Infection

DOI 10.1007/s15010-015-0733-6

ORIGINAL PAPER

Occult hepatitis B virus infection in children born

to HBsAg‑positive mothers after neonatal passive‑active
immunoprophylaxis
Hanan Foaud · Sahar Maklad · Faten Mahmoud ·
Hanaa El‑Karaksy 

Received: 2 November 2014 / Accepted: 19 January 2015

© Springer-Verlag Berlin Heidelberg 2015

Abstract  HBeAg and was positive for anti-HBs (titer 267 mIU/mL)

Background  Occult hepatitis B virus infection (OBI) is a
well-recognized clinical entity characterized by the detec-
tion of HBV DNA in serum and/or liver in the absence of
detectable HBsAg. Diagnosis of OBI requires a sensitive
HBV DNA assay.
Aim  We aimed at determining the frequency of OBI in
infants, born to HBsAg-positive mothers, who received
immunoprophylaxis at birth.
Methods  Sixty-four infants and children, born to HBsAg-
positive mothers, who received hepatitis B immunoglobulin
and HBV vaccine within 48 h after birth, were tested for
HBV serological profile and HBV DNA by real-time PCR
at least 1 month after last dose of HBV vaccine and not
before 6 months of age.
Results  The median age of the studied infants and chil-
dren was 8 months, ranging from 6 to 132 months; 54.7 %
were females. HBV DNA was detected in 2 infants. One
case had OBI; she was negative for HBsAg, anti-HBc total,
H. Foaud (*) 
Department of Pediatrics, National Hepatology and Tropical
Medicine Research Institute, 7101 El‑Kahera Buildings,
El‑Mokattam, Cairo 11415, Egypt
e-mail: hananminaped@gmail.com
S. Maklad  sure passive-active immunoprophylaxis using hepatitis B
Department of Internal Medicine and Hepatology, National
Hepatology and Tropical Medicine Research Institute, Cairo,
Egypt
F. Mahmoud  Occult hepatitis B virus infection (OBI) was defined
Department of Clinical Chemistry, National Hepatology
and Tropical Medicine Research Institute, Cairo, Egypt
H. El‑Karaksy 
Department of Pediatrics, Kasr Alainy Medical School, Cairo
University, Cairo, Egypt
with low level of viremia (HBV DNA 1.13 x 103 IU/mL).
Another infant showed immunoprophylaxis failure with
positive HBsAg, anti-HBc total, HBeAg, negative anti-HBe
and anti-HBs; HBV viral load was 1.7 × 108 IU/mL. Both
mothers were HBsAg and HBeAg-positive.
Conclusion  OBI may occur in infants born to HBsAg-
positive mothers despite the receipt of immunoprophylaxis.
OBI was detected in a low frequency in the present study.
Anti-HBs positivity does not exclude OBI.
Keywords  Egypt · HBsAg-positive mothers · HBV
immunoprophylaxis · Occult HBV infection
Introduction
Perinatal transmission of hepatitis B virus (HBV) from
HBsAg-positive mothers to their infants is the most impor-
tant mode of transmission in highly endemic area [1]. With
no intervention, 40–90 % of these infants will acquire HBV
infection [2–4]. Approximately 90 % of infected infants
develop chronic HBV infection with a 15–25 % risk for
premature death from cirrhosis or hepatocellular carcinoma
(HCC). To prevent perinatal HBV transmission, post-expo-
vaccine and hepatitis B immune globulin (HBIG) is recom-
mended within 12 h of birth, in addition to 3-dose vaccine
series at one and 6 months of age [5].
as the presence of HBV DNA in serum and/or liver tissue
without detectable HBsAg with or without anti-HBc or
anti-HBs antibodies outside the pre-seroconversion win-
dow period [6]. It is classified into seropositive and seron-
egative infections depending on positivity for anti-HBc

13

and anti-HBs antibodies [7]. Diagnosis of OBI requires a
sensitive HBV DNA PCR assay because the serum level of
HBV DNA in these patients is usually < 104 copies/ml [8].
OBI is present worldwide and varies significantly
between geographic regions and populations with increased
prevalence in countries highly endemic for HBV [9]. OBI
has been associated with more advanced fibrosis/cirrhosis,
ultimately leading to HCC [10, 11].
Previous studies on OBI in children suggested that it may
be transmitted to children from occult infected or HBsAg-
positive mothers [12, 13]. The mechanism of OBI despite
vaccine/HBIG prophylaxis is unclear. OBI may represent
the window period after acute infection, persistence of low
level replication after recovery, the occurrence of an escape
mutant in the ‘‘a’’ determinant undetected by current HBsAg
assays [14], or inadequate neutralization against HBV [15].
Other factors that may contribute are host immunosuppres-
sion and co-infection with hepatitis C virus [9, 10, 16].
The present study was undertaken to study the frequency
of OBI in infants born to HBsAg-positive mothers and who
received immunoprophylaxis to prevent perinatal acquisi-
tion of HBV infection at birth.
Materials and methods
This cohort prospective study was carried out in the pedi-
atric and the adult HBV specialized clinics at the National
Hepatology and Tropical Medicine Research Institute and
Cairo University Pediatric Hospital. The study protocol was
approved by the Research Ethics Committee of the General
Organization for Teaching Hospitals and Institutes. The
study included all HBsAg-positive pregnant females who
were followed up at the HBV specialized clinic. The study
as well included children of HBsAg-positive mothers who
were previously immunized at birth. All mother-infant pairs
were enrolled after the mother signed an informed consent.
The study was conducted from June 2012 to June 2014.
Fourty-seven HBsAg-positive pregnant females were
recruited in the study. Pregnant females were investigated
for liver enzymes and viral markers. At the end of the 2nd
trimester of pregnancy, quantitative HBV DNA using PCR
was done to decide on the need for antiviral treatment dur-
ing pregnancy. Females with high viral load; HBV DNA
titre of > 106–7 IU/ml or more were candidates for lamivu-
dine (100 mg/day) or tenofovir (300 mg/day) therapy for
the last trimester of pregnancy, with further reassessment
of the condition after delivery. Females who were receiving
Lamivudine before pregnancy and their HBV DNA became
below detection limit, were continued on lamivudine dur-
ing the whole pregnancy. Females who had low viral load
or below detection limit received no antiviral treatment dur-
ing pregnancy [17].
13
H. Foaud et al.
All enrolled pregnant females and their husbands were
informed to contact a single researcher by cell phone at the
time of delivery to administer immunoprophylaxis against
HBV to their newborns.
Forty-six newborns of the HBsAg-positive mothers, who
contacted the researcher at the time of delivery, received
HBV immunoprophylaxis in the form of 0.5 ml (100 IU)
HBIG (Hepabig, Korea) and 0.5 ml of the pediatric formu-
lation of hepatitis B vaccine (Euvax B, Korea), a recombi-
nant vaccine containing 10 µg of HBsAg. Both were given
as an intramuscular injection at 2 different sites, within
24 h after birth. Second dose of the vaccine was given
1 month later, then the infant received another 3 doses of
the vaccine within the expanded program of immunization
at 2, 4, 6 months of age (the corresponding vaccination
schedule included quadruple vaccine containing DPT plus
hepatitis B). Only one mother contacted the researcher later
than 48 h after delivery, although her baby received immu-
noprophylaxis, still he was excluded from the study.
In addition, 20 previously immunized children who
received immunoprophylaxis against HBV at birth were
included (Fig. 1).
Blood samples were drawn from all enrolled children
at least 4 weeks after the last dose of the vaccine and not
before 6 months of age [5]. For all the children HBV sero-
logical profile (HBsAg, anti-HBc total, HBeAg, anti-HBe
and anti-HBs titre) and HBV DNA quantification were
done. Two immunized infants did not come for post-vacci-
nation testing.
Laboratory tests
A 5 ml non-fasting blood sample was obtained by veni-
puncture and added on EDTA. Samples were centrifuged
and sera were divided into 2 aliquots, one was stored at
−80 °C for HBV DNA assay by PCR and the other was
stored at −20 °C for HBV serology profile assay. HBV
serology was done by ELISA technique (Abbott Murex
was used for HBsAg, Diasorin was used for HBeAg, anti-
HBe, anti-HBc total, and anti-HBs titre, Clinilab).
Interpretation of anti-HBs titer in infants performing post-
vaccination testing after few weeks of the last booster was as
follows, a titer of <10 mIU/ml was considered negative and
revaccination with 3 doses was considered; a titer ranging
between 10 and 100 mIU/ml was considered a poor response
and a booster was given; a titer of 100 mIU/ml or more was
considered immune [18]. Children previously immunized
were considered negative if their titer is <10 mIU/ml.
Quantitative HBV‑DNA by PCR
HBV DNA was determined by real time quantification
MX 400 (Stratagene). Three steps are used: extraction,
Occult B infection in infants of HBsAg mothers

Fig. 1  Flowchart of study
groups Recruited HBsAg-positive
pregnant females
N=47

Previously immunized
Immunized infants who children
completed follow up
N=20
N=44

Enrolled children
N=64

HBsAg
Anti-HBc
HBeAg
Anti-HBe
Anti-HBs titre
HBV DNA

HBsAg +ve HBsAg -ve HBsAg -ve HBsAg -ve

Anti-HBc +ve Anti-HBc –ve Low anti-HBs titre Anti-HBs titre +ve
HBeAg +ve Anti-HBs titre +ve HBV DNA –ve HBV DNA –ve
HBV DNA +ve HBV DNA +ve N=13 N=49

Immunoprophylaxis Booster(s)
Occult HBV Successful
failure
N=1 immunoprophylaxis
N=1
‘’protected ‘’
N=62

amplification and detection. The kit used was designed for
use with all types of probe including Quanti probes (as port
of Quanti Ject Gene expression assays and Quanti Ject cus-
tom assays), Tagman or Operon dual-labeled probe, light
cycler hybridization (FRET) probes and Molecular Bea-
cons. High specificity and sensitivity in PCR were achieved
by the use of the hat start enzyme (Tag DNA polymerase)
together with a specialized buffer. The cutoff for detection
was 4 IU/mL.
Statistical methods
Data were collected and tabulated. Statistical Package for
Social Science (SPSS) program version 17.0 was used
for data analysis. Mean and standard deviation (SD) and
median and interquartile range (IQR) were estimates of
quantitative data while frequency and percentage were esti-
mates of qualitative data.
Results
A total of 64 infants and children born to HBsAg-positive
mothers, who received immunoprophylaxis for HBV at
birth were included. Their median ages were 8 months,
(range 6–132 months) and 54.7 % were females. The first
dose of vaccine and HBV immunoglobulin were given after
a median of 2 h after delivery and the second dose of vac-
cine was given at age of 1 month (67.2 %). Most of the
infants received more than 3 doses of vaccine (98.4 %);
40.6 %. were delivered by normal vaginal delivery and
92 % were breast fed. There was history of previously

13
 H. Foaud et al.

Table 1  Data of infants and children born to HBsAg-positve mothers Table 2  Data of HBsAg-postive mothers (N = 64)
who received immunoprophylaxis (N = 64)
Variable Data
Variable Data
Age in years
Age in months  Mean + SD 26.9 + 4.2
 Median (IQR) 8 (13)  Min-max 20–38
 Min-max 6–132 Positive HBeAg (N = 53); N (%) 16 (30.2)
Sex; N (%) Positive HCV Ab; N (%) 1 (1.6)
 Male 29 (45.3) HBV DNA (N = 58); N (%)
 Female 35 (54.7)  Below detection limit 34 (58.6)
First dose timing in hours  Positive 24 (41.4)
 Median (IQR) 2 (6.75) Antiviral use during pregnancy (N = 24, 37.5 %); N (%)
 Min-max 1–36  Lamivudine 22 (91.7)
Age at second dose; N (%)  Tenofovir 2 (8.3)
 One month 43 (67.2) Duration of therapy during pregnancy (N = 24); N (%)
 Two months 21 (32.8)  Last trimester 8 (33.3)
Total HBV vaccine doses; N (%)  All through pregnancy 16 (66.7)
 Three 1 (1.6)
 More than three 63 (98.4)
Mode of delivery; N (%) with Lamivudine or Tenofovir before pregnancy and con-
 NVD 26 (40.6) tinued on the same treatment during pregnancy. Maternal
 CS 38 (59.4) data are shown in Table 2.
Feeding; N (%) Among our study group, one case developed OBI. She
 Breast 59 (92.2) was a female aged 20 months, delivered by cesarean sec-
 Formula 5 (7.8) tion with no previously affected sibling. She received her
Other affected siblings; N (%) birth dose of HBIG and vaccine within 1 h of delivery
 None 56 (87.5) and completed a total of 4 doses of vaccine before testing.
 One 5 (8.3) She was negative for HBsAg, anti-HBc total, HBeAg and
 Two 3 (5) positive for anti-HBs (titer 267 mIU/mL). Her HBV DNA
Positive HBsAg and HbeAg; N (%) 1 (1.6) was 1,130 IU/mL which was repeated twice at 4-months
HBsAb titer; mIU/mL intervals showing persistent low level of viremia (900
 Median (IQR) 258.5 (518.4) and 2,500 IU/mL, respectively). Her mother was receiv-
 Min-max 3.4–1,303 ing lamivudine for 4 years preceding her pregnancy and all
Positive HBV DNA; N (%) 2 (3.2) through pregnancy. The mother had positive HBeAg with
Fate; N (%) DNA below detection level; she was negative for HCV and
 Immune 62 (96.9) breast fed her baby (Table 3).
 Immunoprophylaxis failure 1 (1.6) Another case showed immunoprophylaxis failure. She
 Occult HBV 1 (1.6) was a female aged 12 months, delivered by cesarean sec-
tion and was artificially fed. She received her birth dose
IQR inter quartile range, NVD normal vaginal delivery, CS cesarean of HBIG and vaccine 7 h after delivery and completed a
section, HBV hepatitis B virus
total of 4 doses of vaccine before testing. She has positive
for HBsAg, anti-HBc total, HBeAg, and was negative for
affected sibling(s) in 13.3 %. Viral markers of HBV, namely anti-HBe, anti-HBs. Her HBV DNA was 1.7 × 108 IU/mL.
HBsAg, anti-HBc total and HBeAg were positive in only Her mother was HBsAg-positive HBeAg-positive at time
one case while HBV DNA was positive in two cases. The of delivery with quantitative HBV DNA of 8 × 107 IU/ml
median anti-HBs titre was 258.5 mIU/ml (range from 3.4 to (Table 3).
1,303 IU/ml). Children’s data are shown in Table 1.
The mean age of the mothers was 26.9 + 4.2 years, with
HbeAg positivity in 30.2 % and detectable HBV DNA in Discussion
41.4 %. Twenty-four (37.5 %) HBsAg-positive mothers
were candidates for antiviral therapy during pregnancy. The present study is the first Egyptian study on OBI in a
Eight out of them had a high viral load and received lami- high risk group of infants born to HBsAg-positive moth-
vudine in the last trimester while 16 females were treated ers and receiving immunoprophylaxis at birth. OBI is

13
Occult B infection in infants of HBsAg mothers

Table 3  Data of the case Occult HBV Infection Immunoprophylaxis Failure

with occult HBV infection
and case with failure of Age in months 20 12
immunoprophylaxis
Gender Female Female
Mode of delivery CS CS
Timing of HBIG in hours 1 7
Total HBV vaccine doses 4 4
Feeding Breast Artificial
HBV DNA in IU/ml 1,130 1.7 × 108
HBsAg Negative Positive
HBcAb Negative Positive
HBeAg Negative Positive
HbsAb titre in mIU/ml 267 <10
BDL below detection limit, Maternal HBeAg Positive Positive
CS cesarean section, HBIG Maternal DNA in IU/ml BDL 8 × 107
Hepatitis B immunoglobulin, Treatment during pregnancy Lamivudine throughout pregnancy No
HBV Hepatitis B virus

Table 4  Prevalence of HBV infection (HBsAg carrier rate) and occult HBV infection in infants born to HBsAg-positive mothers and who
received immunoprophylaxis

Country Overall prevalence of HBV infection Frequency of occult HBV infection in infants born to
HBsAg-positive mothers and who received immunoprophylaxis

Egypt 6.7 (Lehman and Wilson 2009) [30] 1.6% (The present study)
France 0.65 % (Meffre et al. 2010) [26] 2 % (Chakvetadze et al. 2011) [27]
Iran 1.3 % (Moezzi et al. 2014) [28] 28 % (Shahmoradi et al. 2012) [29]
Taiwan 4 % (Sun et al. 2014) [22] 10.9 % (Mu et al. 2009) [23]
China 7.2 % (Lu and Zhuang, 2009) [24] 4.9 % (Su et al. 2013) [25]

recognized as a poosible phase of chronic HBV infection
[17]. It is the persistence of viral genomes in the liver tis-
sue or serum of individuals who are HBsAg-negative [9].
Mostly OBI is related to suppression of viral activities by
host defense mechanisms [19]. The clinical consequence
and transmission risk of OBI in populations remains
unclear. OBI may persist in individuals for years without
obvious symptoms of overt HBV infection or may develop
into hepatitis, cirrhosis and HCC [10].
In the present prospective cohort study; 64 infants born
to HBsAg-positive mothers, who received immunoprophy-
laxis (vaccine and HBIG) against HBV at birth were tested
for the presence of HBV DNA at least 4 weeks after the
last dose of the vaccine and not before 6 months of age [5].
Only one case developed OBI in the presence of adequate
levels of anti-HBs and was anti-HBc negative.
The success rate for immunoprophylaxis among our
studied children was 96.9 %. Postexposure prophylaxis for
infants born to HBsAg-positive mothers protects at least
85 % of infants from perinatally acquired HBV infection
[20]. In a recent study on Egyptian infants born to HBsAg-
positive mothers, who received immunoprophylaxis, the
success rate was 92.6 % [21].
In Taiwan, where the HBV prevalence is estimated to be
4 % [22], OBI was reported in 10.9 % of studied children
born to HBsAg-positive mothers, who received immuno-
prophylaxis at birth [23]. In China where the HBsAg car-
rier rate is 7.18 % [24], OBI was found in 4.9 % of a similar
high risk group [25]. In France, a country with low ende-
micity for HBV (the HBsAg carrier rate is 0.65 %) [26],
OBI was reported in 2 % [27]. Surprisingly in Iran, where
the estimated HBV prevalence is 1.3 % [28], Shahmoradi
et al. [29] reported OBI in 28 % of his immunized children
born to HBsAg-positive mothers (Table 4). Despite that the
present study utilized a highly sensitive method for HBV
DNA detection, we report a very low prevalence of OBI
in our high risk group of infants. Egypt is a region with
intermediate endemicity for HBV, where the overall preva-
lence of HBsAg is 6.7 % and it is 1.6 % among children
[30], with HBV genotype D the most prevalent genotype
among chronic HBV infection [31]. In such an intermedi-
ately endemic area, only one infant (1.6 %) born to HBV
mother and immunized at birth was diagnosed to have OBI.
The reported prevalence of OBI in similar groups of high
risk infants ranged from 2 to 28 %, in different endemic
regions [23, 25, 27, 29]. The fact that almost one third of

13

the HBsAg-positive mothers in the present study were
receiving treatment during pregnancy (37.5 %) and almost
two-thirds were HBeAg negative (58.6 %) has to be put in
consideration and may explain this low frequency; despite
the fact that the mother of the only case that developed OBI
was receiving antiviral treatment during pregnancy. In com-
parison, Su et al. [25] found OBI in 4.9 % of their 186 high
risk infants, whose mothers had positive HbeAg in16.9 %,
high viremia in 29.5 % and none of them received specific
antiviral treatment during pregnancy. It seems that antiviral
treatment during pregnancy combined with at birth passive-
active immunization can ameliorate the rate of mother-
infant transmission and decrease the risk of high maternal
viraemia and HBeAg positivity.
The mother of our OBI case was receiving lamivu-
dine for 4 years preceding her pregnancy and all through
pregnancy. Antiviral drug-associated potential vaccine-
escape mutants can occur with the use of lamivudine and
telbivudine, they have the potential to produce HBV pol/S
gene overlap mutants [32, 33]. Mothers receiving antivi-
ral treatment might develop mutant variants that may not
be detected by conventional HBsAg screening tests (diag-
nostic-escape) [34–36]. Recent studies showed that drug-
resistant viral variants may emerge even with short-term
lamivudine therapy during late pregnancy [37]. Despite this
risk, antiviral treatment in the last trimester of pregnancy is
recommended for mothers with high viral load (107 IU/ml)
[17] as pregnant women with high HBV replication levels
are the most at risk population for perinatal transmission of
HBV despite passive-active immunoprophylaxis [38–40].
To avoid the risk of emergence of resistance mutations
of HBV with antiviral use during pregnancy, administra-
tion of HBIG to HBsAg-positive women during the third
trimester of pregnancy was studied. Some studies reported
satisfactory results regarding efficacy and safety [41, 42]
while others could not demonstrate such efficacy [43].
There are no reported HBV S gene mutations in these
patients [42, 44]. Multicenter controlled trails are needed to
confirm such results.
Previous Egyptian studies on OBI revealed different
rates according to population studied and the method used.
Prevalence of OBI was 1.3 % in blood donors [45], up to
6.3 % in hemodialysis patients [46], 16 % in chronic HCV
patients [11], 22.5 % in patients with HCC [47] and 32.4 %
in thalassemic children [48].
Our OBI case was negative for HBsAg, anti-HBcAB,
HBeAg, anti-HBe and positive only for anti-HBs (titer
276 mIU/mL), thus was considered sero-positive OBI [7].
Alone, an anti-HBs result ≥ 10 mIU/mL does not con-
firm that the infant is protected [49] and HBV infection
could be detected in anti-HBs positive individuals [50].
Su et al. [25] found that 66.7 % of his infants with OBI
were positive for anti-HBs; with titers lower than 100 mIU/
13
H. Foaud et al.
mL. Several studies have suggested that the neutralizing
capacity of low-level anti-HBs was limited [27]. The nega-
tivity of HBsAg, anti-HBcAb and anti-HBs are not suffi-
cient to completely exclude HBV DNA carriers. OBI may
be detected in absence of the anti-HBc. Shahmoradi et al.
[29] reported that 76 % of infants with OBI were anti-HBc
negative.
Our OBI case had low level of viremia (HBV DNA
1,130 IU/ml). Mu et al. [23], found low titre of HBV DNA
among his group of infants with OBI. Many factors may be
related to occult infection in vaccinated children, such as
nonresponse or hyporesponse to HBV vaccination, declin-
ing titers of protective antibody, escape mutations in the
S gene or high maternal viral loads [9, 25]. Occult HBV
infection of infants possibly comes from vertical transmis-
sion or mother-to-infants transmission. Loss of HBsAg or
HBV DNA may occur in a proportion of vertically exposed
infants within the first year of life [25].
Immunoprophylaxis failure occurred in one case whose
mother was HBsAg-positive, HBeAg-positive and her
HBV DNA was 8 × 107 IU/ml. Risk factors associated
with HBV immunoprophylaxis failure include HBeAg-
positive mothers and maternal viral load (HBV DNA
level ≥ 107 copies/mL) [25, 38, 39]. HBV mother-to-infant
transmission still occurs after passive-active immunization.
Pregnant women of high HBV replication levels are the
major risk population of HBV mother-to-infant transmis-
sion [40].
Our study limitation is the small number of mother-
infant pairs included in the present study to allow us to con-
clude on risk factors and prevention recommendations. A
larger number is needed to study the effect of HBV treat-
ment during pregnancy on development of OBI.
Despite the limited number of the studied mother-infant
pairs, the strength of the present study arises from the fact
that previous information about OBI in high risk neonates
in Egypt is lacking. However, our results cannot be gen-
eralized to all neonates at risk of perinatal transmission of
HBV, as more than one third of the mothers were on spe-
cific antiviral treatment for HBV.
In conclusion, OBI was detected in a low percent in the
present study despite the use of a highly-sensitive method
of detection. OBI may occur in infants born to HBsAg-
positive mothers despite receipt of immunoprophylaxis.
Infants born to HBsAg-positive mothers have to be tested
for OBI even if seropositive for anti-HBs. To conclude on
the protective effect of treatment of HBsAg-positive preg-
nant females against OBI, larger numbers of subjects have
to be studied.
Acknowledgments  This work has been funded partially by the
National Hepatology and Tropical Medicine Research Institute, Cairo,
Egypt.
Occult B infection in infants of HBsAg mothers
Conflict of interest  No conflict of interest to declare.
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