ORIGINAL ARTICLE

Hepatitis B virus infection in children of HBV-related chronicliver disease patients: a study of intra-familial HBV transmission

Hartono Gunardi1 • Melanie Y. Iskandar1 • Turyadi2 • Susan I. Ie2 •

Pramita G. Dwipoerwantoro1 • Rino A. Gani3 • David H. Muljono2,4,5

Received: 2 May 2016 / Accepted: 25 August 2016

� Asian Pacific Association for the Study of the Liver 2016

Abstract

Background HBV-infected patients are potential sources

of intra-familial transmission. We studied HBV transmis-

sion and molecular characteristics within families of HBV-

related chronic liver disease (CLD) patients.

Methods Family members [index cases (ICs), spouses, and

1–18-year-old children] of HBV-related CLD patients were

tested for HBsAg, anti-HBc, and anti-HBs. HBsAg-posi-

tive subjects were tested for HBeAg/anti-HBe. Anti-HBc-

positive children together with their family members were

further investigated for HBV DNA. Sequences of positive

isolates were analyzed over surface, precore (PC) and basal

core promoter (BCP) regions.

Results Among 94 children of 46 ICs, the prevalence of

HBsAg, anti-HBc, and anti-HBs was 10 (10.6 %), 19

(20.2 %), and 46 (48.9 %), respectively. Thirty-eight

(40.4 %) children were seronegative, indicating suscepti-

bility to HBV infection. HBV DNA was identified in all

ICs, 4 spouses, and 16 children. Having both parents with

HBsAg positive and at least two HBV carriers in the

households were significant risk factors of intra-familial

transmission. HBV genotype/subtype distributions were

comparable between children and ICs/spouses, with pre-

dominance of genotype B. The majority of HBV DNA

sequences found in children were identical to their corre-

sponding ICs—particularly mothers—including mutation

patterns in the surface, PC, and BCP regions. Recognized

mutations associated with HBsAg detection and/or vacci-

nation failure, T140I, T143S/M, G145R, and Y161F, were

identified in 20 subjects; while mutations linked to HBeAg-

defective variants, PC G1896A and BCP A1762T/G1764A,

were found in 7 and 11 subjects, respectively.

Conclusions Children of HBV-related CLD patients were

at increased risk of HBVinfection through multi-modal

H. Gunardi and M. Y. Iskandar contributed equally.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12072-016-9764-z) contains supplementarymaterial, which is available to authorized users.

& David H. Muljono

davidhm@eijkman.go.id; dhmuljono@gmail.com

Hartono Gunardi

h_gunardi@yahoo.com

Melanie Y. Iskandar

mel97819@gmail.com

Turyadi

yadi@eijkman.go.id

Susan I. Ie

sii@eijkman.go.id

Pramita G. Dwipoerwantoro

pramitagd@yahoo.com

Rino A. Gani

personaly@yahoo.com

1 Department of Child Health, Faculty of Medicine, University

of Indonesia, Jakarta, Indonesia

2 Eijkman Institute for Molecular Biology, Jalan Diponegoro

69, Jakarta 10430, Indonesia

3 Department of Internal Medicine, University of Indonesia,

Cipto Mangunkusumo Hospital, Jakarta, Indonesia

4 Faculty of Medicine, Hasanuddin University, Makassar,

Indonesia

5 Sydney Medical School, University of Sydney, Sydney,

NSW, Australia

123

Hepatol Int

DOI 10.1007/s12072-016-9764-z

transmission routes despite negative parental HBsAg and

HBeAg status.

Keywords Hepatitis B in children � Hepatitis B virus �Intra-familial transmission � Transmission modes of HBV �Mutation

Introduction

Hepatitis B virus (HBV) infection is a major health prob-

lem. An estimated 2 billion people have been infected, and

240 million are chronic carriers with risks of liver disease

progression to cirrhosis and hepatocellular carcinoma

(HCC) and premature mortality in productive age [1]. The

prevalence of HBV infection varies in different regions.

Depending on the prevalence of hepatitis B surface antigen

(HBsAg), HBV endemicity is classified into three cate-

gories: high ([8 %), intermediate (2–8 %), and low

(\2 %) [2]. In HBV endemic countries, most infections

occur in infancy resulting from vertical or mother-to-child

transmission (MTCT), while in low endemic countries,

transmission occurs through horizontal mode, primarily

among unvaccinated adults [3]. Indonesia has a moderate-

to-high hepatitis B endemicity with average HBsAg

prevalence of 9.4 % (4–20.3 %, varying across the archi-

pelago) and anti-HBV core (anti-HBc) of 32.8 % with a

steady increase from 10.04 % in the age group\5 years to

58.05 % in the age group C60 years, indicating high

infection rates through horizontal transmission [4].

Patients with chronic hepatitis B (CHB) are considered

to be major reservoirs for HBV transmission [5, 6]. Within

families, the HBsAg carrier rate in children is closely

related to their parents’ HBsAg status [7, 8]. The acquisi-

tion age of infection and maternal hepatitis B ‘e’ antigen

(HBeAg) status during pregnancy are important factors in

determining chronicity in children [9]; chronic infection

develops in 90 % of infected neonates or infants and

25–50 % of infected children in the age 1–5 years, but only

1–5 % of infected adults [2]. Without HBV immunopro-

phylaxis, more than 90 % of infants of HBeAg/HBsAg-

positive mothers will become chronic HBV carriers, in

contrast to \5 % of those born to HBsAg-positive but

HBeAg-negative mothers [2].

Although clinical histories and HBV serological mark-

ers of family contacts can reveal the possible routes of

intra-familial HBV transmission in most instances, identi-

fication of transmission routes—particularly when both

parents are HBsAg positive—is not possible on the basis of

clinical and serological evidence alone. Instead, HBV

genotyping and phylogenetic analysis followed by

nucleotide sequence comparison from different viral strains

have been used for this purpose [10, 11]. Studies of viral

mutants also help in tracing intra-familial transmission,

since HBV mutants—particularly of the core gene—re-

main stable for more than 2 decades in perinatally acquired

familial infection [12].

This study aimed to investigate the molecular epidemi-

ology of hepatitis B in children of HBV-related chronic

liver disease (CLD) patients in Jakarta, Indonesia, and the

intra-familial mode of HBV transmission by analysis of

HBV genotypes and nucleotide sequences, including the

specific mutations at the surface, precore (PC), and basal

core promoter (BCP) regions of viral isolates of infected

family members.

Materials and methods

This was a cross-sectional study on children of HBV-re-

lated CLD patients (i.e., CHB, liver cirrhosis, or HCC) who

were treated at the Hepatology Outpatient Clinic, Depart-

ment of Internal Medicine, Cipto Mangunkusumo Hospital,

Jakarta, during the period of July–September 2009. Patients

enrolled as the index cases (ICs) were HBsAg positive for a

period of more than 6 months. Study children were

recruited from each patient’s household. Family members

were eligible for inclusion if they had at least one child

aged 1–18 years with no history of blood transfusion,

intravenous drug use, and/or tattooing. After informed

consent was obtained, each family was asked to complete a

questionnaire on factors associated with HBV transmission

to the children that included demography, perinatal events,

and behavioral factors of the children, as well as medical

histories of the ICs [13]. Serum samples were obtained

from the patients/ICs, ICs’ spouses, and their children.

Liver function was assessed directly for serum alanine

transaminase (ALT), and other aliquots were stored at

-70 �C until used. This study was approved by The

Committee of Medical Research Ethics of the Faculty of

Medicine, University of Indonesia (no. 297/PT02.FK/

ETIK/2009).

HBV serological examination

HBsAg, anti-HBs, and anti-HBc were tested on all subjects

(ICs, ICs’ spouses, and children) using commercially

available immunoassays (Elecsys HBsAg, Elecsys anti-

HBs, and Elecsys anti-HBc Immunoassays; Roche Diag-

nostics, Indianapolis, IN, USA) according to the manu-

facturer’s instructions. Anti-HBs level C10 IU/l was

considered positive [1]. All subjects who were positive for

HBsAg and/or anti-HBc were further tested for HBeAg/

anti-HBe status by MonolisaTM HBeAg Ag-Ab PLUS

[Biorad, Marnes-la-Coquette, France] according to the

manufacturer’s instructions.

Hepatol Int

123

HBV DNA detection, genotype determination,

and analysis of S gene

HBV DNA was extracted from 140 ll of serum using

QIAamp DNA-MiniKit (Qiagen, CA, USA) according the

manufacturer’s instructions. The DNA fragment of S gene

that encodes the HBsAg ‘a’ determinant was amplified by

nested PCR using specific primer sets S2-1 (nt 455–474,

sense; 50-CAA GGT ATG TTG CCC GTT TG-30)/S1-2 (nt

704–685, antisense; 50-CGA ACC ACT GAA CAA ATG

GC-30) as outer primers and S88 (nt 462–481, sense; 50-TGT TGC CCG TTT GTC CTC TA-30)/S2-2 (nt 687–668,

antisense; 50-GGC ACT AGT AAA CTG AGC CA-30) asinner primers [14–16]. The amplification product was

purified using a purification column (Qiagen, CA, USA)

and subjected to direct sequencing reactions on DNA

sequence analyzer ABI 3130xl (Applied Biosystems,

Carlsbad, CA, USA). HBV genotypes were determined by

phylogenetic tree analysis based on the S gene sequences

together with HBV sequences of known genotypes

retrieved from GenBank, using the neighbor-joining

method and Kimura-2 parameter in Phylip v.3.68 software,

with 1000 bootstraps (Suppl. Fig. 1). HBsAg subtypes

(adw, adr, ayw, or ayr) were determined using deduced

amino acid sequences of two pairs of mutually exclusive

determinants, d/y and w/r, at amino acids 122 and 160 in

the ‘a’ determinant region, respectively [17, 18]. S gene

mutations were analyzed based on alignment against the

wild-type reference sequence M54923 retrieved from

GenBank. The nucleotide numbering was based on the

EcoRI restriction site within the HBV genome.

The sensitivity of the nested PCR performed in this

study was validated using a panel of sera with various HBV

DNA titers tested by COBAS TaqMan 48 Real-Time PCR

(Roche Molecular System, Branchburg, NJ, USA). The

nested PCR was capable of detecting HBV DNA at titers

lower than the detection limit of the COBAS TaqMan 48

Real-Time PCR (6 IU/ml) and thus met the sensitivity

requirement for detection of occult HBV infection (OBI) of

less than 10 IU/ml (Suppl. Table 1) [19].

Analysis of the BCP and PC region

BCP and PC regions were amplified from the extracted

DNA by nested PCR using specific primer sets PC1 (nt

1554–1573, sense; 50-CTG TGC CTT CTC ATC TGC CG-

30)/PC2 (nt 1972–1949, antisense; 50-AAA GAA GTC

AGA AGG CAA AAA AGA-30) as outer primers and S12

(nt 1679–1699, sense; 50-AAT GTC ACC GAC CGA CCT

TG-30)/S13 (nt 1941–1919, antisense; 50-TCC ACA GAA

GCT CCA AAT TCT AA-30) as inner primers [15]. The

purified products were sequenced as described above. The

sequences were aligned with the wild-type reference

sequence M54923 retrieved from GenBank.

Statistical analysis

The baseline data were summarized descriptively. Chi-

square or Fisher’s exact test was used to determine the

differences in categorical variables. Linear-by-linear asso-

ciation chi-square was used to assess the age-specific trend

of HBV prevalence among the children [20]. Factors

associated with transmission of HBV to the children were

analyzed, and outcomes were reported as odds ratios (OR)

with 95 % confidence intervals (CI). A p value of\0.05

was considered significant. All statistical analyses were

two-sided and performed using the Statistical Program for

Social Sciences (SPSS 15.0 for Windows; SPSS, Chicage,

IL, USA).

Results

Forty-six (male/female 31/15) patients, their spouses, and

94 children (male/female 46/48) were included in this

study. Among 46 ICs, 38 % had CHB and 39 % had cir-

rhosis. The recruitment of study subjects was performed as

shown in Fig. 1.

Serological profile of 94 children

Among 94 children, 10 (10.6 %) were HBsAg positive, 19

(20.2 %) were anti-HBc positive, and 46 (48.9 %) were

anti-HBs positive. Of 19 anti-HBc-positive children, 10

(52.6 %) were HBsAg positive and 9 (47.4 %) HBsAg

negative. Of 46 anti-HBs-positive children, 9 (19.6 %)

were HBsAg negative and anti-HBc positive, while 37

(80.4 %) were negative for both HBsAg and anti-HBc. The

remaining 38 (40.4 %) children were negative for all three

markers, indicating no past or present infection but sus-

ceptibility to HBV infection. The baseline characteristics

and detailed serological profile of 94 children are shown in

Suppl. Table 2.

There was no significant difference in the prevalence of

HBsAg, anti-HBc, and anti-HBs between males and

females. To assess the impact of age on the prevalence of

these serological markers, the children were clustered into

the age groups: 1–5, 6–12, and 13–18 years. In the three

age groups, the prevalence of HBsAg was 10.0 % (2/20),

5.0 % (2/40), and 17.6 % (6/34), while that of anti-HBc

was 15.0 % (3/20), 17.5 % (7/40), and 26.5 % (9/34),

respectively. Although not statistically significant, escala-

tion of anti-HBc-positive rates was seen with increasing

age. The prevalence of anti-HBs among the three age

Hepatol Int

123

groups was 70.0 % (14/20), 47.5 % (19/40), and 38.2 %

(13/34), respectively, showing a decreasing trend of anti-

HBs rates as age increased (p = 0.030) (Fig. 2).

In both univariate and multivariate analyses, there was no

significant relationship between HBV exposure to the chil-

dren (defined by positive anti-HBc) and risk factors for

hepatitis B. However, two factors were associated with

higher risk of HBV transmission: having both parents pos-

itive for HBsAg (p\ 0.001) and having at least two HBV

carriers in the household (p\ 0.001) (Suppl. Table 3).

Analysis of 19 anti-HBc-positive children and their

corresponding parents

Nineteen children of 14 ICs were anti-HBc positive, indi-

cating evidence of HBV exposure. These children together

with the 14 related ICs and ICs’ spouses were further

investigated. Of 19 children (male/female 10/9), 10

(52.6 %) were HBsAg positive and 9 (47.4 %) were

HBsAg negative. History of hepatitis B vaccination was

obtained in 14 children, of whom 12 had completed three-

dose series of vaccination during infancy. Two children

obtained hepatitis B booster-dose vaccines at adolescence

and had high anti-HBs levels ([1000 IU/l). Anti-HBs

positivity was significantly higher in the HBeAg-negative

than in the HBeAg-positive groups (p\ 0.001). The

characteristics of the 19 children are shown in Table 1.

Serological profile, HBV DNA detection,

and molecular analysis of 19 anti-HBc-positive

children and their ICs’ family members

Serological investigation of HBV markers (HBsAg, anti-

HBs, anti-HBc, HBeAg, and anti-HBe) as well as HBV

DNA detection, genotype determination, and mutation

analysis were done in all ICs’ family members of the 19

anti-HBc-positive children. HBV DNA was detected in 14

ICs, 4 ICs’ spouses, and 16 children, with no significant

difference between HBsAg-positive and HBsAg-negative

groups. Two of the ICs’ spouses and six of the children

with positive HBV DNA were HBsAg negative/anti-HBc

positive. HBV genotype B was more prevalent than

genotype C among ICs (10 vs. 4) and children (11 vs. 5),

but equal among the ICs’ spouses. In HBV DNA-positive

subjects, HBeAg-positive and HBeAg-negative cases were

54 HBV-related CLD patients

46 families (46 Index cases [ICs], 46 spouses and 94 children) were included

94 children were tested for HBV serological markers (HBsAg, anti-HBs, and anti-HBc)

9 children with HBsAg (-), anti-HBc (+) from 9 families

75 children with HBsAg (-), anti-

HBc (-) from 32 families

10 children with HBsAg (+), anti-HBc (+) from 7 families

19 anti-HBc-positive children and their respective parents (ICs and spouses) from 14 families†

were available for HBeAg, anti-HBe, and HBV DNA detection and analysis

8 declined to participate

Fig. 1 Recruitment of study

subjects. Nineteen children with

anti-HBc positivity and their

parents and other family

members were further

investigated for intra-familial

study of HBV transmission.

Daggers denote some families

had more than one child with

different HBV markers between

siblings

Fig. 2 Prevalence rates of HBsAg, anti-HBc, and anti-HBs in 94

children of HBV-related CLD patients. The prevalence rates of

HBsAg, anti-HBc, and anti-HBs in the age groups 1–5, 6–12, and

13–18 are shown. No statistically significant difference was found in

the HBsAg and anti-HBc positive rates among the three age groups.

Linear-by-linear association test showed a decreasing trend of anti-

HBs positive rates with increasing age (p = 0.030)

Hepatol Int

123

equally distributed in the IC group; one HBeAg-positive

and three HBeAg-negative among IC’s spouses; and ten

HBeAg-positive, six HBeAg-negative among the children

(p = 0.087). Both genotypes B and C were relatively

comparable between HBeAg-positive and HBeAg-negative

subjects (Suppl. Table 4).

Mutations in the HBV S, PC, and BCP regions

The possible routes of HBV transmission were traced by

investigation of familial relations, HBV serology, geno-

types, HBsAg subtypes, and analysis of nucleotide

sequences of the HBV genome (Fig. 3; Suppl. Table 4).

The majority of HBV DNA sequences found in children

were identical to those of the corresponding ICs and ICs’

spouses, including some mutations in the S, PC, and BCP

regions. Mutations in the S gene causing substitutions in

the HBsAg ‘a’ determinant were detected in 20 subjects.

Certain mutations were associated with genotypes: M133L

with genotype B and T126I, T143S, and Y161F with

genotype C.

In the PC region, G1896A mutation was found in 8

subjects, of whom 7 were HBeAg negative, while in the

BCP region, the A1762T/G1764A mutation was identified

in 12 subjects, with 5 showing HBeAg-negative phenotype.

Significantly higher frequency of HBeAg negativity was

observed in subjects with PC G1896A mutation

(p = 0.029), but not in those with BCP A1762T/G1764A

mutation (p = 0.710). Two subjects in two families

showed deletion patterns within the BCP region (30

nucleotides at nt 1740–1770 and 20 nucleotides at nt

1754–1773, respectively), both with HBeAg positivity. The

sequences generated in this study have been deposited in

the GenBank database (accession no. KP123339-

Table 1 Characteristics of 19 anti-HBc-positive children from 14 families of HBV-related CLD patients

Subject no

Family no.

Index case

Age(year) Gender

Hepatitis B vaccination

HistoryHBsAg Anti-HBs

(IU/L)ALT(U/L)

HBV DNA/Genotype

16 8 Mother 13 M − + 1.99 164 +/C17 9 Father 16 M − + 1.99 12 +/B24 12 Mother 4 F + + 2.31 24 +/B26 13 Mother 13 M + + 1.99 43 +/B27 13 Mother 6 F + + 1.99 44 +/B29 15 Father 18 F + + 1.99 18 +/B56 28 Father 16 M + + 1.99 12 +/B57 28 Father 11 F + + 1.99 20 +/B58 28 Father 3 M + + 2.17 17 +/B88 44 Mother 13 M + + 1.99 22 +/B1 1 Father 16 M − − 12.86 15 +/B7 4 Mother 18 F − − >1000 11 +/C

12 6 Mother 7 M + − 161.8 18 +/B31 15 Father 8 M + − 33.67 12 −53 27 Father 9 F + − 642.8 20 −73 37 Father 18 M − − 50.71 43 +/C78 39 Father 12 F +a − >1000 12 −84 42 Father 6 F +a − 238 14 +/C89 44 Mother 5 F + − 134 13 +/CChildren with positive HBsAg are highlighted. Anti-HBs levels ≥10 IU/l were considered positiveCLD chronic liver disease, HBsAg hepatitis B surface antigen, anti-HBs antibody against HBsAg, ALT alanine transaminase,M male, F femaleaIncomplete vaccination

Children with positive HBsAg are highlighted. Anti-HBs levels C 10 IU/l were considered positive

CLD chronic liver disease, HBsAg hepatitis B surface antigen, anti-HBs antibody against HBsAg, ALT alanine transaminase, M male, F femalea Incomplete vaccination

Hepatol Int

123

KP123372 for S gene sequences and KP959314-KP959347

for BCP/PC sequences).

Discussion

Children with HBV infection are generally asymptomatic

and do not require treatment, but they are at increased risk

of developing complications and may become sources of

infection for their entire lifetime [9, 21]. To our knowl-

edge, this is the first study done in Indonesia exploring

HBV infection among children of HBV-related CLD

patients in the context of intra-familial transmission of this

virus.

This study showed that the overall HBsAg prevalence in

children from the families of HBV-infected CLD patients

was 10.64 % (10/94), higher than in the general population

of Indonesia (9.4 %) [4].

The prevalence of anti-HBc as evidence of HBV expo-

sure was 20.2 % (19/94) with escalating rates by age,

indicating the importance of horizontal transmission. Of 46

families, 14 (30 %) were identified to have at least one

child with positive anti-HBc. Our finding was in accor-

dance with studies on the familial occurrence of HBV

infection reported from Northeastern Egypt, India, and

Brazil, where HBsAg and anti-HBc prevalence ranged

from 10.7 to 47.9 % and 23 to 26 %, respectively, among

family members of CHB patients [22]. The decreasing

trend of anti-HBs prevalence with age could indicate the

waning antibody levels with time [23].

Based on the concordant HBV genotypes between car-

rier children and their parents, HBV infection in the 16

HBV DNA-positive children could be mostly transmitted

from the mothers (8/16), followed by the fathers (3/16)

(Fig. 3). The rest could be from either or both parents (3/

16) or unidentified sources (2/16), as observed in family

Fig. 3 Family trees of 14 HBV-related CLD patients. The transmis-

sion routes were investigated by identifying the concordant HBV

genotype/subtype between carrier children and corresponding parents.

Squares denote males, and circles denote females. B: genotype B; C:

genotype C; adr, subtype adr; adw, subtype adw; ayw, subtype ayw.

Infected: positive HBsAg, anti-HBc, and HBV DNA; occult: negative

HBsAg with positive anti-HBc and HBV DNA; exposed: negative

HBsAg and HBV DNA with positive anti-HBc; uninfected: negative

for HBsAg, anti-HBc, and HBV DNA

Hepatol Int

123

42, in which two distinct HBV clusters were identified, and

family 44, where different HBV genotypes were identified

in two daughters. This finding was similar to a study in

Iranian children that indicated the possible HBV trans-

mission sources as mothers (37.5 %), fathers (15 %), and

the community (33.8 %) [24].

Data regarding the differences of transmission routes

between HBV genotypes are scarce. Two studies in 2007

reported that genotype C had the strongest association with

perinatal transmission compared to genotypes A, B, D, and

F [25, 26]. This could not be demonstrated in our study; of

eight mothers presumed to be the sources of transmission,

six had genotype B and two had genotype C. However, a

study in 2011 showed that both genotypes B and C could

be transmitted from maternal and horizontal origins; the

increased prevalence of genotype C was found because of

the higher rate of breakthrough infection in immunized

children born to genotype C mothers than those with

genotype B [27]. The role of HBV genotypes in trans-

mission routes remains a question and needs further global

studies, considering the association between HBV genetic

diversity with host ethnicity and geographical distribution

[19, 22].

Mutations in the ‘a’ determinant of HBsAg, either singly

or in combination, were identified in several paired sera of

children and their corresponding parents, providing a

stronger indication of intra-familial HBV transmission. The

mutations were observed in both genotype B and C, with

predominance of M133L in genotype B, and T143S and

T126I in genotype C. These mutations were also reported

in another study from Indonesia associated with HBsAg

detection failure in blood donors [28].

The well-known mutation G145R—associated with

vaccine escape and HBsAg detection failure [29]—was

found in two subjects, one of which was HBsAg positive.

Other important mutations associated with HBsAg detec-

tion and vaccination failure—M133L, T140I, T143S/M,

Y161F—were also quite frequent. Intriguingly, the ‘a’

determinant mutations are more prevalent in those with

genotype B than C (p = 0.01). Some of these mutations

were found in six children and two spouses with HBsAg

negative but anti-HBc and HBV DNA positive (OBI). This

finding confirms that individuals with OBI could be ‘hid-

den’ sources of transmission to their spouses, children,

siblings, and even the community [30, 31].

In CLD, HBeAg is a valuable marker for infectivity in

HBV transmission [32]. However, mutations in the PC and

BCP regions may downregulate or abolish HBeAg produc-

tion, which mimics seroconversion to anti-HBe and immune

clearance of the virus, while maintaining the presence of

viremia known clinically as e-negative CHB [33]. The most

common mutation in the PC region is G1896A, which

abolishes HBeAg production by introducing a premature

stop codon [34]. Reports from Europe indicated that

G1896A was more common in genotype A-infected patients

and associated with progressive liver diseases [34]. How-

ever, in Asia, this mutation was found in genotype B, C, and

D and detected in similar percentages in asymptomatic

carriers and patients with cirrhosis and HCC [15, 34–36]. In

our study, a higher frequency of G1896A was observed in

HBeAg-negative than in HBeAg-positive family members

(p = 0.029) (Suppl. Table 4). Among BCP mutations, the

most frequent is the double mutation A1762T/G1764A that

decreases HBeAg expression but enhances viral replication

[15, 37, 38]. This mutation is associated with more severe

diseases, cirrhosis, and HCC [39, 40]. In this study, A1762T/

G1764A was relatively more frequent in HBeAg-negative

subjects and infection with genotype C.

One limitation of the study was the cross-sectional

design that did not make it possible to determine the time

of HBV exposure in the children and the precise duration

of the infection. The closest approximation that could be

made is by comparing the viral characteristics of the

children with other infected family members [6, 10, 11].

Another limitation was the direct sequencing method for

HBV DNA analysis that could not further characterize the

viral quasispecies in the subjects with positive HBsAg but

having mutation types associated with HBsAg detection

failure. However, by performing genotyping/subtyping and

comparison of HBV nucleotide sequences, combined with

investigation of family relationships, this study could

reveal the evidence of multiple transmission patterns of

HBV infection among the family members of HBV-in-

fected patients.

In conclusion, this study confirms that children of HBV-

related CLD patients are at risk of acquiring hepatitis B

infection, despite the negative HBsAg and HBeAg status of

their parents. Having both parents with HBsAg positive

and the presence of at least two HBV carriers in the

household are significant risk factors of intra-familial HBV

transmission. This intra-familial spread of HBV with high

infection rates in children could contribute to the genera-

tion of pools of HBV-infected subjects that may become

sources for further transmission to wider communities.

This study reiterates that efficient preventive strategies that

include routine infant hepatitis B vaccination, particularly

the birth dose vaccine, and catch-up immunization targeted

to unvaccinated children are of great importance for the

children of HBV-related CLD patients.

Acknowledgements The authors would like to thank all children and

parents who participated in this study. Special thanks are extended to

the Division of Hepatology, Department of Internal Medicine, Faculty

of Medicine, University of Indonesia/Cipto Mangunkusumo Hospital,

Jakarta, Indonesia, for recruitment of patients. Sincere gratitude is

expressed to the Eijkman Institute, Jakarta, for the molecular work

Hepatol Int

123

support and to the academic staff of the Department of Pediatrics,

Faculty of Medicine, University of Indonesia, for discussions and

suggestions.

Compliance with ethical standards

Funding This study was partly supported by a research grant from the

Ministry for Research and Higher Education, Republic of Indonesia.

Conflict of interest All authors declare that he/she has no conflict of

interest.

Ethical approval All procedures performed in studies involving

human participants were in accordance with the ethical standards of

the institutional and/or national research committee and with the 1964

Helsinki Declaration and its later amendments or comparable ethical

standards. This study was approved by The Committee of Medical

Research Ethics of the Faculty of Medicine, University of Indonesia

(no. 297/PT02.FK/ETIK/2009).

Informed consent Informed consent was obtained from all individ-

ual participants included in the study.

References

1. World Health Organization. Hepatitis B fact sheet no. 204. 2016.

http://www.who.int/mediacentre/factsheets/fs204/en/. Accessed

20 July 2016. (updated July 2016)2. Chen ST, Chang MH. Epidemiology and natural history of hep-

atitis B in children. In Jonas MM, ed. Viral hepatitis in children.

New York: Humana Press, 2010. 13–28

3. Custer B, Sullivan SD, Hazlet TK, Iloeje U, Veenstra DL,

Kowdley KV. Global epidemiology of hepatitis B virus. J Clin

Gastroenterol 2004;38:S158–S168

4. Ministry of Health Republic of Indonesia. Report on the national

basic health research (RISKESDAS) 2007 Jakarta: The National

Institute of Health Research and Development (NIHRD), 2010

5. Milas J, Ropac D, Mulic R, Milas V, Valek I, Zoric I, Kozul K.

Hepatitis B in the family. Eur J Epidemiol 2000;16:203–208

6. Thakur V, Kazim SN, Guptan RC, Malhotra V, Sarin SK.

Molecular epidemiology and transmission of hepatitis B virus in

close family contacts of HBV-related chronic liver disease

patients. J Med Virol 2003;70:520–528

7. Yao GB. Importance of perinatal versus horizontal transmission

of hepatitis B virus infection in China. Gut 1996;38:S39–S42

8. Takegoshi K, Zhang W. Hepatitis B virus infections in families in

which the mothers are negative but the fathers are positive for

HBsAg. Hepatol Res 2006;36:75–77

9. Abdel-Hady M, Kelly D. Chronic hepatitis B in children and

adolescents: epidemiology and management. Paediatr Drugs

2013;15:311–317

10. Lin CL, Kao JH, Chen BF, Chen PJ, Lai MY, Chen DS. Appli-

cation of hepatitis B virus genotyping and phylogenetic analysis

in intrafamilial transmission of hepatitis B virus. Clin Infect Dis

2005;41:1576–1581

11. Datta S, Banerjee A, Chandra PK, Chowdhury A, Chakravarty R.

Genotype, phylogenetic analysis, and transmission pattern of

occult hepatitis B virus (HBV) infection in families of asymp-

tomatic HBsAg carriers. J Med Virol 2006;78:53–59

12. Bozkaya H, Akarca US, Ayola B, Lok AS. High degree of con-

servation in the hepatitis B virus core gene during the immune

tolerant phase in perinatally acquired chronic hepatitis B virus

infection. J Hepatol 1997;26:508–516

13. Martinson FE, Weigle KA, Royce RA, Weber DJ, Suchindran

CM, Lemon SM. Risk factors for horizontal transmission of

hepatitis B virus in a rural district in Ghana. Am J Epidemiol

1998;147:478–487

14. Okamoto H, Nishizawa T. Non-B non C non-G hepatitis virus

gene, polynucleotide, polypeptide, virion, method for separating

virion, and method for detecting virus. 1992.

15. Turyadi, Thedja MD, Ie SI, Harahap AR, El-Khobar KE, Roni M,

Muljono DH. HBsAg, HBeAg and HBV DNA level changes and

precore/basal core promoter mutations in the natural history of

chronic hepatitis B in Indonesian patients. Hepatol Int

2013;7:969–980

16. Iizuka H, Ohmura K, Ishijima A, Satoh K, Tanaka T, Tsuda F,

Okamoto H, et al. Correlation between anti-HBc titers and HBV

DNA in blood units without detectable HBsAg. Vox Sang

1992;63:107–111

17. Norder H, Courouce AM, Magnius LO. Molecular basis of hep-

atitis B virus serotype variations within the four major subtypes.

J Gen Virol 1992;73:3141–3145

18. OkamotoH, ImaiM, Tsuda F, Tanaka T,MiyakawaY,MayumiM.

Point mutation in the S gene of hepatitis B virus for a d/y or w/r

subtypic change in two blood donors carrying a surface antigen of

compound subtype adyr or adwr. J Virol 1987;61:3030–3034

19. Thedja MD, Muljono DH, Nurainy N, Sukowati CH, Verhoef J,

Marzuki S. Ethnogeographical structure of hepatitis B virus

genotype distribution in Indonesia and discovery of a new

subgenotype, B9. Arch Virol 2011;156:855–868

20. Herbert A. SPSS Notes (SPSS version 15.0) 2008. In Salford

Royal Hospitals NHS Trust. 2008. http://www.salfordresearch.

org.uk. Accessed 1 July 2016

21. Darmawan E, Turyadi, El-Khobar KE, Nursanty NK, Thedja MD,

Muljono DH. Seroepidemiology and occult hepatitis B virus

infection in young adults in Banjarmasin, Indonesia. J Med Virol

2015;87:199–207

22. Ragheb M, Elkady A, Tanaka Y, Murakami S, Attia FM, Hassan

AA, Hassan MF, et al. Multiple intra-familial transmission pat-

terns of hepatitis B virus genotype D in north-eastern Egypt.

J Med Virol 2012;84:587–595

23. van der Sande MA, Waight P, Mendy M, Rayco-Solon P, Hutt P,

Fulford T, Doherty C, et al. Long-term protection against carriage

of hepatitis B virus after infant vaccination. J Infect Dis

2006;193:1528–1535

24. Amiri SS, Amiri MJS, Roushan MRH. Possible sources for

transmission of hepatitis B virus infection in 80 children in Babol,

North of Iran. Casp J Intern Med 2010:50–52

25. Livingston SE, Simonetti JP, Bulkow LR, Homan CE, Snowball

MM, Cagle HH, Negus SE, et al. Clearance of hepatitis B e

antigen in patients with chronic hepatitis B and genotypes A, B,

C, D, and F. Gastroenterology 2007;133:1452–1457

26. Inui A, Komatsu H, Sogo T, Nagai T, Abe K, Fujisawa T.

Hepatitis B virus genotypes in children and adolescents in Japan:

before and after immunization for the prevention of mother to

infant transmission of hepatitis B virus. J Med Virol

2007;79:670–675

27. Wen WH, Chen HL, Ni YH, Hsu HY, Kao JH, Hu FC, Chang

MH. Secular trend of the viral genotype distribution in children

with chronic hepatitis B virus infection after universal infant

immunization. Hepatology 2011;53:429–436

28. Thedja MD, Roni M, Harahap AR, Siregar NC, Ie SI, Muljono

DH. Occult hepatitis B in blood donors in Indonesia: altered

antigenicity of the hepatitis B virus surface protein. Hepatol Int

2010;4:608–614

29. Ghany MG, Ayola B, Villamil FG, Gish RG, Rojter S, Vierling

JM, Lok AS. Hepatitis B virus S mutants in liver transplant

recipients who were reinfected despite hepatitis B immune

globulin prophylaxis. Hepatology 1998;27:213–222

Hepatol Int

123

30. Huang X, Hollinger FB. Occult hepatitis B virus infection and

hepatocellular carcinoma: a systematic review. J Viral Hepat

2014;21:153–162

31. Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, Chen

DS, et al. Asian-Pacific clinical practice guidelines on the man-

agement of hepatitis B: a 2015 update. Hepatol Int 2016;10:1–98

32. Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical rele-

vance of hepatitis B viral mutations. Hepatology

2000;31:1037–1044

33. Tong MJ, Blatt LM, Kao JH, Cheng JT, Corey WG. Precore/basal

core promoter mutants and hepatitis B viral DNA levels as pre-

dictors for liver deaths and hepatocellular carcinoma. World J

Gastroenterol 2006;12:6620–6626

34. Schaefer S. Hepatitis B virus: significance of genotypes. J Viral

Hepat 2005;12:111–124

35. Datta S. An overview of molecular epidemiology of hepatitis B

virus (HBV) in India. Virol J 2008;5:156

36. Fujiko M, Chalid MT, Turyadi, Ie SI, Maghfira, Syafri, Wahyuni

R, et al. Chronic hepatitis B in pregnant women: is hepatitis B

surface antigen quantification useful for viral load prediction? Int

J Infect Dis 2015;41:83–89

37. Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P.

Effect of basal core promoter and pre-core mutations on hepatitis

B virus replication. J Gen Virol 2008;89:901–909

38. Kidd-Ljunggren K, Oberg M, Kidd AH. Hepatitis B virus X gene

1751 to 1764 mutations: implications for HBeAg status and

disease. J Gen Virol 1997;78:1469–1478

39. Yang HI, Yeh SH, Chen PJ, Iloeje UH, Jen CL, Su J, Wang LY,

et al. Associations between hepatitis B virus genotype and

mutants and the risk of hepatocellular carcinoma. J Natl Cancer

Inst 2008;100:1134–1143

40. Liu CJ, Kao JH. Global perspective on the natural history of

chronic hepatitis B: role of hepatitis B virus genotypes A to J.

Semin Liver Dis 2013;33:97–102

Hepatol Int

123