Viral replication in chronic HBV hepatitis
Romanian Journal of Gastroenterology
September 2005 Vol.14 No.3, 219-224
Address for correspondence:Lazãr Cãlin, MD PhD,
Sstr. Moþilor, 68
Evaluation of Viral Replication in Children with Chronic
Hepatitis B with and without Interferon Treatment*
Cãlin Lazãr1, Paula Grigorescu-Sido1, Rodica Manasia1, ªtefan Mireºtean1, Cristina Skorka1, Daniela Neculoiu2,
Geza Molnar3, Stela Cocean3
1) „Axente Iancu” 1stPediatric Clinic. 2) 3rd Medical Clinic.3) „Iuliu Moldovan” Institute of Hygiene, Cluj-Napoca
* This study was funded by a grant (2001-003) from the National
Council of Scientific Research in Higher Education (CNCSIS) and
by the Romanian Branch of Schering-Plough Co.
Background. In chronic infection with hepatitis virus B
the fact that HBeAg becomes negative does not always
mean suppression of viral replication. Method. HBV
replication was assessed in 74 patients with chronic hepatitis
or viral B cirrhosis, in whom diagnosis was made according
to clinical, biological, and histological criteria. The patients
were divided into two groups: group I (36 patients with
interferon-α therapy, 3 million U/m2/dose, 3 doses/week
over a period of 4-6 months) and group II (control group of
38 patients who did not undergo interferon therapy). After
a follow up period of 6 years in which patients underwent
clinical, biochemical and serologic monitorization, HBV
DNA was detected by the hybridization method on solid
medium. Results. During evolution the levels of trans-
aminases became normal in both groups. The HBe Ag/Ab
seroconversion rate at the end of the interferon therapy
was 52.8% and the spontaneous HBe Ag/Ab serocon-
version rate was 72.7% in group II after an average evolution
of 6 years. HBs Ag/Ab seroconversion was not detected in
any patient. Assessment of viral replication by HBV DNA
testing at the end of the follow up period showed higher
levels as compared to the HBeAg testing (69.4% vs. 25% in
group I, 55.2% vs. 7.9% in group II). The absence of viral
replication (HBV DNA negative) had similar rates in both
groups (30.6% in group I vs. 44.8% in group II, p>0.9) and
HBV DNA titers in the two groups were not significantly
different at the end of the follow up period. In both groups,
HBV DNA titers were significantly higher in patients with
positive HBeAg. The concordance between the two viral
markers was 100%. Conclusion. Because of the fluctuating
evolution, long-term follow up and monitorization (including
HBV DNA testing) of patients with chronic hepatitis B and
of inactive HBsAg carriers are necessary.
Chronic hepatitis B - HBV DNA - children - sero-
Premize. In infecþia cronicã cu virus hepatitic B
negativarea AgHBe nu semnificã întotdeauna dispariþia
replicãrii virale. Metodã. Am evaluat stadiul replicãrii VHB la
74 pacienþi cu hepatitã cronicã sau cirozã hepaticã viralã B
la care diagnosticul a fost stabilit pe baza criteriilor clinice,
bioumorale ºi histopatologice. Pacienþii au fost grupaþi în
douã loturi: lotul I (36 pacienþi trataþi cu interferon-α, 3
milioane U/m2/dozã, 3 doze/sãptãmânã, timp de 4-6 luni) ºi
lotul II (lot martor constituit din 38 pacienþi care nu au urmat
terapie cu interferon). La sfârºitul unei perioade de
supraveghere de 6 ani în care pacienþii au fost monitorizaþi
clinic, biochimic ºi serologic, s-a determinat ADN-VHB prin
metoda hibridizãrii pe suport solid. Rezultate. Pe parcursul
evoluþiei transaminazele s-au normalizat la ambele loturi. Rata
de seroconversie Ag/AcHBe la sfârºitul tratamentului cu
interferon a fost de 52,8% iar rata de seroconversie Ag/Ac
HBe spontanã la lotul II dupã o evoluþie medie de 6 ani a
fost de 72,7%. Seroconversia Ag/AcHBs nu a fost
constatatã la nici un pacient. Evaluarea replicãrii virale prin
determinarea ADN-VHB la sfârºitul perioadei de
supraveghere a avut o valoare net superioarã faþã de
determinarea AgHBe (69,4% vs. 25% pentru lotul I, 55,2%
vs. 7,9% pentru lotul II). Absenþa replicãrii virale (ADN-
VHB negativ) a fost înregistratã în rate apropiate la ambele
loturi (30,6% la lotul I vs, 44,8% la lotul II, p >0,9) iar titrurile
ADN-VHB nu au prezentat diferenþe semnificative între cele
douã loturi la sfârºitul perioadei de monitorizare. La ambele
loturi titrurile ADN-VHB au fost semnificativ mai mari la
pacienþii cu AgHBe pozitiv, concordanþa între cei doi markeri
Lazãr et al220
virali fiind de 100%. Concluzie. Datoritã evoluþiei fluctuante
a infecþiei cu VHB este necesarã monitorizarea (inclusiv cu
determinarea ADN-VHB) pe termen lung a pacienþilor cu
hepatitã cronicã viralã B ºi a purtãtorilor inactivi de AgHBs.
According to recent epidemiological data there are over
350 million HbsAg carriers in the world. Romania is ranked
as an area of intermediate endemicity for hepatitis virus B
infection (HBV), according to recent studies which reported
a prevalence rate of HbsAg of 6-8% in the general population
The history of the disease is characterized by an early
HBeAg and a late anti-HBe positive phase. Recent studies
have shown that following anti-HBe seroconversion liver
disease may persist associated with HBV-DNA positivity.
Thus, absence of HBeAg does not always mean the
supression of viral replication. For a correct diagnosis of
HBeAg negative hepatitis it is necessary to evaluate the
HBV-DNA viremia. In Romania, HBV DNA testing has
become available only recently. Before this, viral replication
was presumed to be present only when HbeAg was positive.
The aim of our study was to correlate the outcome of the
disease with the levels of viral replication in treated and
untreated children with chronic HBV hepatitis or cirrhosis.
Material and method
The study was performed in 68 patients with chronic
hepatitis B and 6 patients with viral B liver cirrhosis who
had been diagnosed in the 1st Clinic of Pediatrics over the
period 1998-2001. The diagnosis was based on clinical,
biological, serological and histopathological criteria. Liver
biopsy was performed in 52 patients, with the informed
consent of their parents. The patients were divided into two
groups: group I (36 patients with interferon (IFN) therapy)
and group II (38 patients with no IFN therapy). Patients
were selected for IFN therapy according to the following
criteria: serum aminotransferases of at least twofold the
upper normal limit (40 UI/I); positive HBeAg; necro-
inflammatory activity evidenced in liver biopsy samples
(Knodell score with histologic activity index > 6 and fibrosis
= 3). We did not have the opportunity to use the HBV DNA
testing, as it was not available at that time.
Interferon therapy was initiated and α-2b (Intron) or α-
2a (Roferon) were used in a dose of 3MU/m2 dose, 3 doses/
week, for 4-6 months. Monitorization of patients included
the following: the clinical aspect, serum transaminases
(checked every other two months) and HBV serology
(HBsAg, HBeAg, HBeAb). According to the classical criteria
the therapeutic response when IFN was suppressed was
judged as complete, incomplete, transitory or absent (2).
According to the EASL (3) and AASLD (4) consensus, in group
I the response to IFN was classified as: end-of-treatment
response, end-of-follow-up response and no response.
After a mean follow-up of 6 years, all the patients were
reassessed by testing the transaminases and the serologic
markers of HBV infection. The HBV DNA quantitative testing
was also performed by means of the hybridization method
on solid medium, which became available for 72 patients (34
from group I, and 38 from group II). The ELISA method
(Organon kits of second generation with ISO certified
standardized immunoreactives) was used to determine the
serologic markers of the HBV infection (HBsAg, HBsAb,
HBeAg, HBeAb, HBcAb) as well as the antibodies for the
possible HCV or HDV coinfections.
HBV DNA was evidenced by using Digene Hybrid
Capture testing kits (HBV DNA Assay) (Abott). The method
was applied in 4 stages: the incubation of the serum
(assumed as containing HBV DNA) with RNA specific for
the ad or ay subtypes, thus DNA RNA hybrids being
obtained; the capture of hybrids on a solid medium lined
with DNA RNA hybrid antibodies; the interaction between
hybrids and hybrid antibodies (in liquid state) marked with
alkaline phosphatase molecules; finally the cleavage of a
chemiluminiscent substrate by the alkaline phosphatase
with light signal emission (quantified by chemiluminiscence
and compared to a standard calibration curve). The results
express the HBV DNA concentration in pg/ml or copies/ml.
According to the sensitivity limit of the kit used, positive
HBV DNA was taken into consideration for levels higher
than 0.017 pg/ml (corresponding to 4.7x 103 copies/ml) and
negative HBV DNA for levels below this level.
The results were statistically interpreted using the
Student and χ2 tests.
The age of the patients at diagnosis in the two groups
and the follow up duration are shown in Table I.
The acute onset of the HBV infection (biological and
clinical picture of an acute viral hepatitis) was found in 3
patients of group II. The source of the infection could not
be certain in all patients. Intrafamilial infection was detected
in 13 patients (9 from group I and 4 from group II) who had
at least one case of HBV infection in their families.
The studied patients were 46 boys and 28 girls (M/F
ratio = 1.6/1). The difference between gender repartition in
the two groups was not statistically significant. The serum
transaminase levels in the patients of the two groups, at
diagnosis and at reassessment, as well as the levels found
when IFN therapy was stopped (in group I) are shown in
Fig.1. When patients were reassessed, all had normal levels
of serum transaminases, irrespective of the therapy they
had been on or of their serologic status at that moment.
When patients were reassessed, the physical examination
showed normal findings except for a slight hepatomegaly in
19 of the patients.
The results of the assessment of the viral replication by
HBeAg and HBeAb testing at diagnosis, when the IFN
therapy was suppressed (in the patients of group I) and
when the patients were reassessed are shown in Table II.
Viral replication in chronic HBV hepatitis 221
Table I The age of the patients at diagnosis and duration of the
The age of the
The follow up
with the moment
of IFN therapy
Mean levels of
onset of the
- ALT (UI/l)
- AST (UI/l)
7.6 + 3.5/
6.0 + 3.2 /
mean +DS /
3 -14.6 years
mean +DS /
Fig.1 The transaminase levels in the patients from the two groups: A- at diagnosis; I- when interferon therapy was stopped (group
I); B- when patients were reassessed.
Table II Assessment of viral replication by HBeAg testing
*after 5,13+2,31 years (group I) and after 6,82+3,74 years (group II)
A(I-II)=33,277 → p<0,001; χ2
B( I-II)= 3,981 → p<0,05; χ2
T(A-B)= 31,074 → p<0,001; χ2
I(A-B)= 36,086 → p<0,001; χ2
The serologic response (assessed by HBe Ag/Ab sero-
conversion) when IFN therapy was suppressed, was present
in 23 (63.8%) of the patients in group I. In 4 of these patients
the response was transient: HBeAg became positive after
therapy suppression in an average period of 12.6 months
(limits: 5-20 months) and remained positive over
the whole follow up period. A sustained response at 6
months and at 12 months after IFN was present in 22 (61.1%)
patients and 21 (58.3%) respectively, of the patients in group
I. At the end of the follow up period, HBe Ag/Ab
seroconversion was permanent in 19 patients after IFN
(52.8% of the treated group). Other 8 patients out of the 13
patients in group I (61.5%), who had HBeAg positive when
IFN therapy was suppressed, presented spontaneous HBe
Ag/Ab seroconversion with an annual rate of 13.5% over
the next years of follow up (mean = 4.53 years). When
reassessed, 27 patients in group I had HBe Ag/Ab
seroconversion (representing 75% of the group).
In group II, 8 patients out of the 11 (72.7 %) with positive
HBeAg at diagnosis presented spontaneous HBe Ag/Ab
seroconversion. The spontaneous seroconversion annual
Lazãr et al222
Fig.2 Assessment of viral replication by AgHBe and HBV DNA
in the two subgroups: A - at diagnosis; I - when interferon
therapy was suppressed (for subgroup I); B - when patients
Table III HBV DNA and HBeAg when patients were reassessed
(n = 36)
(n = 38)
(n = 74)
χ2 1,2 = 0,144 → p > 0.05
Out of the two groups, 15 patients (11 from group I and
4 from group II), representing 20.2% of all patients, had
levels over 105 copies/ml. In 6 out of the 15 patients (4 from
group I and 2 from group II) viremia was higher than 105
copies/ml when AgHBe became negative. Initially HCV and
HDV co-infections were detected in one patient each.
The two groups were comparable in terms of age and
gender distribution. They were different only in terms of
duration of follow up, which was longer for group II. This
group did not undergo IFN therapy (Table I). The α-IFN
dose used in the patients of group I (3 MU/m2/ dose) was
lower than the 6 MU/m2/dose recommended in the recent
consensus (3,4). This dose had been established at a national
level by the Ministry of Health in Romania.
The initial levels of serum transaminases were signi-
ficantly higher in group I which had undergone IFN therapy
as compared to those in group II (p<0.005 both for ALT and
for AST- Fig.1). One of the factors of decision for IFN
therapy was the level of aminotransferases, which was more
than double the normal values. Transaminase levels norma-
lized in both groups during the disease evolution (though
at a slower rate in group II, without IFN therapy) (Fig.1).
The initial serologic assessment (Table II) showed
significantly more cases with positive HBeAg in group I as
compared to group II (100% vs 28.9%; p<0.001).
When IFN therapy was suppressed, the incidence of
positive HBeAg cases in group I became significantly lower,
decreasing to 36.1%, showing that therapy is efficient in
rapid HBe Ag/Ab seroconversion. The response was
transient in 4 patients, HBe Ag/Ab seroconversion, with an
incidence of 63.8% when IFN was suppressed, decreasing
to 52.8% by the end. This rate is close to the one we reported
in previous studies (5,6) and also ranks within the limits
reported in the literature (22-78%) (7-19). The differences
seem to depend on: the number of cases in the study (9 -
837), the duration of the follow up period (12-50 months),
the moment of the reassessment (at the end of therapy or at
different therapy intervals), the geographical area (endemic
regions reporting weak responses to IFN), the IFN therapy
duration (6-12 months) and the dose of IFN administered
(3MU/m2 - 10 MU) (7-19).
Spontaneous seroconversion in group II was 72.7% over
a 6-year period. This level was higher than those reported
by other authors, 5%-65% (20,21). The spontaneous annual
rate, calculated by reference to the follow up period (mean
6.82 years) was of 10.6/ 100/ year.
HBs Ag/Ab seroconversion was not detected in any
patient in group I. It was only found in 2 patients of group II
(5.2%). The HBs Ag/Ab seroconversion annual rate was of
Assessment of the viral replication by both HBeAg and
HBV DNA testing was possible only when patients were
Table IV HBV DNA (copies/ml) at patients’ reassessment
with positive HBeAg
and negative HBeAb
23138 + 21915 x 103
73528 + 47656 x 103
35736 + 35126 x 103
with negative HbeAg
and positive HbeAb
173,23 + 526,35 x 103
169,47 + 765,32 x 103
178 + 526 x 103
5008 + 13607 x 103
6023 + 22993 x 103
5612 + 17354 x 103
P a(I-II) > 0.05 ; P b (I-II) > 0.05 ; P c (I-II) > 0,05; P I (a-b) < 0.01*; P II (a-b) < 0.05*; P III (a-b) < 0.01*
* -statistically significant
The HBV DNA testing indicated presence of viremia not
only in the patients with positive HBeAg, but also in some
of those HbeAg negative (Table III).
HBV DNA titers in patients of the two groups who are
grouped according to AgHBe presence or absence at the
moment of patients’ reassessment, are shown in Table IV.
Viral replication in chronic HBV hepatitis223
seroconversion rate in group II was close to that detected
in group I, which did not respond to IFN (10.6% vs. 13.5%)
In our patients, HBs Ag/Ab seroconversion after IFN
was not detected in any patient, which is concordant to the
results reported in adults by other studies (22-24). However,
this is in contrast with the two other observations in Romania,
indicating a rate of 7.25% (18) or 20% (19) in children. The
5.2% incidence of spontaneous HBs Ag/Ab seroconversion
in group II is comparable to that reported by other authors
The patients who had undergone HBe Ag/Ab serocon-
version (spontaneously or after IFN) were ranked as HBsAg
inactive carriers, as they had positive HBsAg, negative
HBeAg, normal levels of transaminases over the whole fol-
low up period, and HBV DNA titers below 105 copies/ml.
However, 20-30% of these patients might present later reacti-
vation of the viral infection and develop chronic hepatitis.
This is the reason why long-term monitoring of the serum
transaminase levels, every 6-12 months, is necessary (3,4).
When patients were reassessed, viral replication
evaluated by the presence of HBeAg was significantly lower
in both groups as compared with the initial assessment.
The rate was higher in group I than in group II (Fig. 2).
When all the patients were reassessed, irrespective of their
HBe serologic status, they all had normal levels of serum
transaminases. Therefore, we can state that at that particular
moment we could not identify any case in which AgHBe
negative chronic hepatitis could be suspected.
When HBV DNA testing became available, in both
groups viral replication as determined by the HBV DNA
titer was at higher rate than in case of HBeAg testing: 69.4%
vs. 25% in group I and 55.2% vs. 7.9% in group II. Persistence
of viremia after HBeAg becomes negative can be explained
by the presence of pre-core mutant strains. The 34 patients
of our study with negative HBeAg and positive HBV DNA
(45.9% of the 74 investigated patients) (Table III) could be
presumed to have an infection with pre-core HBV mutants.
Viral genome testing could confirm this, but it is not available
in Romania. The 45.9% prevalence, that we found, ranks
within the limits that are mentioned in the literature for HBV
pre-core mutants. This also depends on the endemicity of
the reference zone: 35-65% in Japan, India and China (26,27),
40-70% in the Mediterranean region (26,28) and 50-85% in
the Balkans (29, 30).
Absence of viral replication (HBV DNA negative) was
found in both groups: in group I - 30.6% and in group II -
44.8% (Table III). Moreover, HBV DNA titers at the end of
the follow-up period were not different in the two groups
(Table IV). These results indicate that, after an average
evolution of 6 years, the level of viral replication in patients
who had IFN therapy was similar with that detected during
the natural evolution of the disease.
As reported in other studies too (29,31,32), we found
HBV DNA titers significantly higher in cases with positive
HBeAg (Table IV). There was also a 100% concordance
between positive HBeAg and positive HBV DNA.
Viremia over 105 copies/ml persisted in 15 patients even
when liver cytolysis was absent and even in some of the
HBeAg negative patients. This raises questions with regard
to the progress of the disease and the evolution of the
patients, as clinical and biological signs of chronic hepatitis
can occur at some point. Because all our patients did not
show either clinical changes or cytolysis, irrespective of
the level of viremia, we concluded that the level of viral
replication, as a single parameter, is not determinant in
inducing the clinical and biological aspect of chronic
Although it has been accepted that serum HBV DNA is
quite an accurate reflection of the level of viral replication,
there is no certain threshold level of viremia to differentiate
patients with negative HBeAg as either inactive carriers or
having chronic hepatitis B (33, 34). The threshold of 105
copies/ml for viral replication, accepted by most authors
and by EASL and AASLD consensus (3,4,35,36) was
arbitrarily set as the upper limit below which the liver disease
is considered non-progressive. This level actually represents
the detection limit of HBV DNA by methods that do not
make use of PCR. Titers over 105 copies/ml were correlated
with high levels of transaminases and major histologic
changes (37,38). However, some observations showed that
even titers of 25-30,000 copies/ml can have pathologic
significance, being associated with a higher risk of
hepatocellular carcinoma (39,40). At present, the clinical
significance of low HBV DNA titers (at times persistent even
after HBsAg becomes negative) remains uncertain.
Because of the fluctuating evolution of HBV infection, a
single HBV DNA testing is irrelevant, repeated testings
being necessary in order to state the viral replication status.
For the same reasons, all patients infected with HBV (even
those with negative HBeAg and HBV DNA below 105 copies/
ml) need a long follow-up (years or even decades). The
serum transaminases, HbsAg, HBeAg, and HBV DNA
testings must be performed at regular intervals of 3, 6 or 12
The results of our study confirm the effectiveness of
IFN therapy for obtaining rapid HBe Ag/Ab seroconver-
sion in children with chronic hepatitis B. They also show
that the rate of viral replication as assessed by HBeAg
presence is obviously lower than that assessed by HBV
DNA testing. This clearly confirms that this testing must be
used in selecting the patients for IFN therapy, in assessing
their response to therapy and in monitorizing the evolution
in children with chronic hepatitis B. HBV DNA determination
is indispensable for determining the viral replication status
and it must be carried out in all patients including those
with AgHBe negative and normal transaminases. Presence
of the HBV DNA (especially in levels over 105 copies/ml)
requires viremia monitorization at least every 6 to 12 months.
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