International Journal of Infectious Diseases 41 (2015) 83–89

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Chronic hepatitis B in pregnant women: is hepatitis B surface antigen

quantification useful for viral load prediction?
Masita Fujiko a,1, Maisuri T. Chalid a,1, Turyadi b, Susan I. Ie b, Maghfira a, Syafri a,
Ridha Wahyuni a, Martono Roni b, Ilhamjaya Patellongi a, M. Nasrum Massi a,
David H. Muljono a,b,c,*
a
Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
b
Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta Pusat 10430, DKI Jakarta, Indonesia
c
Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

A R T I C L E I N F O S U M M A R Y

Article history: Background: New cases of hepatitis B virus (HBV) infection continue to occur worldwide. Most of these
Received 27 July 2015 are due to mother-to-child transmission (MTCT), with maternal viraemia as the most important
Received in revised form 20 October 2015 contributing factor. The hepatitis B surface antigen (HBsAg) level, which correlates positively with viral
Accepted 4 November 2015
load, has been used for treatment monitoring in chronic hepatitis B. This study evaluated the usefulness
Corresponding Editor: Eskild Petersen, of quantitative HBsAg for viral load prediction in HBsAg-positive pregnant women.
Aarhus, Denmark.
Methods: A total of 943 pregnant women in Makassar, Indonesia, were screened for HBsAg. Sixty-four
women were HBsAg-positive and investigated. HBsAg level and hepatitis B e antigen (HBeAg)/hepatitis B
Keywords: e antibody (anti-HBe) status were determined serologically. Viral load was measured by real-time PCR.
HBsAg level HBV DNA was sequenced and analysed for identification of genotype and basal core promoter (BCP)/
HBV DNA level precore (PC) mutations.
Pregnant women
Results: Of 64 subjects, 12 (18.8%) were HBeAg-positive and 52 (81.3%) were HBeAg-negative. HBsAg
HBV vertical transmission
and HBV DNA levels were significantly higher in the HBeAg-positive group (p < 0.001). HBsAg and HBV
Mother-to-child-transmission
DNA levels were positively correlated in the HBeAg-positive group (r = 0.659; p = 0.02), but not in the
HBeAg-negative group (r = 0.194; p = 0.168). Low HBsAg levels (<3.0 log10 IU/ml) corresponded with
HBV DNA levels < 6.0 log10 IU/ml (r = 0.404; p = 0.001), a recognized threshold for MTCT. Genotype C
was more prevalent than genotype B, but not associated with HBsAg level, viral load, or HBeAg status.
Two-thirds of HBeAg-negative subjects with high HBV DNA levels harboured BCP (A1762T/G1764A) and/
or PC (G1896A) variants.
Conclusions: HBsAg levels provide a good viral load predictor in HBeAg-positive but not HBeAg-negative
pregnant women. The HBeAg-negative group had a frequent occurrence of BCP/PC variants, which may
have contributed to the lack of correlation observed. Samples with a low HBsAg level, which is associated
with a low risk of MTCT, do not require HBV DNA measurement.
ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

1. Introduction from hepatitis B annually. In highly endemic areas of Asia, the

Pacific, and Sub-Saharan Africa, most HBV infection occurs
More than 240 million people worldwide are chronically perinatally or during early childhood. This is associated with a
infected with the hepatitis B virus (HBV), and about 780 000 die high rate of persistent infection and increased risk of morbidity
and mortality from cirrhosis and hepatocellular carcinoma later
in life.1
* Corresponding author. Tel.: +62 21 3148695; fax: +62 21 3147982. Efforts in the prevention of hepatitis B have focused on the
E-mail addresses: [email protected], [email protected] immunization of infants implemented in 183 World Health
(D.H. Muljono).
1 Organization (WHO) member states. As of 2012, 94 member
Authors Masita Fujiko and Maisuri T. Chalid contributed equally to this
manuscript. states including Indonesia had introduced the hepatitis B birth

http://dx.doi.org/10.1016/j.ijid.2015.11.002
1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
84 M. Fujiko et al. / International Journal of Infectious Diseases 41 (2015) 83–89

dose immunization.1 This program has markedly decreased the antibody (anti-HBe) were tested using Monolisa HBeAg-Ab PLUS
disease burden, carrier rate, and HBV-related morbidity and immunoassay (Bio-Rad, Marnes-la-Coquette, France). HBsAg
mortality.2 However, 50 million new cases of HBV infection quantification was done using Elecsys HBsAg Quant II (Roche
continue to be diagnosed annually, with the highest incidence due Diagnostics, Indianapolis, USA) on a Roche Cobas e411 Immunoa-
to mother-to-child transmission (MTCT). Despite the administra- nalyzer following the manufacturer’s protocol.
tion of hepatitis B immunization (active) or active plus hepatitis B
immune globulin (HBIG) at birth, at least 10% of infants born to 2.3. HBV DNA detection and analysis
HBV-carrying mothers still suffer HBV infection.3 Several factors
such as maternal serum HBV DNA level, hepatitis B e antigen The HBV DNA level was determined from 500 ml of serum by
(HBeAg) status, HBV S gene variation, mode of delivery, and quantitative real-time PCR (CobasTaqman HBV Test; Roche
neonatal immune deficiency have been related to MTCT. Of these Diagnostics, Indianapolis, USA) with a range of linearity between
factors, maternal HBV DNA level has been identified as the most 6 and 1.1  108 IU/ml. HBV DNA for molecular analysis was
relevant.4 Practice guidelines from major professional associations obtained by extracting the DNA from 140 ml of serum using the
address the decision for antiviral treatment in pregnant women QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and amplifica-
based on the HBV DNA threshold.4–6 tion by nested PCR using primers S2-1/S1-2 for the first-round and
Assays for HBV DNA quantification with high sensitivity and S88/S2-2 for the second-round (Supplementary Material,
specificity are currently available. However, the routine application Table S1).12 Amplicons were purified using a PCR purification
of these methods for screening pregnant women is hampered by cost column (Qiagen, Valencia, CA, USA) and subjected to direct
and limited resources. In recent years, the hepatitis B surface antigen sequencing on a DNA sequence analyzer ABI 3130xl (Applied
(HBsAg) level, which correlates positively with viral load, has been Biosystems, Carlsbad, CA, USA).
used as a biomarker to predict disease status and to monitor the The HBV genotype was determined by phylogenetic analysis
treatment response in chronic hepatitis B (CHB).7,8 The HBsAg level based on the 226-nucleotide sequences of the S gene compared
correlates with covalently closed circular DNA (cccDNA) in with 70 reference sequences of known genotypes (A–H) retrieved
hepatocytes and can be considered a surrogate marker of infected from GenBank, using Phylip 3.68 software with the Kimura 2-
cells.9 One attraction of the use of the HBsAg level is that the assay is parameter model, neighbour-joining algorithm, and 1000 boot-
less costly, is suitable for high-throughput screening, and its strapping.12
platforms are commonly used in many laboratories. However, some
studies have reported that HBsAg quantification correlates poorly 2.4. Identification of BCP and PC mutations
with the HBV DNA level. HBsAg and HBV DNA levels may vary during
different phases of CHB and the correlation is associated with the Amplification of BCP and PC regions was done by nested PCR
HBeAg status of the patient.10–12 Certain HBV variants with basal using primers PC1/PC2 for the first-round and S012/S013 for the
core promoter (BCP) A1762T/G1764A or precore (PC) G1896A second-round (Supplementary Material, Table S1). Amplicons
mutations could influence the synthesis of HBeAg.13 The presence of were purified and sequenced as described previously.12 The
these variants needs to be taken into account since they often occur sequences were aligned with reference sequence M54923
in endemic areas and may be associated with certain HBV genotypes retrieved from GenBank.
that differ among geographical regions.14,15
This study aimed to evaluate the usefulness of quantitative 2.5. Statistical analysis
HBsAg as a viral load predictor in pregnant women with CHB in
Makassar, Indonesia, and to analyse the association of the HBsAg The baseline data were summarized descriptively. Continuous
level and viral load with the HBeAg status, as well as the molecular and categorical variables were compared between groups using
characteristics of HBV variants defective for HBeAg production. the Mann–Whitney test and Chi-square/Fisher’s exact test,
respectively. Pearson’s correlation coefficient was used to describe
2. Materials and methods the correlation between two continuous, normally distributed
variables. Spearman’s correlation was used for categorical vari-
2.1. Study population ables or continuous variables that were not normally distribut-
ed.10,12,13 Statistical analyses were performed using IBM-SPSS v.
This cross-sectional study was carried out from January to July 20 software (IBM Corp., Armonk, NY, USA). All statistical
2014 in the antenatal care units of Wahidin Sudirohusodo Hospital, significance values were assessed at p < 0.05.
Hasanuddin University Hospital, Fatimah Mother and Child
Hospital, Pertiwi Mother and Child Hospital, Labuang Baji Hospital,
and Ibnu Sina Hospital, as well as several maternity clinics, in 3. Results
Makassar, South Sulawesi. A total of 943 pregnant women were
screened for HBV infection; 64 of them were HBsAg-positive and 3.1. Characteristics of study subjects
considered eligible for enrolment. For inclusion it was required
that the woman had been HBsAg-positive for >6 months without Among 943 pregnant women attending several antenatal
prior antiviral therapy.16 Subjects co-infected with hepatitis A clinics in Makassar, 64 (6.8%) were HBsAg-positive. Of these
virus, hepatitis C virus, or human immunodeficiency viruses, as women, 12 (18.8%) were HBeAg-positive and 52 (81.3%) were
well as those with evidence of liver diseases, were excluded. This HBeAg-negative. HBV DNA levels were significantly higher in the
study was approved by the Ethics Committee of the Faculty of HBeAg-positive group (median 7.43 log10 IU/ml) than in the
Medicine, Hasanuddin University, Makassar, Indonesia. Written HBeAg-negative group (median 1.55 log10 IU/ml) (p < 0.001).
informed consent was obtained from each patient. Similarly, HBsAg levels were significantly higher in the HBeAg-
positive group (median 4.21 log10 IU/ml) than in the HBeAg-
2.2. Serological examination negative group (median 2.91 log10 IU/ml) (p < 0.001). Age, alanine
aminotransferase (ALT) levels, and HBV genotype distribution
HBsAg status was determined by VIDAS HBsAg immunoassay were comparable in the two groups (Table 1 and Supplementary
(bioMérieux SA, Marcy l’Etoile, France). HBeAg and hepatitis B e Material Table S2).
 M. Fujiko et al. / International Journal of Infectious Diseases 41 (2015) 83–89 85

Table 1
Baseline characteristics of HBsAg-positive pregnant womena

Parameter Overall HBeAg-positive HBeAg-negative p-Valueb

(n = 64) (n = 12) (n = 52)

Age (years) 29 (18–42) 28.5 (22–42) 30 (18–41) 0.564

ALT (IU/l) 24.5 (9–129) 27 (16–129) 24.5 (9–88) 0.129
HBV DNA (log10 IU/ml) 1.71 (0.78–8.05) 7.43 (1.54–8.05) 1.54 (0.78–6.48) <0.001
HBsAg (log10 IU/ml) 3.03 (0.70–4.11) 4.21 (3.25–4.91) 2.91 (0.70–4.11) <0.001
Ratio HBsAg/HBV DNA (log10 IU/ml) 1.25 (0.40–5.30) 0.61 (0.56–0.7556) 1.43 (0.90–2.78) <0.001
Genotype (n = 47)
B 8 (17.0%) 2 (22.2%) 6 (15.8%) 0.889
C 39 (83.0%) 7 (77.8%) 32 (84.2%)

HBeAg, Hepatitis B e antigen; ALT, alanine aminotransferase; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen.
a
Results are reported as the median (minimum–maximum), or number (percentage of detected samples).
b
Comparison between HBeAg-positive and HBeAg-negative groups (Mann–Whitney U-test or Chi-square test).

3.2. Distribution of serum HBV DNA levels among HBsAg-positive content). The sequences generated have been deposited in the
pregnant women according to HBeAg status GenBank database (accession numbers KP241791–KP241837).
Median HBsAg and HBV DNA levels were not significantly different
HBV DNA levels were categorized into <3.0 log10 IU/ml (close to in each genotype. Genotype C was more prevalent than genotype B
3.3 log10 IU/ml or 2000 IU/ml, which is the threshold to define (83% vs. 17%), but the proportions were comparable between the
inactive carrier state of CHB),17 3.0–6.0 log10 IU/ml, and 6.0 log10 HBeAg-positive and HBeAg-negative groups (Figure 4).
IU/ml (a level associated with HBV immunoprophylaxis failure).18
The proportions of pregnant women with HBV DNA levels <3.0 3.6. Subgroup analysis for BCP and PC mutations in HBeAg-negative
log10 IU/ml, 3.0–6.0 log10 IU/ml, and 6.0 log IU/ml in the HBeAg- pregnant women
positive group were 9% (1/12), 25% (3/12), and 67% (8/12),
respectively; in the HBeAg-negative group, the proportions were Sequencing of the BCP/PC region of the HBV genome was
83% (43/52), 13% (7/52), and 4% (2/52), respectively (Figure 1A). performed successfully in 12 HBeAg-negative subjects (sequencing
was not possible in the other subjects because of low HBV DNA
3.3. Distribution of serum HBsAg levels among the HBsAg-positive content). The sequences generated have been deposited in
pregnant women according to HBeAg status GenBank (accession numbers KP241838–KP241844 and
KP241846–KP241850). Four subjects had the wild-type HBV
HBsAg levels were categorized into <3.0 log10 IU/ml (a level DNA sequence at both the BCP and PC sites, four had the BCP
associated with a lower risk of CHB outcomes), 3.0–4.0 log10 IU/ml, mutation alone, and two had the PC mutation alone. Concurrent
and 4.0 log10 IU/ml.5 The proportions of subjects with HBsAg BCP and PC mutations were detected in two subjects. All subjects
levels <3.0 log10 IU/ml, 3.0–4.0 log10 IU/ml, and 4.0 log10 IU/ml with the BCP mutation had HBV genotype C, while the PC mutation
were 0% (0/12), 42% (5/12), and 58% (7/12), respectively, in the was detected in two subjects with genotype B and one subject with
HBeAg-positive group, and 58% (30/52), 35% (18/52), and 8% (4/52), genotype C (Table 2).
respectively, in the HBeAg-negative group (Figure 1B).
4. Discussion
3.4. Correlation between HBsAg and HBV DNA levels
This study represents one of few reports of HBV infection in
In all 64 HBsAg-positive subjects, serum HBsAg and HBV DNA pregnant women from Indonesia. Of 943 pregnant women
levels showed a significantly moderate correlation (r = 0.513; p < attending several antenatal clinics in Makassar, 64 (6.8%) were
0.001) (Figure 2A). When analysed separately according to HBeAg HBsAg-positive. This figure is higher than that reported recently
status, there was a strong correlation between HBV DNA and from Jakarta (2.2%),20 and other places in Indonesia reported
HBsAg levels in the HBeAg-positive group (r = 0.659; p = 0.02) around 1985 (4.7% in West Java, 1.9% in Bali, 3.4% in Mataram).21,22
(Figure 2B), but no correlation was observed in the HBeAg-negative The wide variation in HBV infection rates may be associated with
group (r = 0.194; p = 0.168) (Figure 2C).19 the general HBsAg prevalence in Indonesia (3.4–19.5%), geograph-
There were four subjects (M150, M414, M415, and M818) with ical variation, and differences in cultural practices, as well as the
high HBsAg levels (>4.0 log10 IU/ml) but low HBV DNA levels (<3.0 methods used to detect HBV infection.23,24 This fact is of concern,
log10 IU/ml). In contrast, there were four subjects (M167, M173, because it occurs in pregnant women who tend to be in the
M258, and M810) with low HBsAg levels (<3.0 log10 IU/ml) but immune-tolerant phase of CHB with normal physical/laboratory
who had moderate viraemia (3.0–6.0 log10 IU/ml). However, in examinations and high-level viraemia, but unaware of their
most cases, low levels of HBsAg were associated with low levels of HBsAg-positive status.
HBV DNA; HBsAg levels <3.0 log10 IU/ml were significantly Varying thresholds of maternal HBV DNA have been discussed
correlated to HBV DNA levels <3.0 log10 IU/ml (r = 0.363; p = 0.003) in association with MTCT and immunoprophylaxis failure. Wise-
and to HBV DNA levels <6.0 log10 IU/ml (r = 0.404; p = 0.001). No man reported that immunoprophylaxis failure occurred in infants
subjects with HBsAg levels <3.0 log10 IU/ml had HBV DNA 6.0 when the maternal viral load was 8 log10 copies/ml (>1.7  107
log10 IU/ml (Figure 3). IU/ml).25 Zou et al. showed that the immunoprophylaxis failure
increased with higher levels of maternal HBV DNA.18 When the
3.5. HBsAg and HBV DNA levels in genotype B and C mothers’ HBV DNA levels were stratified to <6, 6–6.99, 7–7.99, and
8 log10 copies/ml, the corresponding rates of immunoprophylaxis
HBV genotype was successfully determined in 47 pregnant failure were 0%, 3.2%, 6.7%, and 7.6%, respectively, and it was
women based on the S gene sequences (sequencing was not concluded that an antenatal HBV DNA level >6 log10 copies/ml
possible in the other subjects because of insufficient HBV DNA (>200 000 IU/ml) was the most important predictor of MTCT.18
86 M. Fujiko et al. / International Journal of Infectious Diseases 41 (2015) 83–89

Figure 1. Distribution of (A) HBV DNA and (B) HBsAg levels among pregnant women according to HBeAg status: (i) among the HBeAg-positive group (n = 12) and (ii) among
the HBeAg-negative group (n = 52).

The European Association for the Study of the Liver (EASL) and the first study to evaluate the relationship between HBsAg and HBV
Asian Pacific Association for the Study of the Liver (APASL) DNA levels with regard to HBeAg status in pregnant women with
guidelines recommend treating pregnant women when HBV DNA CHB in Indonesia. This study revealed that among all 64 HBsAg-
levels are >2  106 IU/ml in the third trimester for the prevention positive pregnant women, the HBsAg level was correlated with the
of MTCT.5,6 In the present study, 15.6% (10/64) of all subjects had HBV DNA level regardless of age and viral genotype. When
HBV DNA levels >6.0 log10 IU/ml, distributed in 67% (8/12) of the stratified based on HBeAg status, the correlation was strong in
HBeAg-positive group and 4% (2/52) of the HBeAg-negative group. HBeAg-positive pregnant women but missing in the HBeAg-
The fact that the subjects had a skewed distribution toward the negative women. A possible explanation for this finding is that
higher levels of HBV DNA should be regarded as important, as this HBsAg synthesis has a pathway distinct from HBV DNA synthesis
shows a higher possibility of MTCT. and under the influence of different immune-control mecha-
There are few studies on the potential applications of nisms.27,28 HBsAg is present as a component of HBV virions but also
quantitative HBsAg in the management of hepatitis B during as subviral particles, which exceed the number of virions.9
pregnancy.16,26 To the best of the authors’ knowledge, this is the Pregnant women with an HBeAg-positive status could be in the

Figure 2. Correlation of HBsAg and HBV DNA levels in pregnant women according to HBeAg status: (A) overall correlation in all pregnant women (n = 64); (B) correlation of
HBsAg and HBV DNA levels in HBeAg-positive women (n = 12); (C) correlation of HBsAg and HBV DNA levels in HBeAg-negative women (n = 52). Both the HBV DNA and HBsAg
levels are calculated in log10 IU/ml. The correlation is regarded very weak for r = 0–0.19, weak for r = 0.20–0.39, moderate for r = 0.40–0.59, strong for r = 0.60–0.79, and very
strong for r = 0.80–1.19.
 M. Fujiko et al. / International Journal of Infectious Diseases 41 (2015) 83–89 87

Table 2
BCP/PC mutations, HBV DNA and HBsAg levels, and HBV genotype in 12 HBeAg-
negative pregnant women

Subject BCP PC HBV DNA HBsAg Genotype

code mutation mutation (log10 IU/ml) (log10 IU/ml)
A1762T/ G1896A
G1764A

M177 + + 6.48 3.46 C

M384 + 6.08 3.04 C
M167 + 4.55 2.69 B
M810 3.50 2.70 C
M218 + 3.29 3.56 C
M336 + + 2.70 3.74 C
M150 2.62 4.00 C
M898 + 2.08 3.04 B
M253 1.36 3.05 C
M818 + 1.34 4.11 C
M19A 0.78 2.51 C
M212 + 0.78 3.55 C

Figure 3. Distribution of HBV DNA levels among pregnant women according to BCP, basal core promoter; PC, precore; HBV, hepatitis B virus; HBsAg, hepatitis B
HBsAg levels. In most cases, low levels of HBsAg were associated with low levels of surface antigen; HBeAg, hepatitis B e antigen.
HBV DNA; HBsAg levels <3.0 log10 IU/ml were significantly correlated to HBV DNA
levels <3.0 log10 IU/ml (r = 0.363; p = 0.003) and to HBV DNA levels <6.0 log10 IU/ml
(r = 0.404; p = 0.001). No subjects with HBsAg levels <3.0 log10 IU/ml had HBV DNA
6.0 log10 IU/ml. over the number of virions.8 This implies that a high level of HBsAg
cannot be used to predict the HBV DNA level. The serum HBsAg
level should thus be used together with, but not as a substitute for,
immune-tolerant phase where HBV virions and their antigens are HBV DNA.29 Four other subjects had low HBsAg levels but
minimally subjected to the host immune reaction, while those moderate viraemia (>4 log10 IU/ml). Possible explanations for
with an HBeAg-negative status could be in the low-replicative the decreased detection of HBsAg include (1) differences in
phase of CHB where the number of virions has decreased as a result analytical sensitivity and specificity in HBsAg detection of viruses
of successful immune control.12,27 Therefore, the reduction in of different genotypes; (2) mutations in the pre-S/S gene that cause
HBsAg levels was not proportional to that of HBV DNA levels. The HBsAg detection failure; (3) treatment-associated mutations that
ratio of HBsAg/HBV DNA reflects the association between HBsAg cause derangement of the P gene with subsequent alteration of the
production and HBV replication; this was significantly higher in overlapping S gene; or (4) the concomitant presence of hepatitis B
HBeAg-negative subjects than in the HBeAg-positive group surface antibodies (anti-HBs) leading to the formation of immune
(median 1.43 vs. 0.61 log10 IU/ml, respectively). complexes poorly displaced by HBsAg-capture antibodies.7,30,31 In
Four pregnant women had high HBsAg levels with low HBV the majority of subjects, however, low levels of HBsAg (<3.0 log10
DNA. As explained, this could be due to the larger excess of HBsAg IU/ml) correlated significantly with low levels of HBV DNA (<6.0

Figure 4. Distribution of serum HBV DNA (A) and HBsAg (B) levels in subjects with HBV genotype B (n = 8) and HBV genotype C (n = 39). HBV DNA and HBsAg levels were
comparable in the two genotypes. Median values (max, min; log10 IU/ml) with 95% confidence intervals are shown.
88 M. Fujiko et al. / International Journal of Infectious Diseases 41 (2015) 83–89

log10 IU/ml) and an attendant lower risk of MTCT.6,18 This result Funding: This study was supported by an Operational Support
suggests that HBV DNA quantification may not be necessary for Grant for State University (BOPTN) from the Directorate General of
pregnant mothers with low HBsAg levels. Higher Education, Indonesian Ministry of Education (grant number
Another important finding from this study was the presence of 2058/UN4.20/PL.09/2013). PT Roche Indonesia donated the
HBeAg-negative pregnant women with high viraemia. These reagents used for HBsAg quantification.
women, with no evidence of liver disease, were apparently in the Conflict of interest: No conflict of interest to declare.
inactive carriers of CHB. However, some inactive carriers may
have high HBV DNA levels accompanied by persistently normal
ALT levels.5 Studies have documented that certain HBV variants Appendix A. Supplementary data
with nucleotide substitutions in the PC and/or BCP regions could
abolish or down-regulate HBeAg production. These variants may Supplementary data associated with this article can be found, in
replicate rapidly in HBeAg-negative CHB, where HBsAg and HBV the online version, at http://dx.doi.org/10.1016/j.ijid.2015.11.002.
DNA levels are preserved.13 Analysis for the presence of BCP
(A1762T/G1764A) and PC (G1896A) mutations was performed on
HBeAg-negative subjects who had HBV DNA levels >2000 IU/ml. A References
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