Long Term Efficacy of Hepatitis B Vaccine among High Risk... Transfusion Children in Egypt

Journal of Applied Sciences Research, 5(12): 2504-2510, 2009
© 2009 INSInet Publication
Long Term Efficacy of Hepatitis B Vaccine among High Risk Multiple Blood
Transfusion Children in Egypt
1
5
Mohamed Mokhles, 6 Rasha El Ashry, 2 Mohamed Sedky, 3Nahed Emara, 3Maha Rasheed,
4
Maha EL-Wassef, 5Amal Saad, 6Youcef ElTonbary
Departments of 1Internal Medicine, 2Pediatric, 3Clinical Pathology, 4Medical Biochemistry, and
Environmental & Occupational Medicine, National Research Center, and Department of 6Pediatric,
Mansoura University Hospital, Egypt.
Abstract: The duration of hepatitis B vaccine (HBV) protection is controversial, hence the need for a
booster dose specially in context of high risk population. This research aimed to study the efficacy of
HBV vaccine in high risk children subjected to repeated blood transfusion 10-16 years post vaccination.
The study included 48 patients with hemato-oncological disorders treated in the onc-haematology centre,
at Mansoura university, Egypt, subjected to repeated blood transfusion. They were born after 1992 (year
of mandatory HBV vaccination for newborns in Egypt) and before 1998. The control group comprised
32 apparently healthy children with matched age and sex. All the children were subjected to blood testing
for hepatitis B surface antigen (HBsAg), and hepatitis B surface antibody (HBsAb) titre. The results
revealed that the percentage of cases having HBsAb titres below the protective level (<10 IU/L) (39.6%)
was significantly higher than that of the control group (18.8%). W hile, the percentage of patients positive
for HBsAg (31.3%) did not differ significantly compared to the controls (18.8%). W ithin the cases, there
was no significant difference considering HBsAb titre (HBsAb <10 IU/L or $10 IU/L) between the
positive and negative HBsAg subjects. Also, the presence or absence of protective level of HBsAb titre
could not be attributed to the frequency of blood transfusion in the included cases. Thus, the present study
concluded that high risk groups of children with repeated blood transfusion are significantly risky for HBV
infection secondary to loss of HBsAb protective titre. Thus, booster dose to this group should be
considered.
Key words: HBV vaccination, high risk children, multiple blood transfusion, HBsAg, and HBsAb titre.
INTRODUCTION
Hepatitis B remains a major healthcare problem
worldwide. It is estimated that 350 million people are
chronically infected, and 500,000 to 1 million die each
year [1 ]. Globally, most HBV-related deaths result from
the chronic sequels of infection acquired in the
perinatal and early childhood periods [2 ] . Transmission of
HBV can occur percutaneously, sexually, perinatally,
through blood transfusion and organ trans-plantation.
High-risk groups for HBV infection include healthcare
workers, intravenous drug users, homosexuals,
promiscuous heterosexuals, hemophilics, thalassemics,
or renal failure patients due to frequent blood
transfusions [3 ].
The strategy for the control of hepatitis B virus
(HBV) infection, as outlined by the W orld Health
Organization (WHO) and endorsed by the Advisory
Committee on Immunization Practices (ACIP), is the
introduction of hepatitis B immunization at birth [4 ].
Inclusion of HBV vaccine into national infant
immunization programs could prevent more than 80%
of HBV-related deaths [2 ]. Available vaccines are
prepared from the non-infectious outer surface of the
virus surface antigen (HBsAg), the plasma derived and
the recombinant are equally effective and safe [5 ].
In Egypt, active immunization with one of the
recombinant HBsAg vaccines is delivered as a threedose series at 0, 1, and 6 months. Three doses induce
a protective response ( $ 10 IU/L anti-HBs) in more
then 90% of healthy adults and children [6 ]. Non
responders have peak anti HBs of less than 10 IU/L
and lack protection. Low responders have peak antiHBs levels of 10-100 IU/L and generally lack
detectable anti-HBs levels within 5-7 years and they
may respond to a further booster of double the dose of
the vaccine. Good responders have peak anti-HBs >100
IU/L and usually have long term immunity[5 ].
The duration of hepatitis B vaccine protection is
controversial; where Kenneth et al [7 ] stated that it will
Corresponding Author: Amal Saad, Environmental & Occupational Medicine, National Research Center
2504
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
be very important to follow children into adolescence
and early adulthood for evidence of clinically
significant breakthrough infections indicating a possible
need for routine booster doses. Chang et al [8 ] found
that after universal HBV vaccination, HBV surface
gene variants emerge or are selected under the immune
pressure generated by the host or by administration of
hepatitis B immune globulin and hepatitis B
vaccination. El-Sawy and M ohamed [9 ] recommended
booster inoculations for all previously vaccinated
children. On the other hand Abramowicz and
Twinrix [1 0 ] stated that post-vaccination protection
persists for at least 9 years with more than 50% of the
vaccinated persons, despite the decline of hepatitis B
surface antibody levels (HBsAb) below 10 IU/L. This
was supported by Zanneti et al[1 1 ], they found that
strong immunological memory persists for more than
10 years after immunization of infants and adolescents
with a primary course of vaccination and booster doses
of vaccine do not seem necessary to ensure long-term
protection. Koltan et al[1 2 ] concluded that in children
with cancer vaccinated against HBV according to the
vaccination schedule, the immune response maintains
a protective level of anti-HBs in more than 60% of
cases, despite immuno-suppression. However, a booster
dose should be considered at 5-7 years after the initial
course if the subject is still being exposed to hepatitis
B [1 3 ]. However, Petersen et al[1 4 ] recommended that long
term studies of infants are needed to determine the
duration of protection and the necessity for and timing
of booster doses.
Subjects and M ethodology:
Subjects: A cross-sectional study conducted in the
period between 19/11/2008 to 18/12/2008 in the
pediatric hematology oncology department, at the
Mansourah hospital university, Faculty of Medicine,
Mansourah University, Egypt.
Patients submitted to this study were all patients
carrying benign hematological diseases or hematological
oncological disorders justifying blood components
transfusion in the form of packed RBCs. The selected
patients were all born after the year 1992; the year of
obligatory HBV vaccination for newborns in Egypt,
and before the year 1998 to assure that the period
since vaccination is not less than 10 years.
After obtaining a written consent from the
parent(s), 48 patients 28 males and 20 females with an
age range of 10-16 years were subjected to full
medical history, thorough medical examination, specific
investigations in the form of HBsAg and HBsAb.
M ethods: The viral profile analysis was undertaken by
the clinical pathology laboratory in the National
research Center. The laboratory technique was done
after blood sampling of 5 ml blood undergoing
centrifuge to 5000 RPM with collected serum analyzed
the same day or at most the following day after a
period less than 24 hour as follows:
- Hepatitis B Surface Antigen (HBsAg): Enzyme
immunoassay procedure for determination of Hepatitis
B surface antigen in human serum. Liaison® HBsAg
(code 310100).Liaison ® HBsAg negative and positive
controls (code 310101), produced by DiaSorin
S.p.A.Strada per Crescentino-13040 Saluggia (Vercelli)Italy.
Principle of the procedure: T he H BsAg test is an
in vitro neutralization assay for detection of the
presence of H BsAg in human serum and plasma
samples. The HBsAg test is based on the principle of
neutralization of binding activity. A neutralizing reagent
containing human antibodies to HBsAg is added to one
aliquot of each specimen (neutralized aliquot). As a
control procedure, anti–HBs negative human serum is
added to the other aliquot (non-neutralized aliquot). If
the neutralizing reagent has been added to a sample
containing HBsAg forming an antigen-antibody
complex. If the neutralizing reagent has been added to
a sample containing an interfering substance, the
antibodies in the neutralizing reagent will not bind to
the interfering substance. In the confirmation procedure,
each sample is incubated in a solid matrix coated with
mouse monoclonal antibodies to HBsAg. Next the
antibody conjugate from the screening kit, which
contains sheep antibodies to HBsAg ,is added. If an
antibody –antigen –antibody complex is present , the
antibody conjugate will bind to the complex only
partially. If an antibody-interfering substance complex
is present, the antibody conjugate will bind nonspecifically to the interfering substance. If the signal of
the neutralized aliquot is significantly lower than the
signal of the non-neutraized aliquot, the presence of
HBsAg in the sample is confirmed.
- Antibodies to Hepatitis B Surface Antigen
(HbsAb): Enzyme Immunoassay procedure for
quantitative determination of antibodies to hepatitis B
surface antigen (anti-HBs) in human serum or plasma
samples.ET I-AB-AUK-3 (P001603) produced by
DiaSorin S.p.A.13040 Saluggia (Vercelli)-Italy.
Principle of the procedure: The method for the
quantitative HBsAb determination is a direct, noncompetitive sandwich assay. The presence of HBsAb
allows the enzyme tracer to bind to the solid phase.
The enzyme activity is therefore proportional to the
concentration of HBsAb present in samples or
calibrators. Enzyme activity is measured by adding a
colourless chromogen/substrate solution. The enzyme
action on chromogen/substrate produces a colour which
2505
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
is measured with a photometer.
Calculation of results: Record the absorbance at
450/630nm and for each calibrator and specimen, and
subtract the 630 nm absorbance value from the 450 nm
absorbance values for all calibrators and samples.
Interpolation of results: Plot in log-log coordinates
the absorbance value at 450/630nm for each calibrator
on the ordinate (y axis) as a function of the
concentration of HBsAb expressed as IU/L on the (x
axis). A calibration curve is thus obtained. Directly
from the calibration curve, read the H BsAb
concentration of each sample expressed as IU/L. If the
sample was diluted, the concentration value derived
from the diluted specimen must be multiplied by the
dilution factor. It is generally accepted that an HB sAb
concentration above 10 IU/L is indicative of either.
Statistical M ethods: The collected data was
statistically analyzed using SPSS software, version
14.0 [1 5 ] . Independent t-test was used for comparison
between the quantitative data of two groups. Mannwhitney test was used as a non-parametric test for the
skewness data. Chi-square was used for comparison
between the qualitative data. The significance level was
considered at P-value < 0.05.
Results: Table 1, illustrates the demographic data of
the included children of the two examined groups.
Table 1: D em ographic data of the two exam ined groups
Age
CASES (48)
CO N TR O LS (32)
X±SD
X±SD
Total children
10.1±3.2
9.2±2.1
Fem ales
10.4±3.8
9.5±2.4
M ales
10.0±2.6
9.0±2.0
Gender
N o. (% )
N o. (% )
Fem ales
21 (43.8)
8 (25)
M ales
27 (56.3)
24 (75)
Table 2, showed no significant difference between
the two groups regarding HBsAg. W hile, the percent of
cases with HBsAb titre < 10 IU/L was significantly
higher compared to their controls.
W ithin the high risk group, we compared positive
and negative cases to HBsAg as regards the protective
level of HBsAb $10 IU/L, where we found no
statistical significant difference between the HBsAb
titre of the positive and negative HBsAg, cases as
shown in table 3.
Moreover, there was no significant difference
between positive and negative HBsAg cases as regards
the frequency of blood transfusion (Figure 2).
W e did not find any relationship between the
frequency of blood transfusion and the protective level
of HBsAb ($ 10IU/L) within the high risk group of
children, as shown in Fig.
Discussion: Long-term follow-up of vaccinated children
confirmed that universal HBV vaccination in infancy
has produced adequate protection up to 14 years of
age. Yet, the annual decay rate of hepatitis B surface
antibody (anti-HBs) was 10.2% in children who did not
receive a booster dose [1 6 ]. The protective level of
HBsAb in patients treated for neoplastic diseases has
dropped from 80.5% at 3 months after end of
vaccination program to 78.6% after 18 months of the
end of the program [1 2 ]. Thus, the matter of boosting
after 7-10 years remains questionable, especially in
high risk groups as there was a great variation between
studies that showed high persistence of HBsAb, as the
study carried by W u et al [1 7 ] on high risk infants
showed that, at 10 years, the cumulative persistence of
antibody to hepatitis B surface antigen (HBsAb) was
85%, and the cumulative incidence of HBV infection
was 15%. In another study, Dentico and his
colleagues [1 8 ] showed that the sero-conversion in
vaccinated thalassemics reached 80% after 6 years of
vaccination. Other studies showed a low persistence of
the protective titre in haemodialysis patients that
revealed a sero-prevalence rate of 38% 10 years after
vaccination [1 9 ].
In the present study, after 12-16 years of
vaccination 60.4% of the high risk group children
subjected to repeated blood transfusion due to different
diseases (Thalassemia, sickle cell anaemia, leukaemia)
had a protective level of HBsAb ($10 IU/L). This was
significantly lower than that within the control healthy
vaccinated children of the matched age, as the 81.3%
control group with had protective level of HBsAb ($10
IU/L). This could be attributed to the fact that over
50% of the high risk group included in the present
study (cases) were cases of thalassemia followed by
sickle cell anemia and leukemia. Thalassemics cases
proved to have some alteration in the immune system
as a consequence of iron overload [2 0 ]. Moreover, Hord
et al[2 1 ] found that, the sero-conversion rate was found
to be lower in children with sickle cell disease than
that in the general population, which was also
supported by Keating and Nobel [2 2 ] who found that the
immunogenicity of Hep-B(Eng) was reduced in patients
with conditions associated with impaired immune
function as patients undergoing haemodialysis or being
treated for malignancy.
On the other hand Froutan-Pishbijari et al[2 0 ] found
that the protective HBsAb level in thalassemic patients
was not significantly different from the healthy
subjects, but, it was not a long term evaluation as it
was a post vaccination evaluation. In the long term
evaluation, McMahon and his colleagues[2 3 ] found that
the levels of anti-HBs in the cohort decreased from a
geometric mean concentration of 822 mIU/mL after
2506
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
Fig. 1: Showed the percent of the different blood diseases in the examined cases. The most frequent cases was
â-thalassemia (52.1%) followed by sickle cell anemia and leukemia cases (8.3% of both).
Table 2: Com paring cases and controls as regards HBsAg and HbsAb
CASES
CO N TRO LS
(48)
(32)
N o. (% )
N o. (% )
H BsAg
Positive
15 (31.3)
6 (18.8)
H BsAb
3.879
P< 0.05
$10 IU /L
< 10 IU /L
19 (39.6)
6 (18.8)
Table 3: Com paring of the H BsAb titre between H BsAg positive and negative cases
H BsAg
N egative
Positive
N o. (% )
N o. (% )
H BsAb $ 10 IU /L
19 (57.6)
10 (66.7)
H BsAb < 10 IU /L
14 (42.4)
5 (33.3)
Chi-square
÷2
1.55
P-value
NS
29 (60.4)
26 (81.3)
Chi-square
c2
0.356
P-value
NS
Fig. 2: Comparison of the frequency of blood transfusion betweenpositive and negative HbsAg in children with
blood diseases .
2507
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
Fig. 3: Relationship between the HBsAb and the number of blood transfusion (week) in the diseased children
vaccination to 27 mIU/mL at 15 years. The present
study revealed that after 10-16 years of HBV
vaccination, 39.6% of the high risk children (cases) had
HBsAb below the protective level (< 10 IU/L)
compared to 18.8% in the control group, with
significant difference.
In the present study, 31.3% of the high risk group
was positive to HBsAg, but, was not significantly
different compared to the healthy controls (18.8%).
W e could not attribute the infectivity within the high
risk group to the low protective level of HBsAb, as
there was no significance difference between the
percentage of the cases with HBsAb $10 IU/L and
HBsAb <10 IU/L as regards positively of HBsAg. The
same result was support by Singh et al [2 4 ] who found
that the frequency of HBV infection in â-thalassemic
patients was similar in vaccine responders and nonresponders, where they found a number of mutations in
the S gene, which could have implications for viral
replication as well as virus-host cell interaction.
In fact, HBV surface gene variants are emerged or
selected under the immune pressure generated by the
host or by administration of hepatitis B vaccination.
The appearance of Hepatitis B surface gene mutants in
DNA HBV positive children has been confirmed, and
gradually increased from 7.8% before vaccination to
23.1% 15 years later [2 5 ]. On the contrary an Iranian
study [2 6 ] on high risk group vaccinated infants showed
that infants born to hepatitis B infected mothers had
HBsAg positive detected in 14.3% of children in the
high risk group versus 0% in the unexposed group. It
has been reported that in addition to the seroconversion level, infection rate is also important in the
evaluation of the effectiveness of vaccination [2 7 ].
The present work tried to find a relationship
between the frequency of blood transfusion and the
susceptibility to infection in high risk children, but, we
could not attribute positive HBsAg neither to the low
HBsAb titre nor to the frequency of blood transfusion.
This contradicts with the results of Singh et al[2 4 ], that
found that the prevalence of serological markers
increased with the number of blood transfusions.
The present study concluded that the efficacy of
HBV vaccine after 10-16 years of vaccination in high
risk groups of children with repeated blood transfusion
proved to become significantly risky for HBV infection
secondary to loss of HBsAb protective titre. Thus,
booster dose of Hepatitis B vaccine to high risk groups
is recommended, together with continuous monitoring
of the HBsAb titre to determine the suitable time for
boosting. Also, further epidemiologic studies on a
larger scale of population especially is recommended,
as the present study was a preliminary study.
REFERENCES
1.
2.
2508
Lok, A.S., 2005. The maze of treatments for
hepatitis B. N Engl J Med., 352: 2743-46.
Goldstein, S.T., F. Zhou, S.C. Hadler, B.P. Bell,
E.E. Mast, H.S. Margolis, 2005. A mathematical
model to estimate global hepatitis B disease burden
and vaccination impact. Int J Epidemiol., 34: 132939.
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Chan, H.L., M.G. Ghany, A.S.F. Lok, 1999.
Hepatitis B. In: Schiff ER, Sorrell MF, Maddrey
W C, eds. Shiff's diseases of the liver, 8th ed.
Philadelphia, PA: Lippincott, W illiams & W ilkins.
Shete, P.B., R.S. Daum, 2002. Real versus
theoretical: assessing the risks and benefits of
postponing the hepatitis B vaccine birth dose.
Pediatrics., 109: 701-3.
Sherlock, S., J. Dooley, 2002. Hepatitis B virus
and Hepatitis Delta virus. In: Diseases of the liver
and Biliary system. 7th edition chapter, 17: 292293.
Rivkina, A., D. Pharm, S. Rybalov, 2002. Chronic
Hepatitis B: Current and Future Treatment Options
Pharmacotherapy, 22: 721-37.
Kenneth, M., M.D. Petersen, R. Lisa, M.S.
Bulkow, J. Brian, M.D. McM ahon, B.S. CarolynZanis, R.N. Marilyn-Getty, R.N. Helen-Peters, J.
Alan, 2004. Parkinson, Ph.D. Duration of Hepatitis
B Immunity in Low Risk Children Receiving
Hepatitis B Vaccinations from Birth. Pediatr Infect
Dis J., 23: 650-5.
Chang, M.H., 2006. Hepatitis B virus mutation in
children. Indian J Paediatr, 73: 803-7.
El-Sawy, I.H., O.N. Mohamed, 1999. Long-term
immunogenicity and efficacy of a recombinant
hepatitis B vaccine in Egyptian children. East
Mediterr Health J., 5: 922-32.
Abramowicz, M., A. Twinrix, 2001. A combination
hepatitis A and B vaccine. Med Lett Drugs Ther.,
43: 67-8.
Zanneti, A.R., A. Mariano, L. Romano, R.
Damelio, M. Chironna, R.C. Coppola, M. Cuccia,
R. Manqione, F. Marrone, F.S. Negrone, A.
Parlato, F. Zamparo, C. Zotti, T. Stroffolini, A.
Mele, 2005. Long-term immunogenicity of hepatitis
B vaccination and policy for booster: an Italian
multicentre study. Lancet, 366: 1379-84.
Koltan, S., A. Koltan, M. W ysocki, R. Debski, J.
Styczynski, 2005. Anti-HBs profiles in children
treated for neoplastic disease who had been
vaccinated against hepatitis B postnatal or as
infants. J Hosp Infect, 60: 73-7.
Smedile, A., F. Rosina, G. Saracco, 1991.
H e p a titis B v ir u s r e p lic a tio n m o d u la te s
pathogenesis of Hepatitis D virus in chronic
hepatitis D. Hepatology, 13: 413.
Petersen, K.M., L.R. Bulkow, B.J. McMahon, C.
Zanis, M. Getty, H. Peters, A.J. Parkinson, 2004.
Duration of hepatitis B immunity in low risk
children receiving hepatitis B vaccinations from
birth. Pediatr Infect Dis J., 23: 650-5.
Neter, J., W . W asserman, G.A. W hitmore, 1993.
Applied statistics. A Division of Simon &
Schuster, Inc., USA.
16. Chang. M.H., 2006. Impact of hepatitis B
vaccination on hepatitis B disease and nucleic acid
testing in high-prevalence populations. J Clin
Virol., 36: 45-50.
17. W u, J.S., L.Y. Hwang, K.J. Goodman, R.P.
Beasley, 1999. Hepatitis B vaccination in high-risk
infants: 10-year follow-up. J Infect Dis., 179:
1319-25.
18. Dentico, P., R. Buongiorno, A. Volpe, A.
Zavoianni, D. De M attia, V. Sabato, 1992. Longterm persistence of anti-HBs after hepatitis B
immunization in thalassaemic patients. Infection,
20: 276-8.
19. Peces, R., A.S. Laures, 2001. Persistence of
immunologic memory in long-term hemodialysis
and healthcare workers given hepatitis B vaccine:
role of a booster dose on antibody response.
Nephron, 89: 172-6.
20. Frouta n-Pishbija ri, H .,
H.
G hofra ni,
S.
M irmomenm, S. Kazemi-Asl, M. Nassiri-Toosi,
M.J. Farahvash, H.H. Toroghi, K. Aminian, F.
M ansour-G hanaei, A.H. Bagherzadeh, 2004.
Immunogenicity of hepatitis B vaccine in multitransfused thalassemic patients with and without
hepatitis C infection: a comparative study with
healthy controls. Med Sci Monit., 10: 679-83.
21. Hord, J., B. W indsor, M. Koehler, J. Blatt, J.
Janosky, J. Mirro, 2002. Diminished antibody
response to hepatitis B immunization in children
with sickle cell disease. J Pediatr Hematol Oncol.,
24: 548-9.
22. Keating, G.M., S. Noble, 2003. Recombinant
hepatitis B vaccine (Engerix-B): a review of its
immunogenicity and protective efficacy against
hepatitis B. Drugs, 63: 1021-51.
23. McMahon, B.J., D.L. Bruden, K.M. Petersen, L.R.
Bulkow, A.J. Parkinson, O. Nainan, M. Khristova,
C. Zanis, H. Peters, H.S. Margolis, 2005. Antibody
levels and protection after hepatitis B vaccination:
results of a 15-year follow-up. Ann Intern Med.,
142: 333-41.
24. Singh, H., M. Pradhan, R.L. Singh, S. Phadke,
S.R. Naik, R. Aggarwal, S. Naik, 2003. High
frequency of hepatitis B virus infection in patients
with â-thalassemia receiving multiple transfusions.
Vox Sanguinis, 84: 292-9.
25. Vildozola, H., 2007. Vaccination against Hepatitis
B: 20 years later. Rev Gastroenterol Peru., 27: 5766.
26. Adibi, P., R. Ghassemian, S.M . Alavian, M.
R a njb a r, A .H . M o ha m m ad a liz ad e h, F .
Nematizadeh, M. Mamani, M. Rezazadeh, F.
Keramat, A. Ardalan, A. Esmaeili, M.R. Zali,
2004. Effectiveness of hepatitis B vaccination in
children of chronic hepatitis B mothers. Saudi Med
J., 25: 1414-8.
2509
J. Appl. Sci. Res., 5(12): 2504-2510, 2009
27. Yetgin, S., B. Tavil, S. Aytac, B. Kuskonmaz, G.
Kanra, 2007. Unexpected protection from infection
by two booster hepatitis B virus vaccination in
children with acute lymphoblastic leukemia. Leuk
Res., 31: 493-6.
2510