroconversion, the median peak ALT levels during HBeAg seroconversion was similar to that of patients with subsequent HBsAg seroclearance: 89 U/L (range, 6-819 U/L) versus 136 U/L (range, 8-1806 U/L), respectively (P = .4). The distribution of HBV genotypes is listed in Table 3. Patients with HBsAg seroclearance were more likely to be of genotype B (P = .014).
Table 3. HBV Genotype Distribution of Patients With and Without HBsAg Seroclearance
|Single genotype*|| || |
| A (%)||1 (3.3)||2 (4)|
| B (%)||16 (53.3)||15 (30)|
| C (%)||10 (33.3)||32 (64)|
| D (%)||2 (6.7)||—|
|Mixed genotypes|| || |
| A + D (%)||1 (3.3)||—|
| B + C (%)||—||1 (2)|
HBV DNA in Serum and PBMC.
Only 1 patient (1.1%) had a detectable HBV DNA level of 1870 copies/mL at a median interval of 49.1 months (range, 10-116.77 months) after HBsAg seroclearance. Sixty-six patients had their PBMC isolated for the determination of HBV DNA levels at a median interval of 48.8 months (range, 18.7-126.8 months) after HBsAg seroclearance. None had detectable HBV DNA in the PBMC, though human genomic DNA was detected by real-time PCR for β-actin.
Intrahepatic Total HBV DNA and cccDNA.
Results of the intrahepatic HBV DNA are listed in Table 4. For the 16 patients with HBsAg seroclearance, the median interval between the time of HBsAg seroclearance and the time of liver biopsy was 47.9 months (range, 26.4-127.9 months). Six patients (37.5%) had detectable intrahepatic total HBV DNA levels, 4 of whom were positive for anti-HBs in the serum. There was a significant decreasing trend in median intrahepatic total HBV DNA levels (expressed in copies per nanogram of human genomic DNA) for HBeAg-positive patients (17613.26 copies/ng [range, 370.14-32956.14 copies/ng]); anti-HBe–positive patients (2229.0 copies/ng [range, 25.19-13347.66 copies/ng]); and patients with HBsAg seroclearance (undetectable level [range, undetectable level-12.02 copies/ng]); P < .0001. There was good correlation between the intrahepatic total HBV DNA levels measured by the Invader assay and Cobas Amplicor HBV Monitor Test (r = 0.92, P < .0001).
Table 4. Intrahepatic Total HBV DNA and cccDNA of Patients and Controls
|HBsAg seroclearance||1||12.02 (0.080)||2.51||12.02 (0.080)||100|
| ||2||0.95 (0.006)||0.26||0.95 (0.006)||100|
| ||3||0.96 (0.006)||0.12||0.87 (0.006)||91.07|
| ||4||0.41 (0.003)||Undetectable||0.41 (0.003)||100|
| ||5||2.51 (0.017)||Undetectable||2.52 (0.017)||100|
| ||6||0.85 (0.006)||Undetectable||0.61 (0.004)||71.28|
|HBeAg + ve||17||32956.14 (219.72)||951.81||1118.9 (7.46)||3.4|
| ||18||26544.14 (176.97)||1386.21||1381.63 (9.21)||5.21|
| ||19||17613.26 (117.43)||8991.13||949.69 (6.33)||5.39|
| ||20||7477.43 (49.85)||149.37||479.43 (3.20)||6.4|
| ||21||370.14 (2.47)||56.95||60.99 (0.41)||16.48|
|Anti-HBe + ve||22||13347.66 (88.99)||368.68||536.82 (3.58)||2.56|
| ||23||4527.04 (30.18)||277.57||263.68 (1.76)||5.82|
| ||24||3656.05 (24.37)||107.13||105.26 (0.70)||2.88|
| ||25||2848.33 (18.99)||93.76||142.42 (0.95)||5|
| ||26||1609.66 (10.73)||185.79||107.63 (0.72)||6.69|
| ||27||449.38 (3.00)||40.22||42.74 (0.28)||9.51|
| ||28||74.44 (0.50)||3.31||29.56 (0.20)||39.71|
| ||29||25.69 (0.17)||5.09||14.89 (0.10)||57.96|
|Non-HBV controls (n = 9)|| ||Undetectable||Undetectable||Undetectable||NA|
Intrahepatic HBV DNA in the 6 patients with HBsAg seroclearance and measurable intrahepatic HBV DNA was predominantly in the form of cccDNA, whereas most of the intrahepatic HBV DNA of the 13 control patients had relatively low percentages of cccDNA: median percentage of cccDNA, 100% (range 71.28%-100%) versus 5.82% (range, 2.56%-57.96%), respectively; P < .0001.
The mean age of HBsAg seroclearance for the 14 patients with assessable liver biopsy was 41.96 months (+ 8.34). The median interval between the time of HBsAg seroclearance and liver biopsies was 45.77 months (range, 26.4-66.02 months). The histological features of the liver biopsies are listed in Table 5. Of 14 patients, 12 (85.7%) had nearly normal liver histology. The remaining 2 had mild nonspecific changes. Abnormal histological activity index score was observed in all 4 patients who had HBsAg seroclearance after age 50 and in only 3 of 10 (30%) patients who had HBsAg seroclearance before age 50 (P = .07).
Table 5. Histological Features of the 13 Patients With HBsAg Seroclearance
Cirrhosis-Related Complications and HCC
One patient had esophageal varices after HBeAg seroconversion but before HBsAg seroclearance. Four patients had HCC developing at 20, 21.1, 48, and 65.2 months after HBsAg seroclearance. One patient had ascites at 57 months after HBeAg seroconversion and HCC at 9 months after HBsAg seroclearance. All 6 patients had undetectable HBV DNA in the serum. Of the 5 patients with HCC, all except one had biochemical and radiological evidence of cirrhosis. One patient with HCC had previous interferon-alfa treatment. There were 11 controls (1 positive for HBeAg, 10 positive for anti-HBe) who developed cirrhosis-related complications and/or HCC. Of these, 7 had HCC only, 1 had esophageal varices, ascites, and HCC, and 3 had ascites and/or esophageal varices.
There was no significant difference in the risk of HCC between patients with and without HBsAg seroclearance: 5 of 92 (5.4%) versus 8 of 92 (8.70%), respectively; P = .39]. However, the mean age of HBsAg seroclearance of the 5 patients who subsequently developed HCC was significantly older than that of the remaining 87 patients: 63.16 years (+ 8.27) versus 47.94 years (+ 13.64), respectively; P = .016.
To our knowledge, the current study is the largest series studying the significance and various virological aspects of HBV patients with HBsAg seroclearance, though intrahepatic HBV DNA, liver histology, and HBV genotypes were determined in only a limited number of patients. Liver biochemistry on presentation and peak ALT levels during HBeAg seroconversion were of no predictive value for subsequent HBsAg seroclearance. Although 6 of 92 patients had prior interferon-alfa treatment, we have previously shown in a long-term follow-up study that interferon-alfa treatment does not significantly enhance HBsAg seroclearance in Chinese HBV patients.15 However, earlier HBeAg seroconversion and genotype B (Table 3) were associated with a higher chance of HBsAg seroclearance. Further studies on the effect of genotype in HBV diseases are indicated because some studies have reported that genotype C is associated with more serious HBV disease.16, 17 It may also be worthwhile to study serum HBV DNA levels and/or HBsAg titers after HBeAg seroconversion to see whether they are of predictive value for future HBsAg seroclearance. The majority of our patients (>98%) had undetectable serum HBV DNA levels using a sensitive PCR assay at a median duration of 49 months after HBsAg seroclearance. This confirmed the findings of several studies that serum HBV DNA becomes undetectable 1 to 2 years after HBsAg seroclearance.8, 9, 18–20 The extrahepatic reservoir for HBV DNA was also low since all 66 patients with PBMC testing were negative for HBV DNA. Mason et al. reported that 5 out of 12 patients with HBsAg seroclearance had HBV DNA in PBMC detectable by PCR.21 The difference in the results between our study and Mason et al. might be due to the fact that 9 of the 12 patients studied by Mason et al. had HBV DNA measured within 24 months of HBsAg seroclearance, whereas in our study, the median interval between the measurement of HBV DNA in PBMC and HBsAg seroclearance was 48 months (only 3 of 66 patients had HBV DNA measurement within 24 months of HBsAg seroclearance). It is possible that the viral load in the extrahepatic reservoir decreases with time.
However, a substantial number of these patients (37.5%) still had detectable HBV DNA in the liver, a finding similar to other studies.7–9, 22 We demonstrated quantitatively, that there was a decreasing trend in the amount of total intrahepatic HBV DNA for HBeAg-positive patients, anti-HBe-positive patients, and patients with HBsAg seroclearance (P < .0001). We applied and verified a novel means to quantify the amount of intrahepatic HBV DNA as copies per nanogram of human genomic DNA and copies per cell in order to avoid the error introduced by a gross estimation of tissue weight with HBV DNA expressed as copies per unit weight of tissue.
The present study also showed that, in patients with measurable intrahepatic HBV DNA, it was mainly in the form of cccDNA after HBsAg seroclearance (Table 4). Though we were using the novel Invader assay to measure cccDNA, this assay was stringently verified by synthetic oligonucleotide target specificity assay, S1 nuclease treatment, and intra-assay and interassay variation experiments (data not shown). It has also been verified by PCR technique. cccDNA forms the template for the transcription of viral RNAs. It has been suggested that HBV inside the liver in patients with HBsAg seroclearance is probably in a complete and uninterrupted form.8, 9 The failure to detect mRNA in the liver by hybridization in situ also suggests that viral transcription is minimal or at a rate slower than that of cell turnover.7 But in these previous reports, it was impossible to characterize the HBV DNA in the liver because the investigators were unable to detect HBV DNA by Southern-blot hybridization.7, 8 Our study suggests that the viral HBV DNA was predominantly in the form of cccDNA. However, the role of the integrated HBV DNA is not determined in the present study. The probes of the Invader assay used in this study would probably not detect integrated HBV DNA. Integration often occurs at the 11-base pair DR1 and DR2 regions, leading to breaching of the 2 regions,23, 24 and no hybridization would take place. Nevertheless, the presence of cccDNA in patients with HBsAg seroclearance may have some implications for the potential future development of HCC in patients with HBsAg seroclearance (see below). However, in the majority of these patients, the viral load inside the liver should continue to decrease if the hepatocyte turnover rate exceeds the transcription rate of viral RNAs from the small amount of cccDNA. In theory, therefore, the HBV inside the liver should eventually disappear through natural cell death, though considerable time may be required even after HBsAg seroclearance.
Despite these favorable parameters in patients with HBsAg seroclearance, we were unable to show a significant decrease in the risk for the occurrence of HCC. This finding is similar to other studies.5, 6, 22, 25–28 Of the 5 patients who developed HCC after HBsAg seroclearance, 4 already had evidence of cirrhosis at the time of development of HCC. In addition, the HBsAg seroclearance of these 5 patients occurred at a relatively older age compared to the remaining 87 patients (mean 63.16 years vs. 47.94 years, respectively; P = .016). The longest interval between HBsAg seroclearance and the detection of HCC was 65.2 months. It is possible that cirrhosis of the liver, and probably the process of hepatocarcinogenesis (i.e., the oncogenic effect of the integration of HBV DNA into the host), had already developed at the time of HBsAg seroclearance for these 5 patients. It is also possible that any residual cccDNA in the hepatocytes may still have had integrative capacity. Further studies are required to determine this. HBsAg seroclearance at a younger age is probably associated with a reduced risk of development of HCC. This is supported by the near normal histology of the 14 patients with assessable histology in whom the mean age of HBsAg seroclearance was 42 years. Further longitudinal follow-up of these patients for the occurrence of HBV-related complications is required to prove that HBsAg seroclearance at a relative young age confers good prognosis.
In conclusion, chronic HBV patients with HBsAg seroclearance are usually associated with favorable biochemical, virological, and histological parameters. Intrahepatic HBV DNA levels were extremely low and predominantly in the form of cccDNA. However, development of HCC is still possible, particularly in patients with cirrhosis who have HBsAg seroclearance at an older age.