Pediatrics and Neonatology (2016) 57, 219e224

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ORIGINAL ARTICLE

A Simple and Rapid Test-card Method to

Detect Hepatitis B Surface Antigen and
Antibody: Potential Application in Young
Children and Infants
Fu-Yu Wu, Yu-Wun Liao, Jia-Feng Wu, Huey-Ling Chen,
Hong-Yuan Hsu, Mei-Hwei Chang, Yen-Hsuan Ni*

Department of Pediatrics, National Taiwan University Hospital, Number 8, Chung-Shan South Road,
Taipei 100, Taiwan

Received Apr 10, 2015; received in revised form Jun 25, 2015; accepted Jul 16, 2015
Available online 23 October 2015

Key Words Background: Hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs)
immunochromato- were conventionally determined by enzyme immunoassays. We aimed to apply a rapid, simple,
graphy; and accurate method to detect HBsAg and its antibody.
mass screening; Methods: We collected 1463 serum samples from healthy volunteers, hepatitis B carriers, and
pediatrics children of HBsAg-positive mothers. The test card that we examined is a chromatographic
immunoassay for the qualitative detection of either HBsAg or anti-HBs. We then compared
the results of the test card to the results of the conventional enzyme-immunoassay method,
which is regarded as a standard.
Results: In the use of the test card to check HBsAg, the sensitivity was 88.8% and the specificity
was 100%. The median hepatitis B virus viral load was significantly higher in the true-positive
group [103.71 copies/mL (range, 102e109.03 copies/mL)] than in the false-negative group [102
copies/mL (range, 102 e103.26 copies/mL)] (p Z 0.005). In those who were younger than 2
years, the diagnostic accuracy of the HBsAg test card was 100%. Then, 1272 samples were
tested for anti-HBs rapid test card. The sensitivity was 91.8% and the specificity was 96.5%.
The median anti-HBs titer was significantly higher in the true-positive group (295.8 mIU/mL)
than in the false-negative group (42.3 mIU/mL; p < 0.001).
Conclusion: Because of (1) the limited amount of blood sample required and (2) most of the
young hepatitis B virus carriers having high viremia, and no concerns of false negativity, the

* Corresponding author. Department of Pediatrics, National Taiwan University Hospital, Number 8, Chung-Shan South Road, Taipei 100,
Taiwan.
E-mail address: yhni@ntu.edu.tw (Y.-H. Ni).

http://dx.doi.org/10.1016/j.pedneo.2015.07.003
1875-9572/Copyright ª 2015, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
220
test card is a good rapid screening tool for the detection of HBsAg and anti-HBs in pediatric
group.
Copyright ª 2015, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
by-nc-nd/4.0/).
1. Introduction
Hepatitis B virus (HBV) infection is a global health problem.
It is a major cause of chronic hepatitis, liver cirrhosis, and
hepatocellular carcinoma. Mother-to-infant transmission is
an important route of infection leading to these sequelae in
endemic areas, such as Taiwan.1 The world’s first nation-
wide HBV vaccination program was launched in Taiwan in
1984.2 Based on six sequential seroepidemiology surveys in
Taiwan, the hepatitis B surface antigen (HBsAg) carrier rate
declined significantly from 10% in the prevaccination era to
0.9% after 25 years of universal vaccination.3,4
In spite of immunoprophylaxis with a combination of
passive (hepatitis B immunoglobulin) and active (HBV vac-
cine) immunization, infants of HBsAg-positive mothers,
especially those mothers with positive serum hepatitis B e
antigen (HBeAg) and/or high HBV viral loads, are still at
high risk for the transmission of HBV infection.5,6 A previous
study in Taiwan showed the overall HBsAg-positive rate was
2.46% in all of the children born to HBsAg-positive mothers
despite immunoprophylaxis. The rate was much higher in
the children born to HBeAg-positive mothers (9.26%) than
those born to HBeAg-negative mothers (0.23%).7 The Cen-
ters for Disease Control of Taiwan recommended the post-
vaccination screening from September 2010. The screening
program is to check HBsAg and hepatitis B surface antibody
(anti-HBs) in all children born to HBsAg-positive mothers
when they are about 1 year old. If HBsAg is positive, the
children would be diagnosed and managed as chronic HBV-
infected patients. If HBsAg and anti-HBs are both negative,
a booster vaccine is indicated.
However, the current methods for HBV serological ex-
aminations need sophisticated laboratory work with suffi-
cient amount of blood sample (preferably > 2 mL), and the
results are not immediately available. All of these problems
may hinder the implementation of a screening program.
Thus, a simple and rapid method to detect HBsAg and anti-
HBs is desired and needs to be verified.
2. Methods
2.1. Blood samples
From April 1, 2008 to May 31, 2011, a total of 1463 blood
samples were collected from three sources: (1) stored sera:
samples were acquired from HBV carriers who were regu-
larly followed in the Department of Pediatrics, National
Taiwan University Hospital, Taipei, Taiwan (n Z 237); (2)
stored sera: samples were obtained from healthy volun-
teers who participated in a postvaccination HBV serology
screening program (n Z 50)4; and (3) fresh sera from newly
F.-Y. Wu et al
recruited patients: samples were collected from children
born to HBsAg-positive mothers when they were 1e2 years
old (n Z 1176). Most serum samples were from children of
HBsAg-positive mothers, which is the target group of the
HBsAg and anti-HBs screening program. Informed consents
were obtained from the participants or their legal
guardians.
Approximately 5 mL of whole blood was collected from
each participant. The stored serum samples had been
stored at
80 C for <6 months before the study, and they
had been thawed and mixed completely before testing. The
HBsAg and anti-HBs rapid tests done with fresh sera were
performed within half hour after blood sampling. The study
protocol was approved by the Institutional Review Board of
the National Taiwan University Hospital.
2.2. HBV serologic markers
Serum HBsAg and anti-HBs were measured in all specimens
using enzyme immunoassays (EIAs; Abbott Laboratories,
North Chicago, IL, USA). The serum HBsAg titer 0.05 IU/
mL was considered positive, and that of the anti-HBs 
10 mIU/mL was considered protective.
2.3. HBsAg and anti-HBs rapid tests
The HBsAg One Step Hepatitis B Surface Antigen Test Device
and the HBsAb One Step Hepatitis B Surface Antibody Test
Device were used (General Biologicals Corporation, Hsin-
chu, Taiwan). The kit can be stored at 2e30 C with sta-
bility. It is a qualitative lateral-flow chromatographic
immunoassay. The estimated cost is approximately 10 US
dollars per kit.
The HBsAg One Step Hepatitis B Surface Antigen Test
Device is an HBsAg rapid test utilizing the combination of
monoclonal and polyclonal antibodies. The device must be
placed on a flat surface during the processing. Twenty-five
microliters of serum, plasma, or whole blood is added to
the specimen well, and then the specimen reacts with the
particle coated with monoclonal anti-HBsAg. The mixture
then migrates upward along the membrane by capillary
action, and reacts with polyclonal anti-HBsAg antibodies,
which are precoated on the test line region.
Anti-HBs was checked by HBsAb One Step Hepatitis B
Surface Antibody Test Device, which is an anti-HBs rapid
test utilizing a double-antigen sandwich system. The prin-
ciple is similar to the HBsAg rapid test, except that the
particle and test line region were precoated with HBsAg.
The visual readout was interpreted 15 minutes later by a
trained staff that was blinded to the EIA results. The
invisibility of the control line indicates insufficient spec-
imen volume or incorrect procedure techniques. The
Rapid test of HBsAg and Anti-HBs
presence of one red line in the control region indicates a
negative result, and the presence of two distinct lines in
both the control and test regions indicates a positive result.
A light-red line in the test region, which was recorded as
“weak positive,” was still defined to be a positive result.
The detection limit of the HBsAg One Step Hepatitis B
Surface Antigen Test Device is 1 ng/mL (w0.18 IU/mL), and
the detection limit of the HBsAb One Step Hepatitis B Sur-
face Antibody Test Device is 10 mIU/mL, as per the manu-
facturer’s specification.
2.4. HBV DNA quantification by real-time
polymerase chain reaction
In those HBsAg that tested positive by the EIA method, we
randomly selected 74 samples of which the amount was
good enough for HBV DNA quantification. The detailed
procedures were described previously.8,9 In brief, HBV DNA
was extracted from 50 mL of serum, and the nucleic acids
were redissolved in 50 mL of H2O. The polymerase chain
reaction was performed in a total volume of 10 mL, con-
taining 2 mL of DNA template, 1 mL of LightCycler FastStart
DNA Master Hybridization Mixture (Roche Diagnostics
Applied Science, Mannheim, Germany), 0.8 mL of 25 mmol/
L MgCl2, 0.3 mmol/L each of the anchor and sensor probes,
and 5 mmol/L of each primer. The primers covered nucle-
otide positions 1261e1279 and 1600e1580, the anchor
probe was nucleotide positions 1552e1576, and the sensor
probe was nucleotide positions 1533e1550. The measure-
ment was performed by using the LightCycler analysis
software 3.5 (Roche Diagnostics Applied Science). The
detection limit of this method was 102 copies/mL.
2.5. Statistical analysis
The statistical analysis was done with the software PASW
Statistics v18.0 (SPSS Inc., IBM, Armonk, NY, USA). Using the
serum HBsAg and anti-HBs results by the EIA method as gold
standard, the clinical sensitivity, specificity, positive pre-
dictive value (PPV), and negative predictive value (NPV)
were calculated. Diagnostic accuracy was defined as the
proportion of concordant results. The receiver-operating-
characteristic (ROC) curve analysis was used to evaluate
the performance of each rapid test. The HBV DNA, and
HBsAg and anti-HBs titer were compared between the true-
Table 1 Results of the HBsAg One Step Hepatitis B Surface Antigen Test Device (n Z 1463) and HBsAb One Step Hepatitis B
Surface Antibody Test Device (n Z 1272).
HBsAg rapid test* EIA HBsAg
Positive Negative Total
Positive 183 0 183
Negative 23 1257 1280
Total 206 1257
anti-HBs Z hepatitis B surface antibody; EIA Z enzyme immunoassay; HBsAg Z hepatitis B surface antigen.
* Sensitivity, 88.8%; specificity, 100%; positive predictive value, 100%; negative predictive value, 98.2%; and diagnostic accuracy,
98.4%.
y
Sensitivity, 91.8%; specificity, 96.5%; positive predictive value, 99.5%; negative predictive value, 60.2%; and diagnostic accuracy,
92.4%.
221
positive group and the false-negative group using Man-
neWhitney U test. A p value < 0.05 was considered sta-
tistically significant.
3. Results
3.1. HBsAg rapid test
Of the 1463 samples tested with conventional EIA for
HBsAg, 206 were HBsAg positive and 1257 were HBsAg
negative. The 1257 samples with HBsAg seronegativity by
the EIA method all showed negative results in HBsAg rapid
test. However, among the 206 samples with HBsAg sero-
positivity by the EIA method, 183 showed positive results
(true-positive group) and 23 showed negative results (false-
negative group) in the HBsAg rapid test. The mean age of
the true-positive group was 23.1  10.47 years (range,
1.01e43.06 years), and that of the false-negative group was
27.66  6.25 years (range, 16.78e38 years). If we took the
EIA result as the gold standard, the sensitivity and speci-
ficity of the HBsAg rapid test were 88.8% and 100%,
respectively. The PPV and NPV were 100% and 98.2%,
respectively. The diagnostic accuracy was 98.4% (Table 1).
The area under ROC for HBsAg rapid test was 0.99. By
analyzing the ROC, the sensitivity and specificity may
achieve 99.4% and 99%, respectively, when HBsAg is 11.76
IU/mL.
3.2. Correlation of HBV DNA level and HBsAg rapid
test
In those HBsAg that tested positive by the EIA method, we
randomly selected 74 samples of which the amount was suf-
ficient for HBV DNA quantification. Among them, 63 samples
were from the stored sera of HBV carriers, and 11 samples
were from the children born to HBsAg-positive mothers. There
were 64 samples from the true-positive group (36 males and
28 females) and 10 samples from the false-negative group (8
males and 2 females). The mean age of the true-positive
group was 20.82  10.91 years (range, 1.01e43.06 years),
and that of the false-negative group was 25.77  6.77
years (range, 16.78e34.6 years). The true-positive group
had significantly higher median serum HBV DNA
level [103.71 copies/mL (range, 102e109.03 copies/mL)] than
the false-negative group [102 copies/mL (range,
Anti-HBs rapid testy EIA Anti-HBs
Positive Negative Total
Positive 1036 5 1041
Negative 92 139 231
Total 1128 144
222
102e103.26 copies/mL); p Z 0.005]. Similarly, the median
HBsAg titer was significantly higher in the true-positive group
[291.1 IU/mL (range, 50.1e481.3 IU/mL)] than in the
false-negative group [10.4 IU/mL (range, 3.9e85.2 IU/mL);
p < 0.001]. According to the ROC analysis, the specificity
was 100% when the HBV DNA level was higher than 103.27
copies/mL.
3.3. HBsAg rapid test in offspring of HBsAg-positive
mothers
We further analyzed the subgroup of children born to
HBsAg-positive mothers (n Z 1176). The mean age was
1.22  0.16 years (range, 1e1.65 years). There were 19
(1.6%) samples of HBsAg positive and 1157 samples of HBsAg
negative by the EIA method. The HBsAg rapid test showed
concordant results. The diagnostic accuracy was 100%
(Table 2). The median HBsAg titer was 220.6 IU/mL (range,
132.6e387.2 IU/mL). The median HBV DNA level was 107.8
copies/mL (range, 106.64e109.03 copies/mL).
3.4. Anti-HBs rapid test
Of the 1272 samples tested with the EIA for anti-HBs, 46
samples were acquired from HBV carriers, 50 samples were
obtained from healthy volunteers, and the rest were
collected from children born to HBsAg-positive mothers.
Among the 1128 samples with anti-HBs seropositivity by the
EIA method, 1036 showed true-positive results (541 males
and 495 females) and 92 showed false-negative results (50
males and 42 females) in the anti-HBs rapid test. The mean
age of the true-positive group was 1.43  1.82 years (range,
1e23.38 years), and that of the false-negative group was
2.1  3.98 years (range, 1e24.16 years). Using the EIA
method as the gold standard, the sensitivity and specificity
of the anti-HBs rapid test were 91.8% and 96.5%, respec-
tively. The PPV and NPV were 99.5% and 60.2%, respec-
tively. The diagnostic accuracy was 92.4% (Table 1). The
area under ROC for anti-HBs rapid test was 0.92. By
analyzing the ROC, the sensitivity and specificity may
achieve 90.1% and 83.3%, respectively, when the anti-HBs is
57.6 mIU/mL.
Comparing the true-positive group and the false-negative
group, the former had significantly higher median serum
anti-HBs titer [295.8 mIU/mL (range, 10e16446 mIU/mL) vs.
42.3 mIU/mL (range, 10.3e1000 mIU/mL); p < 0.001].
Table 2 Results of the HBsAg One Step Hepatitis B Surface Antigen Test Device and HBsAb One Step Hepatitis B Surface
Antibody Test Device in children born to hepatitis B surface antigen-positive mothers (n Z 1176).
HBsAg rapid test* EIA HBsAg
Positive Negative Total
Positive 19 0 19
Negative 0 1157 1157
Total 19 1157
anti-HBs Z hepatitis B surface antibody; EIA Z enzyme immunoassay; HBsAg Z hepatitis B surface antigen.
* Sensitivity, 100%; specificity, 100%; positive predictive value, 100%; negative predictive value, 100%; and diagnostic accuracy,100%.
y
Sensitivity, 92.1%; specificity, 95.9%; positive predictive value, 99.7%; negative predictive value, 44.6%; and diagnostic accuracy,
92.3%.
F.-Y. Wu et al
Among the 144 samples with anti-HBs seronegativity by
the EIA method, five showed positive results in the anti-HBs
rapid test (false-positive group). All of the samples in the
false-positive group showed weak positive results in the
anti-HBs rapid test.
3.5. Anti-HBs rapid test in the offspring of HBsAg-
positive mothers
We further analyzed the data of children born to HBsAg-
positive mothers (n Z 1176), and the results were similar to
the whole group. The sensitivity and specificity of the anti-
HBs rapid test were 92.1% and 95.9%, respectively. The PPV
and NPV were 99.7% and 44.6%, respectively. The diagnostic
accuracy was 92.3% (Table 2). The median serum anti-HBs
titer was 236 mIU/mL (range, 0.1e16,446 mIU/mL) in this
subgroup.
4. Discussion
Most of the children with chronic HBV infection were in the
immune-tolerance phase with high HBV DNA level and high
HBsAg titer.10e12 In our study, the false-negative group of
the HBsAg rapid test had a median HBV DNA level of
102 copies/mL. The rapid test may yield 100% diagnostic
accuracy when the HBV DNA level is higher than 104 copies/
mL. We further verified the speculation in our subgroup of
children born to HBsAg-positive mothers, of whom the
median HBV DNA level was as high as 107.8 copies/mL.
Indeed, the HBsAg rapid test had excellent performance in
this subgroup. It is appropriate to use this HBsAg rapid test
for screening the HBsAg carrier status of young children in
the immune-tolerance phase, but it may not be adequate
for the diagnosis and/or follow-up otherwise.
Many HBsAg rapid tests are commercially available
worldwide. According to the assessment report from the
World Health Organization Blood Safety and Clinical Tech-
nology Department, most tests were immunochromato-
graphic assays with 98e100% sensitivity and 95e100%
specificity.13,14 These HBsAg rapid tests can be provided as
relatively inexpensive and technically undemanding diag-
nostic tools. In previous studies, these rapid tests were
mainly used to survey the prevalence of chronic HBV
infection, and improve the treatment of human-immuno-
deficiency-virus-and-HBV-coinfected patients in developing
countries. However, these rapid tests showed high
Anti-HBs rapid testy EIA Anti-HBs
Positive Negative Total
Positive 1016 3 1019
Negative 87 70 157
Total 1103 73
Rapid test of HBsAg and Anti-HBs
specificity (95e100%), but only moderate sensitivity
(43e70%) in the study population.15,16 Apparently lower
prevalence of HBV chronic infection was observed if a rapid
test was used alone. The study concluded that a rapid test
may need to be supplemented by the EIA method particu-
larly for blood-bank screening.15 By contrast, our study
showed both high sensitivity and specificity of the HBsAg
One Step Hepatitis B Surface Antigen Test Device, and
validated the excellent diagnostic accuracy in a pediatric
group. It can serve as an ideal screening tool in endemic
regions for the detection of HBV carriers if they are in the
immune-tolerance phase.
The anti-HBs titer may decline with age after HBV im-
munization.17,18 In addition, a small proportion (5e10%) of
immunocompetent vaccinees failed to develop protective
anti-HBs level after completing the scheduled HBV vacci-
nation.19 Thus, screening of anti-HBs titers in early child-
hood may be essential, especially in those who were born to
HBsAg-positive mothers. These hyporesponders may reach
100% protective response rate after an additional vaccine
booster.20,21
To our knowledge, only one paper published in English
has evaluated the performance of anti-HBs rapid tests so
far.22 In the aforementioned article, 3739 samples were
tested with Quick Profile (LumiQuick Diagnostics, Inc.,
Santa Clara, CA, USA). The specificity (97.8%) and PPV
(97.1%) were high, while the sensitivity (58.3%) and NPV
(64.9%) were low. The results were considered reliable only
when the tests were positive. In the false-negative group,
the median anti-HBs titer was 58 mIU/mL (range,
10e1000 mIU/mL). In our study, not only the specificity
(96.5%) and PPV (99.5%), but also the sensitivity (91.8%) of
the HBsAb One Step Hepatitis B Surface Antibody Test De-
vice was high. Similarly, the median anti-HBs titer was
significantly lower in the false-negative group (42.3 mIU/
mL) than in the true-positive group. This anti-HBs rapid test
card is a good screening tool in a high-risk group of HBV
infection, such as babies born to HBsAg-positive mothers or
health care workers, and it can be used as a reference for
HBV vaccine booster.
Most of the commercially available HBsAg rapid tests
need more than 50 mL specimens, and can only be used with
serum or plasma.13e16,23 Only a 25-mL sample is needed in
this rapid test. For children who are difficult in venipunc-
ture, blood from fingers or heel puncture can be alternative
ways. The kit can be also stored at room temperature
(2e30 C), and can be tested with whole blood, which fa-
cilitates the usage in local clinics or screening stations
without centrifuges or refrigerators.
This study has several limitations. First, the HBV DNA
quantification was not performed in all of the serum sam-
ples. We only randomly selected 74 out of 206 HBsAg-
positive serum samples by the EIA method. In addition,
according to the package insert of the rapid test, it can be
used for the detection of HBsAg/anti-HBs in serum, plasma,
or whole blood. Further validation of the application in
whole-blood specimen may be conducted in a future study.
In conclusion, our study verified the application of the
rapid tests of HBsAg and anti-HBs. The rapid tests do not
require sophisticated equipment, and are easy to perform
and interpret. It is an excellent screening tool in young
children. It is expected that HBV infection and its
223
complications can be further reduced under the extensive
application of the mass postvaccination screening.
Conflicts of interest
The authors have no conflicts of interest relevant to this
study.
Acknowledgments
This work was supported by the Ministry of Health and
Welfare of Taiwan (DOH98-DC-1101) and the National
Taiwan University Hospital (99-S1311). The authors would
like to thank General Biologicals Corporation for providing
the rapid test devices.
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