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Hepat Mon. In Press(In Press):e92317.
Published online 2019 December 17.
doi: 10.5812/hepatmon.92317.
Brief Report
Evaluation of Occult Hepatitis B Infection in Individuals with Chronic
Hepatitis C Before Treatment with Oral Direct-Acting Antivirals
Nathalia Alves Araujo de Almeida 1, Jose Junior F de Barros1, Catarina Goes de Santana 2, Natalia Spitz
1, Leticia Bomfim Campos 1, Marcia Amendola Pires2, Carlos Eduardo Brandao Mello 2and Vanessa
Salete de Paula 1,*
1Laboratory of Molecular Virology, Oswaldo Cruz Institute/Fiocruz, Rio de Janeiro, Brazil
2Gaffrée and Guinle Universitary Hospital, Ambulatory of Liver Disease, Rio de Janeiro State Federal University/UniRio, Rio de Janeiro, Brazil
*Corresponding author: Laboratory of Molecular Virology, Oswaldo Cruz Institute/Fiocruz, CEP 21040-900 Rio de Janeiro,Brazil. Email: vdepaula.fiocruz@gmail.com
Received 2019 April 15; Revised 2019 September 20; Accepted 2019 September 27.
Abstract
Occult hepatitis B virus infection (OBI) is one of the most challenging entities in the field of viral hepatitis. The virological and
clinical relevance of OBI in patients treated with novel direct-acting antivirals (DAA) for hepatitis C virus (HCV) infections is currently
a topic of hot debate. In cases where hepatitis B surface antigen (HBsAg) is not detected, DAA treatment is often initiated without
examining for the presence of hepatitis B virus (HBV) DNA. In this study, the incidence of OBI was investigated in 114 HCV patients
prior to application of DAAs who did not respond to pegylated interferon and ribavirin (PEG-INF and RBV) treatment. Serum samples
were screened for HBV serological markers (antibody to hepatitis B core antigen [anti-HBc] and HBsAg). Samples positive for anti-
HBc without HBsAg were further examined via real-time PCR (qPCR), nested PCR and S-gene mutational analyses. Overall, anti-HBc
was detected in 37.7% chronic HCV patients and 2.6% had OBI with a baseline HBV DNA viral load < 2000 IU/mL before DAA therapy.
One patient was identified as HBV genotype A1 without mutations in surface protein. Our collective data highlight the importance of
clinicians being aware of potential anti-HBc positivity in patients with hepatitis C and the issues surrounding OBI screening before
initiation of treatment with novel DAAs.
Keywords: Hepatitis C, Occult Hepatitis B, DAA Treatment
1. Background
Hepatitis B virus (HBV) infection remains a global pub-
lic health problem, even following the development of an
effective vaccine. Brazil is classified as a region with low
to intermediate prevalence of HBV surface antigen (HBsAg)
carriers (< 2%), including areas of high HBV endemicity (>
8%) (1). Occult HBV infection (OBI) is the most challeng-
ing issue in the field of viral hepatitis, with its virological
and clinical relevance in patients undergoing treatment
for hepatitis C being a hot topic of debate at present. Occult
HBV infection is determined by the absence of HBsAg and
presence of HBV DNA in liver or serum of infected patients
(2,3) and classified as: (1) seropositive OBI (positive for anti-
body to hepatitis B core antigen [anti-HBc] and/or antibody
to hepatitis B surface antigen [anti-HBs]), (2) seronegative
OBI (negative for anti-HBc and anti-HBs), and (3) “false” OBI
due to the presence of HBV variants with mutations in the S
gene (escape mutants) producing modified HBsAg that are
not recognized by some or all commercially available de-
tection assays (2,3). The frequency of OBI in Brazil ranges
from 0% to 24% (4-9) and has significant relevance in a clin-
ical context, since it can lead to the development of severe
hepatic diseases, such as cirrhosis and hepatocellular car-
cinoma, and eventually, death. Viral factors of OBI induc-
tion may be associated with mutations, especially in the S
protein, and co-infection with other viruses such as hep-
atitis C virus (HCV) (10). Reactivation of HBV has been re-
ported in patients with chronic HCV or resolved HBV in-
fection under treatment with newer direct-acting antivi-
rals (DAA) (11), resulting in fulminant hepatitis, liver failure
and in some cases, death. According to the Brazilian Clini-
cal Protocol and Therapeutic Guidelines for Hepatitis C and
Coinfections, individuals detected with HBsAg prior to ini-
tiation of DAA are recommended HBV therapy to prevent
reactivation owing to hepatitis C treatment. However, in
cases where HBsAg is not detected, treatment with DAAs is
released without examining for the presence of HBV-DNA
(12). Although hepatitis B reactivation in patients is known
to occur during hepatitis C treatment with direct-acting
antivirals, only a few cases have been described to date.
Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
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de Almeida NAA et al.
Moreover, no data are available on OBI/HCV coinfection in
patients scheduled for DAA treatment in Brazil.
2. Objectives
In this study, we evaluated the prevalence of anti-HBc
and examined for OBI via real-time and nested PCR analy-
ses in patients with chronic hepatitis C before commence-
ment of DAA therapy. We additionally investigated the
presence of mutations in the S gene associated with cases
of false OBI.
3. Methods
Our retrospective cross-sectional study was conducted
in patients prior to commencement of DAA treatment who
had attended the Outpatient Clinic of Liver Disease at the
Gaffrée and Guinle University Hospital (Rio de Janeiro,
Brazil) from January to December 2018. Clinical, demo-
graphic as well as HBV and HCV treatment data were ac-
quired from medical records. Serum samples were col-
lected from a cohort of 114 consecutive HCV patients. The
patients included for study had failed pegylated interferon
and ribavirin (PEG-INF and RBV) therapy. All serum samples
were HCV RNA-positive and examined for total HBc and HB-
sAg via immunoenzymatic assays (EIA). Samples positive
for anti-HBc with no HBsAg were further examined using
real-time PCR (qPCR) and nested PCR (13). The HBV DNA
was additionally sequenced for genotyping and detection
of S gene mutations. Notably, the presence of OBI was cor-
related with aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) levels.
4. Results and Discussion
In a serological test, 37.7% (43/114) patients were iden-
tified as positive for anti-HBc, consistent with a previ-
ous report of antibody prevalence of up to 35% in HCV-
infected populations (11). Occult HBV infection was de-
tected in three (2.6%) male patients with a mean age of
69.3 years classified as HCV genotypes 1 and 3. The pa-
tients had received treatment with pegylated interferon
(80 mcg) and ribavirin (1.0 g per day) for 24 weeks without
HCV clearance. No patients developed ALT and AST flare-
ups and all were anti-HB-positive, with viral loads rang-
ing from 207.14 to 1547 IU/mL (1.16 ×103to 8.51 ×104
copies/mL) (Table 1). Patients with the highest viral loads
were positive for S-region amplification (GenBank acces-
sion number MK360178) via nested-polymerase chain re-
action (nested-PCR) and HBV in these cases was classified
as genotype A1 (Figure 1), the most prevalent in Brazil (14).
Deletion or insertion of nucleotides in the S region was not
associated with non-detection of HBsAg with commercial
kits or non-expression of HBV surface antigen. The patient
had chronic active hepatitis C, grade 4, with cirrhosis (F4,
38.5 kPa; fibroscan). All patients were re-treated with sofos-
buvir (400 mg) + daclatasvir (60 mg) + ribavirin (1.0 gram),
following which HCV remained undetectable. The pres-
ence of HBV DNA has been reported among patients with
hepatitis C treated with DAAs. However, in patients with
resolved (anti-HBs and anti-HBc) HBV infections, no HBV
reactivation-related hepatitis is documented (3). In the
current study, all three patients had HBV DNA viral loads
< 2000 IU/mL before DAA therapy. A systematic review
and meta-analysis published in 2018 provided evidence of
significantly lower relative risk of HBV reactivation-related
hepatitis in patients with HBV DNA below the lower limit
of quantification at baseline than those with quantifiable
HBV DNA (11).
Figure 1. Phylogenetic tree of the HBV S-gene inferred using the neighbor-joining
method. Values in the branches indicate the percentage of 1000 bootstrap repli-
cates supporting the group. The sequence generated in this study is represented by
a black dot (GenBank accession number MK360178). Reference sequences are indi-
cated by their accession number,followed by subgenotype and geographical origin.
Since the patient populations at risk for HBV reactiva-
tion and management options are currently unclear, these
cases clearly highlight the importance of HBV DNA testing
prior to initiation of DAA therapy. Several studies assess-
ing the prevalence of OBI in patients with chronic HCV in-
fection have reported dissimilar rates of HBV DNA positiv-
ity, which may be explained by differences in the preva-
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de Almeida NAA et al.
Table1. Viral Load of HBV, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) Levels in Patients Infected with HCV and Occult Hepatitis B
ID Copies/mL IU/mL ALT AST HCV Genotype HBV Genotype Fibrosis Stage
001 1.16 ×103207.14 59 102 1a ND F4
042 8.5 ×1041517.8 87 137 3a A1 F4
268 1.29 ×104230.357 74 100 1b ND F4
Abbreviation: ND, not determined
lence of HBV infection in different geographic regions, de-
tection limits of HBV DNA assays and/or the biological ma-
terial analyzed (liver tissue or serum) (9). Low levels of
anti-HBs and decrease to < 12 mIU mL after treatment are
significant risk factors for HBV reactivation or reappear-
ance (15). We observed no differences in anti-HBs levels be-
fore and after treatment with DAAs and the small eleva-
tion of ALT/AST observed in patients with OBI HBV reactiva-
tion could be prevented with pretreatment screening and
prophylactic treatment where necessary (16). It can occur
spontaneously but is triggered by various factors and often
transient without clinical symptoms in most cases other
than causing a hepatitis flare. Moreover, HBV reactivation
may occur regardless of HCV genotype and type of DAA reg-
imen (16).
While OBI and risk of HBV reactivation are relatively
rare, it is important to account for the possibility that in
some instances, subjects undergoing new treatments are
infected with HBV viral DNA, since HCV and previous treat-
ments can inhibit HBV replication and influence evolution
of the disease. Based on findings from the current study,
we recommend that clinicians should be aware of possible
anti-HBc positivity in patients and examine for hepatitis B
infection along with the issues surrounding OBI screening
before initiating treatment with novel DAAs. To our knowl-
edge, this is the first study in Brazil to focus on OBI in in-
dividuals with chronic hepatitis C before DAA treatment.
Our collective results should contribute to formulating ef-
fective therapeutic guidelines for treatment of hepatitis C
and concomitant infections and support routine evalua-
tion of OBI/HCV coinfections before and during treatment
with DAAs.
Footnotes
Conflict of Interests: The authors declare that they have
no conflicts of interest.
Ethical Approval: The study protocol was approved by
the Research Ethics Committee of the Institute Oswaldo
Cruz (CAAE 34246914.4.1001.5248 number 2.927.747/18).
Funding/Support: This work was supported by Coordina-
tion for the Improvement of Higher Education Personnel
(CAPES) and the Oswaldo Cruz Foundation.
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