Mol Biol Rep DOI 10.1007/s11033-014-3339-7
Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients Bhupesh Singla • Anuradha Chakraborti • Bal Krishan Sharma • Shweta Kapil • Yogesh K. Chawla • Sunil K. Arora • Ashim Das • Radha K. Dhiman • Ajay Duseja
Received: 2 October 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naı¨ve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naı¨ve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive
and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p \ 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3339-7) contains supplementary material, which is available to authorized users.
Keywords Hepatitis B virus HBV cccDNA Intrahepatic total HBV DNA Real time PCR Liver biopsy
B. Singla B. K. Sharma S. Kapil Y. K. Chawla (&) R. K. Dhiman A. Duseja Department of Hepatology, Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India e-mail: [email protected] A. Chakraborti Department of Experimental Medicine & Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India S. K. Arora Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India A. Das Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India
Introduction Chronic hepatitis B (CHB) is the most prevalent form of chronic viral hepatitis and constitutes a major health burden, despite the accessibility of an effective vaccine. It is a major cause of morbidity and mortality. CHB infection is found in more than 400 million people across the world [1]. Hepatitis B virus (HBV) accounts for approximately 50–80 % hepatocellular carcinoma (HCC) patients worldwide [2]. HBV life cycle involves the formation of intracellular replicative intermediate, covalently closed circular DNA (cccDNA) through repairing of plus strand of the relaxed circular DNA (rcDNA) within the nuclei of hepatocytes. The cccDNA acts as a template for the synthesis of
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viral and pregenomic messenger RNA. HBV replicates by employing HBV reverse transcriptase enzyme which lacks a proofreading function, hence the rate of mutations in HBV genome is high and creates drug resistant strains which contribute to the viral cccDNA pool [3]. The cccDNA being, the resource of new HBV progeny, is believed to be the main reason for HBV relapse after stoppage of antiviral therapy [4]. Therefore, HBV cccDNA level is considered direct indicator of HBV replication in the liver of HBV infected patients [5]. The majority of studies on estimation of HBV cccDNA levels have been performed with liver tissue samples either from duck or woodchucks [6–8]. As quantitation of HBV cccDNA levels require liver biopsies, so there are only small sample sized reports that have evaluated the role of antiviral therapy in the reduction of HBV cccDNA levels [9–11]. Few reports have studied the role of circulating cccDNA in plasma [12, 13]. There are some studies that have demonstrated the quantitation of HBV cccDNA levels in sera of chronic HBV patients [14–17]. A study by Chen et al. [18] has concluded the presence of HBV cccDNA in the serum of CHB patients as an early signal of liver injury. Earlier studies have shown detectable serum HBV cccDNA in 60–75 % HBV infected patients [15, 18]. However, the data on serum cccDNA levels in chronic HBV patients is scanty. In this report, we aimed to quantify the HBV cccDNA levels in a large number of serum samples from CHB patients. We also investigated intrahepatic levels of HBV DNA and cccDNA in available liver tissue samples of CHB patients.
Patients and methods Patients The study was conducted after getting approval from the Institutional Ethics committee of Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India and a written consent was obtained from each participating subjects. Eighty one chronic hepatitis B treatment naı¨ve patients characterized by positivity for HBsAg in serum for more than 6 months, who consulted the liver clinic at the Department of Hepatology, PGIMER, Chandigarh from January 2009 and June 2011, were enrolled in this study. HBV DNA levels were greater than 2000 IU/mL in HBeAg negative patients, while it was greater than 20000 IU/mL in case of HBeAg positive patients [19]. All subjects were negative for human immunodeficiency virus (HIV), and antibodies for hepatitis C virus (anti-HCV). Blood samples were collected from all enrolled subjects and 23 synchronous liver biopsies were collected out of
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enrolled patients. Liver biopsy was done in HBeAg negative chronic hepatitis B patients with normal (40 IU/mL) or 1–1.5 9 ULN ALT levels. Two liver biopsy samples were available from HBeAg positive chronic hepatitis B patients with family history of advanced liver disease (cirrhosis and HCC). Knodell scoring system was used to calculate histological activity index and fibrosis stage [20]. Three liver biopsies were also collected from patients with disease other than HBV infection (two males and one female, aged 24–48 years) and used as negative controls. All enrolled patients were evaluated for serum bilirubin, ALT, AST, serum total protein, albumin, globulin, HBeAg, antibodies to the HBV e antigen (anti-HBe), and anti-HCV. Liver biopsy samples were examined for fibrosis stage and necroinflammatory activity. HBsAg, HBeAg, anti-HBe and anti-HCV were detected using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The levels of serum HBV DNA were quantified by Cobas Amplicor HBV monitor test (Roche Diagnostics GmbH, Mannheim, Germany). Genotyping of hepatitis B virus For the extraction of HBV DNA from the serum samples, QiaAmp DNA mini kit (Qiagen GmbH, Hilden, Germany) was used according to the manufacturer’s instructions. The quality of DNA was determined by reading the extracted DNA samples at 260 and 280 nm (ratio at 260/280 = 1.8) using UV spectrophotometer (Beckman Coulter, Brea, USA). A multiplex PCR involving genotype-specific primers described previously, was performed to determine HBV genotype of all patients [21]. Multiplex PCR was carried out using Takara PCR Thermal Cycler DiceTM (Takara Bio Inc., Shiga, Japan) and was done with a total volume of 50 lL. Annealing temperature for multiplex PCR was 61 °C. The amplicons were electrophoresed on a 2.5 % agarose in TBE buffer containing 0.5 lg/mL ethidium bromide at 50 V for 1 h and evaluated under a UV transilluminator (UVP, Upland, USA). The sizes of the amplicons were determined by using a standard 50 bp DNA ladder (GeneDirex, Taipei, Taiwan). Standard preparation for HBV cccDNA quantification To prepare the standards for HBV cccDNA level determination, a fragment of 420 base pairs was amplified using cccDNA specific primers. The Integrity of amplicon was verified through size determination using agarose gel electrophoresis and bidirectional sequencing. The concentration of the amplified product was measured with a spectrophotometer (GeneQuant, Amersham, Germany) after purification using a Qiagen purification kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s
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instructions. The number of copies per unit volume (mL) was calculated using the formula described elsewhere [22]. Serial dilutions of this stock solution served as quantification standards to plot the standard curve. No: of copies=mL ¼
6:023 1023 C OD260 Mwt
where 6.023 9 1023 = Avogadro’s no., C = 5 9 10-5 g/mL for DNA, Mwt = bp 9 6.58 9 102 g, OD260 = Absorbance at 260 nm. Quantification of HBV cccDNA and intrahepatic total HBV DNA HBV DNA was extracted from serum and liver tissue samples by the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Real time PCR was performed on the LightCycler480 machine (Roche Diagnostics GmbH, Mannheim, Germany) using the SYBR Green detection method to determine the quantity of HBV cccDNA and intrahepatic total HBV DNA. The 20 lL reaction mixture contained 5 lL of the plasmid-safe ATP-dependent DNase treated DNA sample (for cccDNA quantification), 10 lL of 29 Fast Start SYBR Green master mix (Roche Diagnostics, Mannheim, Germany), 10 pmol of each primers and double distilled water. Forward and reverse primers were 50 GTGCCTTCTCATCTGCCGG- 30 and 50 - GGAAA GAAGTCAGAAGGCAA-30 for HBV cccDNA amplification, respectively, and 50 -GTATGTTGCCCGTTTGTCC30 and 50 -CCCTACGAACCACTGAAC- 30 for intrahepatic total HBV DNA quantification, respectively. Real time PCR was programmed to first incubate the samples for 10 min at 95 °C, followed by 45 cycles consisting of 94 °C
for 25 s, 52 °C for 26 s and 72 °C for 15 s to quantify HBV cccDNA levels. Annealing temperature for quantification of intrahepatic total HBV DNA was 55 °C for 20 s. In order to estimate cell number in liver tissues, b-globin gene was taken as internal control. LightCycler Control Kit (Roche Diagnostics GmbH, Mannheim, Germany) containing b-globin primers and human genomic DNA standards, was used according to manufacturer’s instructions to estimate the levels of genomic DNA and to calculate the number of cells in the liver tissue samples. Statistical analysis SPSS software (version 16) was used to perform all statistical analyses. The categorical variables were expressed as number and percent. Non-skewed continuous variables were summarized as mean ± SD, otherwise, as median along with range. The Chi square test was used for evaluating the significance of nominal categorical variables. Intergroup comparison of continuous skewed data was evaluated by Mann–Whitney U test. For determination of correlation between different variables Spearman’s correlation coefficient was used, whenever appropriate. Certain outliers and extreme values were not shown in graphs for their better and comprehensible presentation. A p value less than 0.05 indicated statistically significant difference.
Results Patient clinical features The clinical data of 81 treatment naı¨ve CHB patients is shown in Table 1. Of these, 63 (77.8 %) were males and 18
Table 1 Clinical characteristics of the study population HBeAg positive (n = 27)a
HBeAg negative (n = 54)b
Total (n = 81)
p value (a vs b)
Gender (M/F)
22/5
41/13
63/18
0.571
Age in years ± SD HBV genotype n (%)
28 ± 8.1
37.0 ± 11.9
34 ± 11.5
0.002
A
0 (0)
4 (7.40)
4 (4.93)
0.146
C
7 (25.92)
1 (1.85)
8 (9.87)
\0.001
D Mean bilirubin in mg/dl (± SD)
20 (74.07)
49 (90.74)
69 (85.18)
0.046
0.8 ± 0.6
0.9 ± 0.4
0.9 ± 0.5
0.179
Median AST (IU/L) (Range)
44.0 (21–343)
35.5 (17–988)
37.0 (17–988)
0.259
Median ALT (IU/L) (Range)
66.0 (26–293)
45.0 (17–730)
52.0 (17–713)
0.126
Median serum HBV DNA (log10IU/mL) (Range)
7.8 (4.4–10.0)
5.9 (3.8–9.8)
6.6 (3.8–10.0)
\0.001
Median Knodell HAI grade (Range)*
1.5 (1–2)
2 (0–6)
2 (0–6)
0.287
Median Ishak Fibrosis stage (Range)*
0
1 (1–3)
1 (1–3)
0.158
* Liver biopsy was done in 21 HBeAg negative and 2 HBeAg positive patients
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Fig. 1 a Correlation between ALT levels and fibrosis stage. b Correlation between ALT levels and serum HBV DNA levels
(22.2 %) were females. Fifty four patients (66.7 %) were HBeAg negative. The mean age was lesser in HBeAg positive patients (28 ± 8.1 years) than in patients with HBeAg negative disease (37.0 ± 11.9 years). The median serum HBV DNA level was 7.8 log10 copies/mL (range 4.4–10.0 log10 copies/mL) in HBeAg positive patients and 5.9 log10 copies/mL (range 3.8–9.8 log10 copies/mL) in HBeAg negative patients. The median values of ALT, AST, Knodell HAI grade, and Ishak fibrosis stage were comparable between HBeAg positive and HBeAg negative groups. A significant positive correlation was found between HBeAg positive status and serum HBV DNA levels (r = 0.416, p \ 0.001), and a significant negative correlation was observed between HBeAg positivity and age of patients (r = -0.354, p = 0.001). The ALT levels were found to correlate positively with fibrosis stage (r = 0.461, p = 0.031) and HBV DNA levels (r = 0.310, p = 0.005) (Fig. 1). Genotyping of hepatitis B virus Multiplex PCR performed using genotype-specific primers showed differences in the lengths of the amplicons according to the respective HBV genotypes. HBV genotype D was the most prevalent genotype and accounted for 85.2 % (69) patients. Genotype C and A were found in 9.9 % (8) and 4.9 % patients (4), respectively. HBV genotype did not show any correlation with ALT levels, fibrosis stage, serum HBV DNA levels, gender, and HAI grade. Quantification of intrahepatic total HBV DNA and intrahepatic HBV cccDNA The liver tissues of three patients with disease other than HBV infection were negative for total HBV DNA and
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HBV cccDNA. The levels of intrahepatic total HBV DNA and HBV cccDNA were determined in 23 liver tissue samples collected from chronically infected HBV patients (Table 2). The median of total intrahepatic HBV DNA was 819.26 copies/cell (range 129.59–1508.92 copies/cell) in HBeAg positive group, while it was 10.32 copies/cell (range 0.09–1164.50 copies/cell) in HBeAg negative group, which were significantly different (p = 0.038). The median intrahepatic HBV cccDNA was 0.31 copies/cell (range 0.14–0.49 copies/cell) and 0.20 copies/cell (range 0.01–1.63 copies/cell) in HBeAg positive and HBeAg negative cases, respectively. A significant positive correlation was observed between levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009) by combining the results of all patients regardless of HBeAg status. The ratio of HBV cccDNA to total HBV DNA was found negatively associated with intrahepatic total HBV DNA levels (r = -0.756, p \ 0.001) (Table 3; Fig. 2a). Quantification of serum HBV cccDNA Sera from all recruited patients were evaluated for the detection of HBV cccDNA by real time PCR method (Figs. S1–S3). In serum samples, HBV cccDNA was measurable in 49 patients (60.5 %). Out of these, 23 patients (28.4 %) were HBeAg positive and 26 patients (32.1 %) were with HBeAg negative status. Serum HBV cccDNA was measureable in 85.2 % (23/27) HBeAg positive and 48.1 % (26/54) HBeAg negative patients. The median level of serum HBV cccDNA was 46000 copies/mL (range 1,780–747,000 copies/mL) in HBeAg positive cases, while it was 26,350 copies/mL (range 530–73000 copies/mL) in HBeAg negative disease, which were significantly different (p = 0.013) (Table 2). A significant positive correlation was detected between serum HBV cccDNA and serum HBV DNA levels in both HBeAg positive and negative
Mol Biol Rep Table 2 Intrahepatic HBV cccDNA, total HBV DNA levels and serum HBV cccDNA levels in study population HBeAg positive (2)a
HBeAg negative (21)b
Total (23)
p value (a vs b)
Median intrahepatic total HBV DNA in copies/cell (Range)
819.26 (129.59–1508.92)
10.32 (0.09–1164.50)
13.98 (0.09–1508.92)
0.038
Median intrahepatic cccDNA in copies/cell (Range)
0.31 (0.14–0.49)
0.20 (0.01–1.63)
0.20 (0.01–1.63)
0.585
Median cccDNA/total DNA (%) (Range)
0.19 % (0.01–0 .37)
1.45 % (0.03–22.06)
1.28 % (0.019–22.06)
0.038
Median serum HBV cccDNA level in copies/mL (Range)
46,000 (n = 23) (1,780–747,000)
26,350 (n = 26) (530–73,000)
41,000 (n = 49) (530–747,000)
0.013
Table 3 Observed significant correlation among different parameters Correlation coefficient (r)
P value
HBeAg negative patients Serum HBV DNA ALT (n = 54)
0.449
0.001
Intrahepatic total HBV DNA (n = 21)
0.588
0.005
Serum cccDNA (n = 26)
0.865
\0.001
0.575
0.006
0.850
\0.001
Serum HBV DNA (n = 81)
0.310
0.005
Fibrosis stage (n = 23) AST (n = 81)
0.461 0.814
0.031 \0.001
Intrahepatic cccDNA Intrahepatic total HBV DNA (n = 21) HBeAg positive patients Serum HBV DNA Serum cccDNA (n = 23) Overall ALT
Intrahepatic total HBV DNA Intrahepatic cccDNA (n = 23) Ratio intrahepatic cccDNA/ intrahepatic total DNA (n = 23) Serum HBV DNA (n = 23)
0.533
0.009
-0.756
\0.001
0.683
\0.001
0.871
\0.001
Serum cccDNA Serum HBV DNA (n = 49)
patients (Table 3; Fig. 2b, c). The details of observed significant correlation among different parameters are given in Table 3.
Discussion The pool of HBV cccDNA present in the nuclei of hepatocytes is responsible for the chronicity of HBV infection [23, 24]. The mechanism of HBV cccDNA elimination is still not clear. Hepatocytes turnover may be responsible for
the elimination of HBV cccDNA. Major obstacles to determine the cccDNA levels include (1) requirement of liver biopsies; (2) lack of ‘‘gold standard’’ method for quantification of cccDNA levels. In this study, we have developed and employed SYBR Green detection method for the estimation of HBV cccDNA and intrahepatic total HBV DNA using real time PCR in liver biopsy and serum samples of chronic HBV patients. The primers used in this study for determination of cccDNA levels in serum and tissue samples, amplified the conserved region flanking the direct repeats DR1 and DR2, therefore, it avoided the nonspecific signal caused by amplification of rcDNA of hepatitis B virus. The present study found a positive correlation between HBeAg positive status and serum HBV DNA levels (r = 0.416, p \ 0.001) and a negative correlation between HBeAg positivity and age of patients (r = -0.354, p = 0.001) in chronic HBV patients. Similar correlations have been observed in earlier report [25]. A significant positive correlation was observed between the fibrosis stage and ALT levels (r = 0.461, p = 0.031). Previous study has also observed the positive correlation of ALT with fibrosis stage and HAI grade (r = 0.744, p = \ 0.01) in HBeAg positive patients [25]. The present study found a positive correlation of ALT levels with serum HBV DNA levels (r = 0.310, p = 0.005) in HBeAg negative patients. A study by Thompson et al. [11] has shown the association of ALT levels with serum HBV DNA concentration. A strong correlation has also been revealed between the serum HBV DNA and ALT levels in Japanese HBeAg negative subjects [26]. The intrahepatic total HBV DNA and HBV cccDNA were determined in 23 liver tissue samples (21 HBeAg negative and 2 HBeAg positive) of CHB patients. The median intrahepatic total HBV DNA was observed 819.26 and 10.32 copies/cell in HBeAg positive and HBeAg negative patients, respectively. Earlier studies have also shown higher median value of intrahepatic HBV DNA in HBeAg positive patients in comparison to HBeAg negative chronic HBV patients [9, 15]. The observed ranges of total
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Fig. 2 a Correlation between intrahepatic total HBV DNA and ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA. b Correlation of intrahepatic total HBV DNA with serum HBV DNA levels. c Correlation of serum HBV DNA levels with serum HBV cccDNA levels
intrahepatic HBV DNA were 129.59–1508.92 and 0.09–1164.50 copies/cell in HBeAg positive and negative patients, respectively. These observed ranges of total intrahepatic HBV DNA were also in concordance with previous studies [9, 15]. In the present study, median intrahepatic HBV cccDNA was not significantly higher in HBeAg positive cases compared to HBeAg negative patients (0.31 vs 0.20 copies/cell, p = 0.585). Thompson et al. [11] has found median intrahepatic HBV cccDNA levels of 3.65 and 0.2 copies/cell in HBeAg positive and negative patients, respectively (p \ 0.001) . A study from Hong Kong observed median intrahepatic HBV cccDNA of 15.8 copies/cell (0.1–141.8 copies/cell) and 2 copies/cell (0.02–213.0 copies/cell) in HBeAg positive and HBeAg negative CHB patients, respectively (p \ 0.001) [27]. This difference in the findings may be because of less liver tissue samples from HBeAg positive CHB patients in our study. The present study found a positive correlation between intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009) and an inverse correlation
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between the ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA and intrahepatic total HBV DNA levels (r = -0.756, p \ 0.001). A recently published study by Wang et al. [28] has observed the similar correlation among these parameters (r = 0.432 for intrahepatic total HBV DNA and intrahepatic HBV cccDNA; r = -0.812 for the ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA and intrahepatic total HBV DNA levels) in treatment naı¨ve chronic hepatitis B patients. These results suggest that the presence of the lower level of intrahepatic HBV DNA would increase the HBV cccDNA/ total HBV DNA ratio, entirely in the form of non-replicative HBV cccDNA. Similar results have been obtained in previous study involving patients with chronic hepatitis B [14]. The source of cccDNA in serum of HBV infected patients is unclear. It is believed that a majority of serum cccDNA originates from the destruction of HBV infected hepatocytes, thereby releasing HBV cccDNA present in the nuclei of hepatocytes into the blood circulation. Peripheral blood mononuclear cells make some contribution to the
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pool of cccDNA in serum of HBV infected patients [29]. There is a study which has shown the loss of intrahepatic cccDNA during the process of cell division, this can also be responsible for presence of cccDNA in serum [30]. This is the first report from India, which evaluated the levels of HBV cccDNA in serum samples of 81 CHB patients. Serum HBV cccDNA was measureable in 60.5 % CHB patients. Previous studies have detected serum HBV cccDNA in 66–75 % CHB subjects [14, 15]. Approximately 85 % HBeAg positive and 48 % HBeAg negative patients showed the presence of HBV cccDNA in sera of patients. The levels of serum HBV cccDNA ranged from 1,780 to 747,000 copies/mL in HBeAg positive cases, which was higher than that observed in HBeAg negative patients. These results are in concordance with previous studies [14, 15]. As mentioned earlier, there was a positive correlation between serum HBV cccDNA and serum HBV DNA levels in all enrolled subjects [14, 15]. However, the current study detected lower serum cccDNA levels than those reported by Wong et al. [14]. This difference in results may reflect differences in method used for the quantification of HBV cccDNA. Wong et al. (2004) used invader assay to quantify total HBV DNA and HBV cccDNA in sera of CHB patients. It is considered that this method detected rcDNA rather than HBV cccDNA. Till date, there is only one study from China evaluating the effect of treatment (lamivudine) on serum HBV cccDNA [17]. The authors have performed the combined estimation of serum HBV DNA and serum HBV cccDNA during the course of lamivudine treatment. The authors observed a decline of 1.96 log and 1.8 log in median serum HBV cccDNA after 6 and 12 months of lamivudine treatment, respectively. However, treatment with lamivudine led to 4.37 log and 3.96 log decrease in median serum HBV DNA level after 6 and 12 months of treatment, respectively. They have concluded that monitoring the levels of total HBV DNA along with cccDNA in the serum could provide more information about the drop in both the replicative and non-replicative forms of virus. Hence, the data on combined estimation of serum HBV DNA and serum HBV cccDNA is limited. Six HBeAg negative CHB patients had serum HBV DNA levels greater than 8 log10 IU/mL in the present study, which is the possible reason for high median serum HBV DNA (5.9 log10 IU/mL) in HBeAg negative CHB patients. A previous study involving HBeAg negative patients has also shown that HBV DNA level in HBeAg negative CHB patients may be higher than 9 log10 copies/mL (1 IU/mL = 5.82 copies/mL) [31]. Similarly, another study has shown that 0.6 % of HBeAg negative CHB patients can have HBV DNA levels greater than 8 log10 copies/mL [32]. In conclusion, the present study showed the estimation of the levels of HBV cccDNA both in the liver biopsies and
serum samples of chronic HBV patients. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. HBeAg positive patients had superior serum cccDNA levels than HBeAg negative patients. The combined measurement of serum HBV DNA and serum cccDNA during the treatment can provide useful information about the stage of the disease and duration of therapy needed. Acknowledgments The authors have no competing interests. This work was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. (ICMR No: VIR/28/2010-ECD-I). Authors are grateful to ICMR for giving Junior Research Fellowship to Bhupesh Singla (3/1/3JRF-2008/MPD, dated 1/9/2008).
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24. Newbold JE, Xin H, Tencza M et al (1995) The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 69:3350–3357 25. Shao J, Wei L, Wang H et al (2007) Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B. World J Gastroenterol 13:2104–2107 26. Sakugawa H, Nakasone H, Nakayoshi T et al (2001) Correlation between serum transaminase activity and virus load among patients with chronic liver disease type B. Hepatol Res 21:159–168 27. Wong DK, Seto WK, Fung J et al (2013) Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency. Clin Gastroenterol Hepatol 11:1004–1010 28. Wang M, Qiu N, Lu S et al (2013) Serum hepatitis B surface antigen is correlated with intrahepatic total HBV DNA and cccDNA in treatment-naı¨ve patients with chronic hepatitis B but not in patients with HBV related hepatocellular carcinoma. J Med Virol 85:219–227 29. Cabrerizo M, Bartolome´ J, Caramelo C, Barril G, Carreno V (2000) Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen-negative cases. Hepatology 32:116–123 30. Moraleda G, Saputelli J, Aldrich CE, Averett D, Condreay L, Mason WS (1997) Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol 71:9392–9399 31. Lampertico P, Vigano` M, Manenti E, Iavarone M, Lunghi G, Colombo M (2005) Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology 42:1414–1419 32. Chen CJ, Yang HI, Su J et al (2006) Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 295:65–73
Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients Bhupesh Singla • Anuradha Chakraborti • Bal Krishan Sharma • Shweta Kapil • Yogesh K. Chawla • Sunil K. Arora • Ashim Das • Radha K. Dhiman • Ajay Duseja
Received: 2 October 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naı¨ve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naı¨ve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive
and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p \ 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3339-7) contains supplementary material, which is available to authorized users.
Keywords Hepatitis B virus HBV cccDNA Intrahepatic total HBV DNA Real time PCR Liver biopsy
B. Singla B. K. Sharma S. Kapil Y. K. Chawla (&) R. K. Dhiman A. Duseja Department of Hepatology, Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India e-mail: [email protected] A. Chakraborti Department of Experimental Medicine & Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India S. K. Arora Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India A. Das Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India
Introduction Chronic hepatitis B (CHB) is the most prevalent form of chronic viral hepatitis and constitutes a major health burden, despite the accessibility of an effective vaccine. It is a major cause of morbidity and mortality. CHB infection is found in more than 400 million people across the world [1]. Hepatitis B virus (HBV) accounts for approximately 50–80 % hepatocellular carcinoma (HCC) patients worldwide [2]. HBV life cycle involves the formation of intracellular replicative intermediate, covalently closed circular DNA (cccDNA) through repairing of plus strand of the relaxed circular DNA (rcDNA) within the nuclei of hepatocytes. The cccDNA acts as a template for the synthesis of
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viral and pregenomic messenger RNA. HBV replicates by employing HBV reverse transcriptase enzyme which lacks a proofreading function, hence the rate of mutations in HBV genome is high and creates drug resistant strains which contribute to the viral cccDNA pool [3]. The cccDNA being, the resource of new HBV progeny, is believed to be the main reason for HBV relapse after stoppage of antiviral therapy [4]. Therefore, HBV cccDNA level is considered direct indicator of HBV replication in the liver of HBV infected patients [5]. The majority of studies on estimation of HBV cccDNA levels have been performed with liver tissue samples either from duck or woodchucks [6–8]. As quantitation of HBV cccDNA levels require liver biopsies, so there are only small sample sized reports that have evaluated the role of antiviral therapy in the reduction of HBV cccDNA levels [9–11]. Few reports have studied the role of circulating cccDNA in plasma [12, 13]. There are some studies that have demonstrated the quantitation of HBV cccDNA levels in sera of chronic HBV patients [14–17]. A study by Chen et al. [18] has concluded the presence of HBV cccDNA in the serum of CHB patients as an early signal of liver injury. Earlier studies have shown detectable serum HBV cccDNA in 60–75 % HBV infected patients [15, 18]. However, the data on serum cccDNA levels in chronic HBV patients is scanty. In this report, we aimed to quantify the HBV cccDNA levels in a large number of serum samples from CHB patients. We also investigated intrahepatic levels of HBV DNA and cccDNA in available liver tissue samples of CHB patients.
Patients and methods Patients The study was conducted after getting approval from the Institutional Ethics committee of Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India and a written consent was obtained from each participating subjects. Eighty one chronic hepatitis B treatment naı¨ve patients characterized by positivity for HBsAg in serum for more than 6 months, who consulted the liver clinic at the Department of Hepatology, PGIMER, Chandigarh from January 2009 and June 2011, were enrolled in this study. HBV DNA levels were greater than 2000 IU/mL in HBeAg negative patients, while it was greater than 20000 IU/mL in case of HBeAg positive patients [19]. All subjects were negative for human immunodeficiency virus (HIV), and antibodies for hepatitis C virus (anti-HCV). Blood samples were collected from all enrolled subjects and 23 synchronous liver biopsies were collected out of
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enrolled patients. Liver biopsy was done in HBeAg negative chronic hepatitis B patients with normal (40 IU/mL) or 1–1.5 9 ULN ALT levels. Two liver biopsy samples were available from HBeAg positive chronic hepatitis B patients with family history of advanced liver disease (cirrhosis and HCC). Knodell scoring system was used to calculate histological activity index and fibrosis stage [20]. Three liver biopsies were also collected from patients with disease other than HBV infection (two males and one female, aged 24–48 years) and used as negative controls. All enrolled patients were evaluated for serum bilirubin, ALT, AST, serum total protein, albumin, globulin, HBeAg, antibodies to the HBV e antigen (anti-HBe), and anti-HCV. Liver biopsy samples were examined for fibrosis stage and necroinflammatory activity. HBsAg, HBeAg, anti-HBe and anti-HCV were detected using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The levels of serum HBV DNA were quantified by Cobas Amplicor HBV monitor test (Roche Diagnostics GmbH, Mannheim, Germany). Genotyping of hepatitis B virus For the extraction of HBV DNA from the serum samples, QiaAmp DNA mini kit (Qiagen GmbH, Hilden, Germany) was used according to the manufacturer’s instructions. The quality of DNA was determined by reading the extracted DNA samples at 260 and 280 nm (ratio at 260/280 = 1.8) using UV spectrophotometer (Beckman Coulter, Brea, USA). A multiplex PCR involving genotype-specific primers described previously, was performed to determine HBV genotype of all patients [21]. Multiplex PCR was carried out using Takara PCR Thermal Cycler DiceTM (Takara Bio Inc., Shiga, Japan) and was done with a total volume of 50 lL. Annealing temperature for multiplex PCR was 61 °C. The amplicons were electrophoresed on a 2.5 % agarose in TBE buffer containing 0.5 lg/mL ethidium bromide at 50 V for 1 h and evaluated under a UV transilluminator (UVP, Upland, USA). The sizes of the amplicons were determined by using a standard 50 bp DNA ladder (GeneDirex, Taipei, Taiwan). Standard preparation for HBV cccDNA quantification To prepare the standards for HBV cccDNA level determination, a fragment of 420 base pairs was amplified using cccDNA specific primers. The Integrity of amplicon was verified through size determination using agarose gel electrophoresis and bidirectional sequencing. The concentration of the amplified product was measured with a spectrophotometer (GeneQuant, Amersham, Germany) after purification using a Qiagen purification kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s
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instructions. The number of copies per unit volume (mL) was calculated using the formula described elsewhere [22]. Serial dilutions of this stock solution served as quantification standards to plot the standard curve. No: of copies=mL ¼
6:023 1023 C OD260 Mwt
where 6.023 9 1023 = Avogadro’s no., C = 5 9 10-5 g/mL for DNA, Mwt = bp 9 6.58 9 102 g, OD260 = Absorbance at 260 nm. Quantification of HBV cccDNA and intrahepatic total HBV DNA HBV DNA was extracted from serum and liver tissue samples by the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Real time PCR was performed on the LightCycler480 machine (Roche Diagnostics GmbH, Mannheim, Germany) using the SYBR Green detection method to determine the quantity of HBV cccDNA and intrahepatic total HBV DNA. The 20 lL reaction mixture contained 5 lL of the plasmid-safe ATP-dependent DNase treated DNA sample (for cccDNA quantification), 10 lL of 29 Fast Start SYBR Green master mix (Roche Diagnostics, Mannheim, Germany), 10 pmol of each primers and double distilled water. Forward and reverse primers were 50 GTGCCTTCTCATCTGCCGG- 30 and 50 - GGAAA GAAGTCAGAAGGCAA-30 for HBV cccDNA amplification, respectively, and 50 -GTATGTTGCCCGTTTGTCC30 and 50 -CCCTACGAACCACTGAAC- 30 for intrahepatic total HBV DNA quantification, respectively. Real time PCR was programmed to first incubate the samples for 10 min at 95 °C, followed by 45 cycles consisting of 94 °C
for 25 s, 52 °C for 26 s and 72 °C for 15 s to quantify HBV cccDNA levels. Annealing temperature for quantification of intrahepatic total HBV DNA was 55 °C for 20 s. In order to estimate cell number in liver tissues, b-globin gene was taken as internal control. LightCycler Control Kit (Roche Diagnostics GmbH, Mannheim, Germany) containing b-globin primers and human genomic DNA standards, was used according to manufacturer’s instructions to estimate the levels of genomic DNA and to calculate the number of cells in the liver tissue samples. Statistical analysis SPSS software (version 16) was used to perform all statistical analyses. The categorical variables were expressed as number and percent. Non-skewed continuous variables were summarized as mean ± SD, otherwise, as median along with range. The Chi square test was used for evaluating the significance of nominal categorical variables. Intergroup comparison of continuous skewed data was evaluated by Mann–Whitney U test. For determination of correlation between different variables Spearman’s correlation coefficient was used, whenever appropriate. Certain outliers and extreme values were not shown in graphs for their better and comprehensible presentation. A p value less than 0.05 indicated statistically significant difference.
Results Patient clinical features The clinical data of 81 treatment naı¨ve CHB patients is shown in Table 1. Of these, 63 (77.8 %) were males and 18
Table 1 Clinical characteristics of the study population HBeAg positive (n = 27)a
HBeAg negative (n = 54)b
Total (n = 81)
p value (a vs b)
Gender (M/F)
22/5
41/13
63/18
0.571
Age in years ± SD HBV genotype n (%)
28 ± 8.1
37.0 ± 11.9
34 ± 11.5
0.002
A
0 (0)
4 (7.40)
4 (4.93)
0.146
C
7 (25.92)
1 (1.85)
8 (9.87)
\0.001
D Mean bilirubin in mg/dl (± SD)
20 (74.07)
49 (90.74)
69 (85.18)
0.046
0.8 ± 0.6
0.9 ± 0.4
0.9 ± 0.5
0.179
Median AST (IU/L) (Range)
44.0 (21–343)
35.5 (17–988)
37.0 (17–988)
0.259
Median ALT (IU/L) (Range)
66.0 (26–293)
45.0 (17–730)
52.0 (17–713)
0.126
Median serum HBV DNA (log10IU/mL) (Range)
7.8 (4.4–10.0)
5.9 (3.8–9.8)
6.6 (3.8–10.0)
\0.001
Median Knodell HAI grade (Range)*
1.5 (1–2)
2 (0–6)
2 (0–6)
0.287
Median Ishak Fibrosis stage (Range)*
0
1 (1–3)
1 (1–3)
0.158
* Liver biopsy was done in 21 HBeAg negative and 2 HBeAg positive patients
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Fig. 1 a Correlation between ALT levels and fibrosis stage. b Correlation between ALT levels and serum HBV DNA levels
(22.2 %) were females. Fifty four patients (66.7 %) were HBeAg negative. The mean age was lesser in HBeAg positive patients (28 ± 8.1 years) than in patients with HBeAg negative disease (37.0 ± 11.9 years). The median serum HBV DNA level was 7.8 log10 copies/mL (range 4.4–10.0 log10 copies/mL) in HBeAg positive patients and 5.9 log10 copies/mL (range 3.8–9.8 log10 copies/mL) in HBeAg negative patients. The median values of ALT, AST, Knodell HAI grade, and Ishak fibrosis stage were comparable between HBeAg positive and HBeAg negative groups. A significant positive correlation was found between HBeAg positive status and serum HBV DNA levels (r = 0.416, p \ 0.001), and a significant negative correlation was observed between HBeAg positivity and age of patients (r = -0.354, p = 0.001). The ALT levels were found to correlate positively with fibrosis stage (r = 0.461, p = 0.031) and HBV DNA levels (r = 0.310, p = 0.005) (Fig. 1). Genotyping of hepatitis B virus Multiplex PCR performed using genotype-specific primers showed differences in the lengths of the amplicons according to the respective HBV genotypes. HBV genotype D was the most prevalent genotype and accounted for 85.2 % (69) patients. Genotype C and A were found in 9.9 % (8) and 4.9 % patients (4), respectively. HBV genotype did not show any correlation with ALT levels, fibrosis stage, serum HBV DNA levels, gender, and HAI grade. Quantification of intrahepatic total HBV DNA and intrahepatic HBV cccDNA The liver tissues of three patients with disease other than HBV infection were negative for total HBV DNA and
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HBV cccDNA. The levels of intrahepatic total HBV DNA and HBV cccDNA were determined in 23 liver tissue samples collected from chronically infected HBV patients (Table 2). The median of total intrahepatic HBV DNA was 819.26 copies/cell (range 129.59–1508.92 copies/cell) in HBeAg positive group, while it was 10.32 copies/cell (range 0.09–1164.50 copies/cell) in HBeAg negative group, which were significantly different (p = 0.038). The median intrahepatic HBV cccDNA was 0.31 copies/cell (range 0.14–0.49 copies/cell) and 0.20 copies/cell (range 0.01–1.63 copies/cell) in HBeAg positive and HBeAg negative cases, respectively. A significant positive correlation was observed between levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009) by combining the results of all patients regardless of HBeAg status. The ratio of HBV cccDNA to total HBV DNA was found negatively associated with intrahepatic total HBV DNA levels (r = -0.756, p \ 0.001) (Table 3; Fig. 2a). Quantification of serum HBV cccDNA Sera from all recruited patients were evaluated for the detection of HBV cccDNA by real time PCR method (Figs. S1–S3). In serum samples, HBV cccDNA was measurable in 49 patients (60.5 %). Out of these, 23 patients (28.4 %) were HBeAg positive and 26 patients (32.1 %) were with HBeAg negative status. Serum HBV cccDNA was measureable in 85.2 % (23/27) HBeAg positive and 48.1 % (26/54) HBeAg negative patients. The median level of serum HBV cccDNA was 46000 copies/mL (range 1,780–747,000 copies/mL) in HBeAg positive cases, while it was 26,350 copies/mL (range 530–73000 copies/mL) in HBeAg negative disease, which were significantly different (p = 0.013) (Table 2). A significant positive correlation was detected between serum HBV cccDNA and serum HBV DNA levels in both HBeAg positive and negative
Mol Biol Rep Table 2 Intrahepatic HBV cccDNA, total HBV DNA levels and serum HBV cccDNA levels in study population HBeAg positive (2)a
HBeAg negative (21)b
Total (23)
p value (a vs b)
Median intrahepatic total HBV DNA in copies/cell (Range)
819.26 (129.59–1508.92)
10.32 (0.09–1164.50)
13.98 (0.09–1508.92)
0.038
Median intrahepatic cccDNA in copies/cell (Range)
0.31 (0.14–0.49)
0.20 (0.01–1.63)
0.20 (0.01–1.63)
0.585
Median cccDNA/total DNA (%) (Range)
0.19 % (0.01–0 .37)
1.45 % (0.03–22.06)
1.28 % (0.019–22.06)
0.038
Median serum HBV cccDNA level in copies/mL (Range)
46,000 (n = 23) (1,780–747,000)
26,350 (n = 26) (530–73,000)
41,000 (n = 49) (530–747,000)
0.013
Table 3 Observed significant correlation among different parameters Correlation coefficient (r)
P value
HBeAg negative patients Serum HBV DNA ALT (n = 54)
0.449
0.001
Intrahepatic total HBV DNA (n = 21)
0.588
0.005
Serum cccDNA (n = 26)
0.865
\0.001
0.575
0.006
0.850
\0.001
Serum HBV DNA (n = 81)
0.310
0.005
Fibrosis stage (n = 23) AST (n = 81)
0.461 0.814
0.031 \0.001
Intrahepatic cccDNA Intrahepatic total HBV DNA (n = 21) HBeAg positive patients Serum HBV DNA Serum cccDNA (n = 23) Overall ALT
Intrahepatic total HBV DNA Intrahepatic cccDNA (n = 23) Ratio intrahepatic cccDNA/ intrahepatic total DNA (n = 23) Serum HBV DNA (n = 23)
0.533
0.009
-0.756
\0.001
0.683
\0.001
0.871
\0.001
Serum cccDNA Serum HBV DNA (n = 49)
patients (Table 3; Fig. 2b, c). The details of observed significant correlation among different parameters are given in Table 3.
Discussion The pool of HBV cccDNA present in the nuclei of hepatocytes is responsible for the chronicity of HBV infection [23, 24]. The mechanism of HBV cccDNA elimination is still not clear. Hepatocytes turnover may be responsible for
the elimination of HBV cccDNA. Major obstacles to determine the cccDNA levels include (1) requirement of liver biopsies; (2) lack of ‘‘gold standard’’ method for quantification of cccDNA levels. In this study, we have developed and employed SYBR Green detection method for the estimation of HBV cccDNA and intrahepatic total HBV DNA using real time PCR in liver biopsy and serum samples of chronic HBV patients. The primers used in this study for determination of cccDNA levels in serum and tissue samples, amplified the conserved region flanking the direct repeats DR1 and DR2, therefore, it avoided the nonspecific signal caused by amplification of rcDNA of hepatitis B virus. The present study found a positive correlation between HBeAg positive status and serum HBV DNA levels (r = 0.416, p \ 0.001) and a negative correlation between HBeAg positivity and age of patients (r = -0.354, p = 0.001) in chronic HBV patients. Similar correlations have been observed in earlier report [25]. A significant positive correlation was observed between the fibrosis stage and ALT levels (r = 0.461, p = 0.031). Previous study has also observed the positive correlation of ALT with fibrosis stage and HAI grade (r = 0.744, p = \ 0.01) in HBeAg positive patients [25]. The present study found a positive correlation of ALT levels with serum HBV DNA levels (r = 0.310, p = 0.005) in HBeAg negative patients. A study by Thompson et al. [11] has shown the association of ALT levels with serum HBV DNA concentration. A strong correlation has also been revealed between the serum HBV DNA and ALT levels in Japanese HBeAg negative subjects [26]. The intrahepatic total HBV DNA and HBV cccDNA were determined in 23 liver tissue samples (21 HBeAg negative and 2 HBeAg positive) of CHB patients. The median intrahepatic total HBV DNA was observed 819.26 and 10.32 copies/cell in HBeAg positive and HBeAg negative patients, respectively. Earlier studies have also shown higher median value of intrahepatic HBV DNA in HBeAg positive patients in comparison to HBeAg negative chronic HBV patients [9, 15]. The observed ranges of total
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Fig. 2 a Correlation between intrahepatic total HBV DNA and ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA. b Correlation of intrahepatic total HBV DNA with serum HBV DNA levels. c Correlation of serum HBV DNA levels with serum HBV cccDNA levels
intrahepatic HBV DNA were 129.59–1508.92 and 0.09–1164.50 copies/cell in HBeAg positive and negative patients, respectively. These observed ranges of total intrahepatic HBV DNA were also in concordance with previous studies [9, 15]. In the present study, median intrahepatic HBV cccDNA was not significantly higher in HBeAg positive cases compared to HBeAg negative patients (0.31 vs 0.20 copies/cell, p = 0.585). Thompson et al. [11] has found median intrahepatic HBV cccDNA levels of 3.65 and 0.2 copies/cell in HBeAg positive and negative patients, respectively (p \ 0.001) . A study from Hong Kong observed median intrahepatic HBV cccDNA of 15.8 copies/cell (0.1–141.8 copies/cell) and 2 copies/cell (0.02–213.0 copies/cell) in HBeAg positive and HBeAg negative CHB patients, respectively (p \ 0.001) [27]. This difference in the findings may be because of less liver tissue samples from HBeAg positive CHB patients in our study. The present study found a positive correlation between intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009) and an inverse correlation
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between the ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA and intrahepatic total HBV DNA levels (r = -0.756, p \ 0.001). A recently published study by Wang et al. [28] has observed the similar correlation among these parameters (r = 0.432 for intrahepatic total HBV DNA and intrahepatic HBV cccDNA; r = -0.812 for the ratio of intrahepatic HBV cccDNA/intrahepatic total HBV DNA and intrahepatic total HBV DNA levels) in treatment naı¨ve chronic hepatitis B patients. These results suggest that the presence of the lower level of intrahepatic HBV DNA would increase the HBV cccDNA/ total HBV DNA ratio, entirely in the form of non-replicative HBV cccDNA. Similar results have been obtained in previous study involving patients with chronic hepatitis B [14]. The source of cccDNA in serum of HBV infected patients is unclear. It is believed that a majority of serum cccDNA originates from the destruction of HBV infected hepatocytes, thereby releasing HBV cccDNA present in the nuclei of hepatocytes into the blood circulation. Peripheral blood mononuclear cells make some contribution to the
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pool of cccDNA in serum of HBV infected patients [29]. There is a study which has shown the loss of intrahepatic cccDNA during the process of cell division, this can also be responsible for presence of cccDNA in serum [30]. This is the first report from India, which evaluated the levels of HBV cccDNA in serum samples of 81 CHB patients. Serum HBV cccDNA was measureable in 60.5 % CHB patients. Previous studies have detected serum HBV cccDNA in 66–75 % CHB subjects [14, 15]. Approximately 85 % HBeAg positive and 48 % HBeAg negative patients showed the presence of HBV cccDNA in sera of patients. The levels of serum HBV cccDNA ranged from 1,780 to 747,000 copies/mL in HBeAg positive cases, which was higher than that observed in HBeAg negative patients. These results are in concordance with previous studies [14, 15]. As mentioned earlier, there was a positive correlation between serum HBV cccDNA and serum HBV DNA levels in all enrolled subjects [14, 15]. However, the current study detected lower serum cccDNA levels than those reported by Wong et al. [14]. This difference in results may reflect differences in method used for the quantification of HBV cccDNA. Wong et al. (2004) used invader assay to quantify total HBV DNA and HBV cccDNA in sera of CHB patients. It is considered that this method detected rcDNA rather than HBV cccDNA. Till date, there is only one study from China evaluating the effect of treatment (lamivudine) on serum HBV cccDNA [17]. The authors have performed the combined estimation of serum HBV DNA and serum HBV cccDNA during the course of lamivudine treatment. The authors observed a decline of 1.96 log and 1.8 log in median serum HBV cccDNA after 6 and 12 months of lamivudine treatment, respectively. However, treatment with lamivudine led to 4.37 log and 3.96 log decrease in median serum HBV DNA level after 6 and 12 months of treatment, respectively. They have concluded that monitoring the levels of total HBV DNA along with cccDNA in the serum could provide more information about the drop in both the replicative and non-replicative forms of virus. Hence, the data on combined estimation of serum HBV DNA and serum HBV cccDNA is limited. Six HBeAg negative CHB patients had serum HBV DNA levels greater than 8 log10 IU/mL in the present study, which is the possible reason for high median serum HBV DNA (5.9 log10 IU/mL) in HBeAg negative CHB patients. A previous study involving HBeAg negative patients has also shown that HBV DNA level in HBeAg negative CHB patients may be higher than 9 log10 copies/mL (1 IU/mL = 5.82 copies/mL) [31]. Similarly, another study has shown that 0.6 % of HBeAg negative CHB patients can have HBV DNA levels greater than 8 log10 copies/mL [32]. In conclusion, the present study showed the estimation of the levels of HBV cccDNA both in the liver biopsies and
serum samples of chronic HBV patients. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. HBeAg positive patients had superior serum cccDNA levels than HBeAg negative patients. The combined measurement of serum HBV DNA and serum cccDNA during the treatment can provide useful information about the stage of the disease and duration of therapy needed. Acknowledgments The authors have no competing interests. This work was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. (ICMR No: VIR/28/2010-ECD-I). Authors are grateful to ICMR for giving Junior Research Fellowship to Bhupesh Singla (3/1/3JRF-2008/MPD, dated 1/9/2008).
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