Intrahepatic Hepatitis B Virus cccDNA Research Articles

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.

Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis.

Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA is the key molecule responsible for viral persistence within infected hepatocytes. The evaluation of HBV cccDNA is crucial for the management of patients with chronic HBV infection and for the personalization of treatment. However, the need for liver biopsy is the principal obstacle for the assessment of intrahepatic HBV cccDNA. In the last decade, several studies have investigated the performance of hepatitis B core-related antigen (HBcrAg) as a surrogate of HBV cccDNA amount in the liver. In this meta-analysis, we collected 14 studies (1271 patients) investigating the correlation between serum HBcrAg and intrahepatic HBV cccDNA. Serum HBcrAg showed a high correlation with intrahepatic HBV cccDNA (r = 0.641, 95% confidence interval (CI) 0.510–0.743, p < 0.001). In a head-to-head comparison, we observed that the performance of HBcrAg was significantly superior to that of hepatitis B surface antigen (r = 0.665 vs. r = 0.475, respectively, p < 0.001). Subgroup analysis showed that the correlation between HBcrAg and intrahepatic HBV cccDNA was high, both in hepatitis B e antigen-positive and -negative patients (r = 0.678, 95% CI 0.403–0.840, p < 0.001, and r = 0.578, 95% CI 0.344–0.744, p < 0.001, respectively). In conclusion, the measurement of serum HBcrAg qualifies as a reliable non-invasive surrogate for the assessment of an intrahepatic HBV cccDNA reservoir.

Serum HBV RNA composition dynamics as a marker for intrahepatic HBV cccDNA turnover.

Current therapies can suppress the replication of hepatitis B virus (HBV) but cannot clear chronic HBV infection, which afflicts hundreds of millions worldwide. HBV persistence is sustained by the viral covalently closed circular DNA (cccDNA), an episome in the nucleus of infected hepatocytes. cccDNA is refractory to current therapies and its clearance is the holy grail for HBV cure. However, it has been difficult to monitor the fate of cccDNA in the liver directly. The current study takes a novel approach to this critical issue by monitoring the dynamic change in the genetic composition of the serum HBV RNA, which appears to reflect the dynamics of intrahepatic cccDNA turnover.

Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infection

The accurate diagnosis of occult hepatitis B virus (HBV) infection (OBI) requires the demonstration of HBV DNA in liver biopsies of hepatitis B surface antigen-negative individuals. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the hepatitis B core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive individuals. The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/105 cells, respectively). A total of 52% (52/100) of the individuals studied were found to have OBI. cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/105 cells (95% CI 5-25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (17.0 [7.0-39.2] vs. 5.7 [3.6-9.7] cut-off index [COI], respectively, p = 0.007). By multivariate analysis, an anti-HBc IgG value above 4.4 COI was associated with the finding of intrahepatic HBV cccDNA (odds ratio 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%. With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients. The covalently closed circular DNA (cccDNA) form of the hepatitis B virus (HBV) sustains the persistence of the virus even decades after resolution of the symptomatic infection (occult HBV infection). In the present study we developed a highly sensitive method based on droplet digital PCR technology for the detection and quantitation of HBV cccDNA in the liver of individuals with occult HBV infection. We observed that the amount of HBV cccDNA may be inferred from the titer in serum of the IgG class antibody to the hepatitis B core antigen. The quantitation of this antibody may represent a surrogate to determine which patients are at the highest risk of HBV reactivation following immunosuppressive therapies.

Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B

Recently, hepatitis B core-related antigen (HBcrAg) has been suggested as an additional marker of hepatitis B virus (HBV) infection. This study aimed to investigate whether serum quantitative HBcrAg (qHBcrAg) was a satisfactory surrogate marker of intrahepatic covalently closed circular DNA (cccDNA). A total of 139 patients with liver biopsy were enrolled, consisting of 59 patients in immune tolerance (IT) phase, 52 patients in immune clearance (IC) phase, 18 patients in low-replication (LR) phase, and 10 patients in reactivation phase. All patients in IC phase have received entecavir (ETV) therapy, and 32 of them undergone a second liver biopsy at 24 months. Among those patients, qHBcrAg was strongly correlated with intrahepatic cccDNA, which is superior to that of qHBsAg and HBV DNA. And similar findings were also observed in patients in IT, IC, LR and reactivation phases. Among the 32 ETV-treated patients with a second liver biopsy in IC phase, the decline of intrahepatic cccDNA was accompanied by changes in both qHBcrAg and qHBsAg. However, as compared to qHBsAg, the change of qHBcrAg was more strongly associated with intrahepatic cccDNA-decline. In summary, serum qHBcrAg should be a satisfactory surrogate of intrahepatic HBV cccDNA in CHB patients.

Detection of HBV DNA and antigens in HBsAg-positive patients with primary hepatocellular carcinoma

Hepatitis B virus (HBV) markers include HBV deoxyribonucleic acid (DNA) and HBV antigens. The former involves HBV covalently closed circular DNA (cccDNA) as well as total HBV DNA, whereas the latter involves HBsAg, HBcAg, and HBx. Samples of tumor and adjacent non-tumor liver tissue were collected from 28 HBV-associated HCC patients. Intrahepatic total HBV DNA and cccDNA were measured using the real-time PCR Taqman assay. HBV antigens in hepatocytes were detected using immunohistochemical staining. Intrahepatic levels of total HBV DNA or cccDNA in HCC patients with different intrahepatic HBV antigen expression patterns were compared, and the correlation between serum HBV DNA and intrahepatic HBV DNA was analyzed. No significant differences in intrahepatic cccDNA levels were observed between tumor and non-tumor liver tissue (median -3.00 vs. -2.30 log copies/cell, P=0.298). However, the tumor tissue had significantly higher levels of total HBV DNA (median -0.60 vs. -1.24 log copies/cell, P=0.045) but significantly lower proportion of intrahepatic HBV DNA in the form of cccDNA (median 0.25% vs. 4%, P=0.023) than the corresponding values in the non-tumor tissue. Also, HBV antigen levels were lower in the tumor tissue than in the non-tumor tissue. Analysis of the correlation between serum HBV DNA and intrahepatic HBV DNA indicated that the viral status in the tumor tissue was more complicated in HBV-HCC patients-the detected serum HBV DNA failed to accurately reflect intrahepatic viral load. HBV DNA may play an important role in hepatocarcinogenesis, and cccDNA was not the predominant form of HBV DNA in the tumor tissue.

Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a key to viral persistence in chronic hepatitis B infection. Serum hepatitis B core-related antigen (HBcrAg) is a novel marker for HBV disease. We aimed to determine whether HBcrAg could be a surrogate marker for intrahepatic cccDNA. Three hundred and five liver biopsies and the corresponding sera collected from 138 nucleos(t)ide analogues-treated patients were analysed. 124 patients had paired liver biopsies at baseline and 1-year post-treatment, and 43 patients had a third biopsy after 6-12years of treatment. Serum HBcrAg, HBV DNA and hepatitis B surface antigen (HBsAg), and intrahepatic HBV DNA and cccDNA were measured. HBcrAg strongly correlated with cccDNA (r=.70), intrahepatic total HBV DNA (r=.67) and serum HBV DNA (r=.69; all P<.0001). In the 130 samples with undetectable serum HBV DNA, HBcrAg was detectable in 101 (78%) samples, and HBcrAg levels still correlated positively with cccDNA (r=.42, P<.0001). At ≥6years of therapy, the median logarithmic reduction in HBcrAg was 2.7log kU/mL, which was comparable to the magnitude of reduction in cccDNA. Twenty-one patients had undetectable cccDNA after ≥6years of treatment, in whom 15 (71%) had detectable HBcrAg (range: 1.2-537kU/mL). Serum HBcrAg is a reliable surrogate marker for intrahepatic cccDNA. HBcrAg could be a very sensitive marker to reflect the cccDNA content and persistence of disease even with the cccDNA levels below the detection limit of assays.

Quantitative intrahepatic HBV cccDNA correlates with histological liver inflammation in chronic hepatitis B virus infection

The aim of this study was to determine the role of baseline hepatitis B virus (HBV) forming covalently closed circular DNA (HBV cccDNA) in liver inflammation in patients infected with HBV with serum alanine aminotransferase (ALT) levels under two times the upper limit of normal (2×ULN). After liver biopsy and serum virological and biochemical marker screening, patients diagnosed with chronic HBV infection with serum ALT levels under 2×ULN and histological liver inflammation of less than grade G2 were prospectively recruited into this study. Recruitment took place between March 2009 and November 2010 at the Center of Infectious Disease, Sichuan University. Patient virological and biochemical markers, as well as markers of liver inflammation, were monitored. A total of 102 patients were recruited and 68 met the inclusion criteria; the median follow-up was 4.1 years (range 3.9-5.2 years). During follow-up, 41 patients (60.3%) exhibited signs of inflammation. Baseline HBV cccDNA >1 copy/cell (odds ratio 9.43, p=0.049) and liver inflammation grade ≥G1 (odds ratio 5.77, p=0.046) were both independent predictors of liver inflammation. A higher baseline intrahepatic HBV cccDNA level may increase the risk of liver inflammation. Further investigations will be required to validate HBV cccDNA as an intrahepatic virological marker of patients who require extended outpatient management.

Intrahepatic hepatitis B virus replication and liver histology in subjects with occult hepatitis B infection

We studied the intrahepatic hepatitis B virus (HBV) replicative status in 40 people with occult hepatitis B infection (OBI) and 40 patients with chronic hepatitis B (CHB). Intrahepatic HBV DNA, covalently closed circular DNA (cccDNA), and pre-genomic RNA (pgRNA) were quantified. Patients with OBI had median necroinflammation and fibrosis scores of 1 and 0, respectively. Intrahepatic total HBV DNA, cccDNA and pgRNA were detectable in 30 (77%), one (3%) and five (13%) of the participants with OBI, respectively. People with OBI had lower median intrahepatic total HBV DNA than the patients with CHB (p < 0.0001). They had nearly normal liver histology and low intrahepatic HBV replication.

The effect of peginterferon alpha-2a vs. interferon alpha-2a on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients

The covalently closed circular DNA (cccDNA), as the template of HBV transcription, plays a key role in the virus infection. The present study aimed to compare the effect of pegylated interferon (IFN)-α-2a with that of conventional IFN-α-2a on intrahepatic covalently closed circular (ccc)DNA in patients with chronic hepatitis B. Seventy-six HBeAg-positive chronic hepatitis B patients were randomly divided into two groups (n=38): group A was treated with interferon alpha-2a (IFN-α-2a) and group B was treated with peginterferon alpha-2a (peg IFN-α-2a). The intrahepatic level of cccDNA and its detection rate, levels of hepatitis B virus (HBV) DNA in liver and serum, histologic inflammation and some biochemistry parameters (alanine aminotransferase, aspartate aminotransferase and total bilirubin levels) were measured. The outcome of 48 weeks therapy showed that the mean level of intrahepatic HBV cccDNA level and its detection rate, the levels of HBV DNA and the histology and biochemistry parameters were significantly decreased following therapy in two groups (P<0.05). While, the reductions in the group treated with peg IFN-α-2a were greater (P<0.05). It may be concluded that the ability of the peg IFN-α-2a to clear and suppress cccDNA and HBV DNA was superior compared with that of conventional IFN-α-2a. Furthermore, the effects of peg IFN-α-2a on histology and biochemistry parameters were also more obvious than conventional IFN-α-2a.

T- and B-cell responses and previous exposure to hepatitis B virus in 'anti-HBc alone' patients.

A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n=27), resolved HBV (n=21), chronic HBV (n=24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P=0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Intrahepatic hepatitis B virus covalently closed circular DNA correlation with serum HBV DNA, serum HBsAg, alanine aminotransferase and age

To investigate the correlation between intrahepatic eovalently closed circular (ccc)DNA of hepatitis B virus (HBV) and pathogen-and patient-related parameters. Ultrasound-guided liver biopsies were obtained from 60 patients with chronic HBV infection. Levels of intrahepatic HBV cccDNA and serum HBV DNA were measured by quantitative fluorescence PCR. Level of serum hepatitis B surface antigen (HBsAg) was measured by chemiluminescence immunoassay. Clinical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (AKP), albumin, globulin (GLO), white blood cell, platelet, prothrombin-international normalized ratio, were measured by standard assay. Demographic information was recorded.The correlation between intrahepatic HBV cccDNA and pathogen-and patient-related parameters was assessed. Intrahepatic HBV cccDNA level was negatively correlated with age, GLO, ALT and grade of necroinflammation. Patients with age of 30 years or more showed significantly higher level of HBV cccDNA level than patients under 30 years-old (7.44±0.58 and 5.66±1.35; t=7.157, P less than 0.001). Intrahepatic HBV cccDNA level was positively correlated with serum HBV DNA level (r=0.916, P less than 0.001) and serum HBsAg level (r=0.727, P less than 0.001). The median ratio of HBV cccDNA to HBV DNA was 1.18, and of HBV cccDNA to HBsAg was 1.67. Intrahepatic HBV cccDNA levels decrease with age, level of ALT, level of GLO and grade of liver necroinflammation, but increase with level of serum HBV DNA and level of serum HBsAg. To a certain extent, serum HBV DNA and serum HBsAg levels may be a sufficient marker of intrahepatic HBV cccDNA levels.

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients

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 naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve 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.

Serum HBV RNA as a possible marker of HBV replication in the liver during nucleot(s)ide analogue therapy

We read with interest the article by Tsuge et al. [1] published in the recent issue of the Journal of Gastroenterology. Treatment with nucleot(s)ide analogue (NUC) strongly suppresses the replication of hepatitis B virus (HBV) leading to a high rate of serum HBV DNA negativity. However, the incidence of relapse after the cessation of NUCs is high. Criterion for safe discontinuation of NUC therapy after long term therapy is not established to date. In HBe antigen positive patients, seroconversion, HBV DNA negativity and consolidation therapy of[6 months may be a consensus criteria but 30–50 % of patients fulfilling this criteria experience a relapse. In HBe antigen negative patients, NUC therapy is generally recommended until HBs antigen becomes undetected. Tsuge et al. [1] measured serum HBV RNA plus DNA by real time PCR and showed that the serum HBV DNA ? RNA titer following 3 months of NUC treatment was a significant predictor of early (within 24 weeks) HBV DNA rebound after discontinuation of NUC. The serum HBV DNA ? RNA titer was also associated with ALT rebound in HBe antigen positive patients. The results of the study by Tsuge et al. indicate that serum HBV DNA ? RNA titer may serve as predictor of relapse after discontinuation of NUC. The high rate of relapse after discontinuation of NUC is due to the persistence of HBV replication in the liver even during the NUC therapy. The replicative intermediate form of HBV, covalently closed circular DNA (cccDNA), may not be eliminated by NUC therapy and serves as a template for viral pre-genomic messenger RNA [2]. This concept was proved by a study showing that quantification of intrahepatic HBV cccDNA had a high accuracy of predicting sustained virological response after NUC discontinuation [3]. Still, we need a non-invasive and clinically usable marker for the assessment of HBV replication in the liver during NUC therapy. The measurement of HBV core related antigen may be an alternative [4]. The rationale of measuring HBV RNA in serum was that immature HBV particles including HBV RNA are released from hepatocytes during NUC treatment under the circumstances that pre-genomic HBV RNA are transcribed from HBV cccDNA, packaged into HBV core particles, but not reverse transcribed into plus-strand HBV DNA due to strong interference by NUC, and the excessive amounts of these immature particles are accumulated in hepatocytes [5, 6]. Tsuge et al. showed that serum HBV DNA ? RNA titer following 3 months of NUC treatment was significantly lower in patients with no rebound of HBV DNA. By using a cut-off value of 4.8 log copies/mL, the cumulative incidence of HBV DNA rebound was significantly lower in patients with serum HBV DNA ? RNA titer \ 4.8 log at 3 months of NUC treatment. The same groups previously showed that HBV RNA levels at 3 months of lamivudine treatment were predictor of early emergence of resistant mutations [7]. Taken together, serum HBV DNA ? RNA titer may be linked to the level of HBV replication in the liver during NUC therapy. Monitoring of serum HBV DNA ? RNA response may be utilized in various decision makings in treatment of HBV patients with NUC therapy. Based on these important findings, several questions may remain for future elucidation. Commercially available transcription-mediated amplification and hybridization assay An answer to this letter to the editor is available at doi:10.1007/s00535-013-0801-6.

Association of intrahepatic cccDNA reduction with the improvement of liver histology in chronic hepatitis B patients receiving oral antiviral agents

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is difficult to eradicate using current antiviral therapy. This study compares cccDNA reduction with relation to liver histology in nucleoside/nucleotide-naïve chronic hepatitis B patients receiving oral antiviral monotherapy (n=35), including entecavir (ETV, n=13), adefovir dipivoxil (ADV, n=22) or placebo (n=14). Serum HBV DNA, intrahepatic total HBV DNA and cccDNA are quantified. Histological hepatic examination is performed at baseline and at 48 weeks of treatment. Treatment with ETV or ADV shows significant median reduction in serum HBV DNA (-6.21 and -4.27 log(10) copies/mL) and intrahepatic total HBV DNA (-1.69 and -1.23 log(10) copies/cell). Intrahepatic cccDNA levels are reduced slightly in the ETV and the ADV groups, but do not differ statistically from the placebo group (-0.17 vs. -0.01 vs. 0.02 copies/cell). Only the level of intrahepatic cccDNA correlates with Knodell necroinflammation activity (r=0.527, P<0.001) and Ishak fibrosis severity (r=0.348, P=0.015) before treatment. Multivariate logistic regression analysis indicates that treatment-induced cccDNA reduction is associated with improved necroinflammation (P=0.041) and fibrosis (P=0.026). In conclusion, baseline intrahepatic cccDNA loads correlate with histologic activity. Although one-year ETV or ADV treatment is insufficient for cccDNA eradication, oral antiviral therapies may improve liver histology, probably by suppressing intrahepatic cccDNA.

Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA

To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

Occult hepatitis B virus infection of donor and recipient origin after liver transplantation despite nucleoside analogue prophylaxis

Liver grafts from donors positive for antibody to hepatitis B core antigen (anti-HBc) may be used for transplantation in patients with hepatitis B virus (HBV)-related liver disease, and an occult HBV infection may develop from either source. Liver biopsy was performed for 31 patients who remained seronegative for hepatitis B surface antigen for a median of 44.5 months (range = 13.6-126.4 months) and received nucleoside analogue prophylaxis post-transplant. Nineteen of these recipients (61%) had received anti-HBc-positive grafts. Intrahepatic total HBV DNA and covalently closed circular DNA (cccDNA) levels were quantified, and the sequence was analyzed. Intrahepatic total HBV DNA and cccDNA were detectable in 26 (84%) and 16 (52%) of the 31 recipients, respectively, and they were more common when the donor was positive for anti-HBc (95% versus 67%, P = 0.038). The intrahepatic HBV DNA level correlated with the recipient pretransplant serum HBV DNA level (P = 0.06), and the intrahepatic HBV cccDNA level correlated with the donor intrahepatic HBV cccDNA level (P = 0.06). A phylogenetic analysis of the isolated HBV DNA sequence revealed HBV infections of both donor and recipient origins. In conclusion, an occult HBV infection after liver transplantation can originate from both the donor and recipient despite prolonged nucleoside analogue prophylaxis. The presence of intrahepatic HBV cccDNA is attributable more to the persistence of preexisting intrahepatic HBV cccDNA from a donor with previous exposure.

Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for viral persistence in the natural course of chronic HBV infection and during prolonged antiviral therapy and serves as the template for the production of HBV pregenomic RNA (pgRNA), the primary step in HBV replication. In this study, we have developed and applied sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays for the measurement of intrahepatic concentration, pgRNA production, and replicative activity of cccDNA in liver biopsy samples from 34 non-treated patients with chronic hepatitis B (CHB); 12 hepatitis B e antigen (HBeAg)(+) and 22 HBeAg(-). Median copy number for cccDNA was 1.5 per cell and for pgRNA significantly higher, 6.5 copies per cell, with a good correlation between cccDNA and pgRNA levels in all samples. In HBeAg(-) patients, median values of cccDNA and pgRNA levels were 10-fold and 200-fold lower than in HBeAg(+), respectively, reflecting the differences in viral activity and clinical characteristics of the two groups. Furthermore, the replicative activity of intrahepatic cccDNA was significantly lower in HBeAg(-) patients harboring mutant HBV strains than in HBeAg(+) patients: median 3.5 versus 101 pgRNA copies per cccDNA molecule. In conclusion, the levels of both HBV cccDNA, a marker of HBV persistence, and pgRNA, an indicator of viral replication, in the liver of chronically infected patients correlate with viral activity and the phase of HBV infection. The combined measurement of cccDNA and pgRNA levels provides valuable information on the presence and replicative activity of intrahepatic HBV cccDNA.

One-Year Entecavir or Lamivudine Therapy Results in Reduction of Hepatitis B Virus Intrahepatic Covalently Closed Circular DNA Levels

Entecavir and lamivudine are potent nucleoside analogues that can suppress hepatitis B virus (HBV) replication. However, the effects of these two antiviral agents on intrahepatic total HBV DNA and covalently closed circular DNA (cccDNA) are not known. In this study, we aimed to assess the effect of 48 weeks of entecavir/lamivudine therapy on intrahepatic total HBV DNA and cccDNA levels. Forty chronic hepatitis B patients, participating in two Phase III entecavir trials at our centre, were randomized to receive 48 weeks of either 0.5 mg once daily of entecavir (n = 21) or 100 mg once daily of lamivudine (n = 19). Their serological, virological and biochemical responses, as well as intrahepatic HBV DNA levels were monitored. There was no significant difference between entecavir and lamivudine therapy in terms of post-treatment serological, virological and biochemical responses. Both nucleoside analogues reduced serum viral load, intrahepatic total HBV DNA, and cccDNA by about 4.8 logs, 2 logs, and 1 log respectively. An increase in the proportion of intrahepatic HBV DNA in the form of cccDNA was seen after 48 weeks of therapy. In conclusion, both entecavir and lamivudine can successfully reduce intrahepatic HBV DNA and cccDNA. CccDNA becomes the dominant form of HBV DNA during viral suppression and is possibly responsible for viral rebound after short-term antiviral therapy.

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.