Elsevier

Journal of Hepatology

Volume 69, Issue 2, August 2018, Pages 301-307
Journal of Hepatology

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

https://doi.org/10.1016/j.jhep.2018.03.021Get rights and content

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.

Introduction

Occult hepatitis B virus (HBV) infection (OBI) refers to the presence of intrahepatic HBV DNA in the absence of detectable hepatitis B surface antigen (HBsAg).1 OBI is secondary to overt HBV infections; it guarantees the persistence of the virus in a cryptic form protected from the immune response of the host. The virologic key is the covalently closed circular DNA (cccDNA), an HBV DNA form generated as a plasmid-like episome from the protein-linked relaxed circular DNA genome; it resides in the nucleus of infected cells and gives rise to viral sequences, which act as a transcription template for all viral RNAs.2

OBI has clinical significance; it can reactivate hepatitis B when the immune response of the host is compromised, as in liver transplant patients or those undergoing chemotherapy. It may also accelerate the progression of hepatic fibrosis in patients with chronic hepatitis C and has been considered a risk factor for hepatocellular carcinoma.3

An accurate diagnosis of OBI would require a liver biopsy to measure the intrahepatic HBV DNA. Yet obtaining liver specimens is difficult. In practice, recognition of OBI is based on the binding of the antibody to the hepatitis B core antigen (anti-HBc), on the premise that this reactivity represents a serological scar to a clinically resolved exposure to HBV and may therefore be an indirect marker of a latent HBV infection.4 Intrahepatic HBV cccDNA is resistant to antivirals and cannot be eradicated,5 but it is possible to prevent OBI reactivation by prophylaxis with HBV antivirals.6 This strategy is recommended for anti-HBc-positive patients undergoing pharmacological immunosuppression; in default of virologic data, the individual indication to prophylaxis is not determined by parameters of HBV infectivity but by the immunosuppressive potential of therapy.

Herein, we developed and present a new assay for HBV cccDNA measurement based on droplet digital PCR (ddPCR), an emerging technique which exhibits improved sensitivity and accuracy to detect low DNA concentrations compared with conventional quantitative real-time PCR (qPCR).7 We used this assay to determine the prevalence and quantity of HBV cccDNA in individuals with OBI recruited among anti-HBc-positive liver donors and to assess the relationship between the viral findings in the liver and the markers of HBV infection in serum.

Section snippets

Patients

From November 2010 to December 2016, 112 consecutive HBsAg-negative/anti-HBc-positive deceased heart-beating liver donors were recruited at the Liver Transplant Center of the University of Turin. Eighteen liver biopsies from liver donors without any HBV marker were collected as negative controls. No organ came from executed prisoners or other institutionalized individuals. A serum sample and a liver needle biopsy were obtained from each donor; the latter was collected in RNA-later solution and

Liver donor features

One hundred of the 112 anti-HBc-positive donors (median age 68.2 [65.5–71.5] years, 64 males and 36 females) were included. Reasons for exclusion were a positive test for antibodies to the hepatitis C virus (n = 4), the finding of an unknown gastrointestinal stromal tumor (n = 1), donor age <18 years (n = 1) and serum/plasma samples unavailable (n = 5). A donor was dismissed for the subsequent finding of HBsAg-HQ positivity (57 mIU/ml). Six donors had HBV DNA detectable in blood (all

Discussion

The hallmark of OBI is the presence of intrahepatic HBV DNA, therefore an accurate diagnosis of this condition requires a liver biopsy. OBI is an asymptomatic condition of viral latency and it would be unethical to submit healthy individuals to an invasive procedure; in clinical practice the finding of anti-HBc in serum with standard immunoassays is considered sufficient evidence to diagnose OBI, with the corollary that all HBsAg-negative persons displaying this marker are candidates for

Financial support

Supported by University of Torino, Research Fund ID # CIAA_RIC_LOC_15_01.

Conflict of interest

The authors declare no conflicts of interest that pertain to this work.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

Study concept and design: Gian Paolo Caviglia, Antonina Smedile.

Performing experiments: Gian Paolo Caviglia, Maria Lorena Abate.

Analysis and interpretation of data: Gian Paolo Caviglia, Maria Lorena Abate, Francesco Tandoi, Alessia Ciancio, Mario Rizzetto, Renato Romagnoli.

Drafting the manuscript: Gian Paolo Caviglia, Maria Lorena Abate, Mario Rizzetto.

Critical revision of the manuscript for important intellectual content: Antonio Amoroso, Mauro Salizzoni, Giorgio Maria Saracco, Renato

Acknowledgements

Authors thank Dr. Corinna Orsini (Fujirebio Italia) for supplying HBsAg-HQ, HBcrAg and anti-HBc IgG test reagents and Dr. Marta Varotto (Bioclarma s.r.l.) for her technical support in the ddPCR assay. Fujirebio Italia had no role in study design, in data collection, analysis and interpretation, in manuscript writing and decision to submit for publication.

References (24)

  • M. Nassal

    HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

    Gut

    (2015)
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