Highlights
- •HBV covalently closed circular DNA (cccDNA) is responsible for viral persistence decades after liver disease resolution.
- •A latent occult HBV infection is identified by the presence of the antibody to the HB-core antigen (anti-HBc).
- •We developed a highly sensitive droplet digital PCR assay for intrahepatic HBV cccDNA quantitation.
- •HBV cccDNA is detectable and quantifiable in 27 out of 100 anti-HBc-positive liver donors.
- •Serum anti-HBc IgG levels are associated with the finding of intrahepatic HBV cccDNA.
Background & Aims
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.
Methods
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).
Results
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%.
Conclusions
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.
Lay summary
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.
Graphical abstract
Graphical Abstract
Keywords
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References
Author names in bold designate shared co-first authorship
- Statements from the Taormina expert meeting on occult hepatitis B virus infection.J Hepatol. 2008; 49: 652-657
- Occult hepatitis B infection.J Hepatol. 2014; 61: 688-689
- Clinical relevance of the study of hepatitis B virus covalently closed circular DNA.Liver Int. 2016; 36: 72-77
- Prediction of occult hepatitis B virus infection in liver transplant donors through hepatitis B virus blood markers.Dig Liver Dis. 2014; 46: 1020-1024
- HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984
- Hepatitis B reactivation associated with immune suppressive and biological modifier therapies: current concepts, management strategies, and future directions.Gastroenterology. 2017; 152: 1297-1309
- Absolute quantification by droplet digital PCR vs. analog real-time PCR.Nat Methods. 2013; 10: 1003-1005
- Occult hepatitis B virus infection in patients with chronic hepatitis C treated with antiviral therapy.Hepat Mon. 2012; 12e7292
- Wild-type e antigen-minus hepatitis B viruses and course of chronic hepatitis.Proc Natl Acad Sci U S A. 1991; 88: 4186-4190
- Hepatitis B Virus (HBV) Core-Related Antigen During Nucleos(t)ide Analog Therapy Is Related to Intra-hepatic HBV Replication and Development of Hepatocellular Carcinoma.J Infect Dis. 2016; 213: 1096-1106
- Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.J Clin Microbiol. 2013; 51: 3484-3491
- Hepatitis B core-related antigen kinetics in chronic hepatitis B virus genotype D-infected patients treated with nucleos(t)ide analogues or pegylated-interferon-α.Hepatol Res. 2016; ([In press])https://doi.org/10.1111/hepr.12811
- Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758
- Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.Liver Int. 2016; ([In press])https://doi.org/10.1111/liv.13346
- Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.J Hepatol. 2016; 65: 683-691
- Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.J Hepatol. 2017; 66: 275-281
- Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.Biosci Biotechnol Biochem. 2016; ([In press])https://doi.org/10.1080/09168451.2016.1196576
- A sensitive and accurate method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.Biotechnol Lett. 2015; 37: 2063-5073
- Baseline value of intrahepatic HBV DNA over cccDNA predicts patient's response to interferon therapy.Sci Rep. 2017; 7: 5937
- Occult hepatitis B virus in liver tissue of individuals without hepatic disease.J Hepatol. 2008; 48: 743-746
- Close monitoring of serum HBV DNA levels and liver enzymes levels is most useful in the management of patients with occult HBV infection.J Hepatol. 2009; 51: 824-825
- Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients.J Med Virol. 2009; 81: 27-33
- Performance evaluation of four dominant anti-hepatitis B core antigen (HBcAb) kits in Japan for preventing de novo hepatitis B virus (HBV) infection.Clin Lab. 2015; 61: 77-85
Article info
Publication history
Published online: April 02, 2018
Accepted:
March 13,
2018
Received in revised form:
March 10,
2018
Received:
November 14,
2017
Identification
Copyright
© 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
