Secondary Logo

Share this article on:

Determination of antihepatitis B antibodies titer among a sample of compulsory fully vaccinated children and adolescents in Cairo, Egypt

El-Melligy, Dalal M.a; Shaalan, Ashraf H.b

doi: 10.1097/01.MJX.0000437957.73502.88
MICROBIOLOGY & IMMUNOLOGY

Objective This study aims to determine the persistence of protective hepatitis B surface antibody (HBsAb) titer till adolescence following a full neonatal compulsory vaccination, whether booster doses are needed or not, and the best age for boosting if needed.

Participants and methods A cross-sectional study was carried out on 189 fully vaccinated healthy children 2–16 years of age. The sample was divided into preschool (2 to ≤6 years) and school age (6–16 years) groups. Quantitative determination of HBsAb was carried out for all children by competitive enzyme-linked immune sorbent assay.

Results The results showed that the protective level of HBsAb (≥10 mIU/ml) was evident in 95.2% of children younger than 6 years of age in contrast to 41.7% of children older than 6 years of age. The χ 2-test showed highly significant differences between the two groups (χ 2=21.5, P<0.0001). Both study groups were classified according to the level of anti-HBs and the likelihood ratio was applied (51.9, P<0.0001), indicating a highly significant difference between the levels of HBsAb in children younger than 6 years of age compared with those older than 6 years of age. The study sample older than 6 years of age was further classified into five age subgroups (6 to <8, 8 to <10, 10 to <12, 12 to <14, and 14–16 years of age), which were analyzed according to the level of HBsAb and compared with the level of children younger than 6 years of age by the χ 2-test (χ 2=40.3, P<0. 0001), indicating a highly significant difference. The decreasing protective percentage was evident among the different age subgroups, indicating that the older the child, the lower the protective ability of the vaccine. All groups were compared according to the level of HBsAb and the likelihood ratio was calculated with a result of 87.26 and P less than 0.0001. The titer of HBsAb was significantly higher in the children younger than 8 years of age compared with all the other children and in children younger than 6 years compared with those aged 6–8 years. The correlation coefficient (r) between the HBsAb titer and the age of children younger than 6 years of age showed almost no correlation (r=0.1, P>0.05), whereas in children older than 6 years of age, there was a significant negative correlation (r=−0.2, P<0.005).

Conclusion The results suggest the need for a booster dose of hepatitis B vaccine at the age of 6 years to be included in the vaccination program in Egypt.

aDepartment of Microbiology and Immunology, Ahmed Maher Teaching Hospital, General Organization for Teaching Hospitals and Institutes, Cairo

bDepartment of Biological Anthropology, National Research Centre, Medical Research Division, Giza, Egypt

Correspondence to Ashraf Shaalan, Department of Biological Anthropology, National Research Centre, Medical Research Division, Elbohouth Street, PO Box 2311, Dokki, Giza, Egypt Tel: +20 100 140 2126; fax: +202 37601877; e-mail: ashrafshaalan@hotmail.com

Received June 15, 2013

Accepted August 25, 2013

Back to Top | Article Outline

Background

Viral hepatitis has remained a major public health problem worldwide. With the advancement in technologies, eight distinct types of hepatitis viruses have been described so far: hepatitis A, B, C, D, E, G, TT, and SEN viruses 1. Hepatitis B virus (HBV) infection is a blood-borne infection and is the most prevalent chronic infectious disease worldwide 2. Over 2 billion of the world’s population has been exposed to this virus. About 350 million of these, 5% of the world’s population, are chronic carriers. Annually, up to 1 million of this population dies because of the consequences of this infection, such as cirrhosis and hepatocellular carcinoma. More than three-quarters of HBV infections occur in Asia, the Middle East, and Africa 3.

HBV is an enveloped, double-stranded DNA virus. It is the smallest DNA virus known to infect humans. The virus is very infectious in nonimmune individuals. The incubation period for acute infection is 45–160 days, with a mean incubation period of 90 days 3. HBV is a major contributor to the disease burden worldwide and one of the most important infectious agents causing acute and chronic morbidity and mortality worldwide 4. It is generally agreed that the control of hepatitis B is a high-priority public health need on a worldwide basis, the only practical solution to which is large-scale vaccination 5. Neonatal HBV vaccination is the most effective measure for prevention of HBV infection in countries with intermediate to high levels of HBV endemicity 6. In 1992, following the recommendation of the WHO that childhood hepatitis B vaccination should be included in immunization programs of all countries, a compulsory vaccination program against hepatitis B infection among infants was initiated in Egypt with a schedule 2, 4, and 6 months of age 7. Seroprotection is ensured when hepatitis B surface antibody (HBsAb) levels are at least 10 mIU/ml 8. Persistence of protective immunity is a vital issue for any vaccine. Some individuals vaccinated as infants have been shown to lose protection from infection during adolescence, leading to recommendations for a booster dose of these vaccines during adolescence. Decreased serum level of HBsAb with age has been reported in some studies 9. If the immunity induced by hepatitis B vaccine decreases in older adolescents, young adults, and high-risk groups, then, HBV infection could occur in adolescence and adulthood 10.

Several Egyptian studies have discussed the long-term efficacy of hepatitis B vaccine in children. However, some researchers included wide age ranges in their studies without classification (6–12-year-old children 7, 6–11-year-old children 11, and 10–16-year-old high-risk children 12). Other researchers included a certain age group in their studies (5–6-year-old children 13), whereas another research included two age groups (one at 5 years age of age and the other at 11 years of age 14). Accordingly, in order to clearly trace the level of the HBV vaccine in Egyptian children and adolescents, a new study with a wider age range and narrower classification is urgently needed.

The aim of this study is to determine HBsAb titer among a sample of Egyptian children and adolescents 2–16 years of age who were vaccinated against HBV under the compulsory vaccination program (1.5–15.5 years after receiving a full vaccination dose) to assess the persistence of long-term immunity to HBV or a need for recompulsory vaccination as a booster dose. In addition, if a booster dose is recommended, the age of inoculation will be suggested according to the age of decrease in the antibody titer below the protective level.

Back to Top | Article Outline

Participants and methods

This cross-sectional study was carried out between October 2010 and October 2011 on children attending the pediatric clinic of Ahmad Maher Teaching Hospital and the Medical Services Unit of National Research Centre in Cairo. Approval was obtained from the General Organization of Teaching Hospitals and Institutes to carry out this study. All children included in the study were healthy, 2–16 years of age, and had received a full course of hepatitis B vaccine as recorded on the back of the child’s birth certificate. Immune-compromised children (e.g. those receiving corticosteroid therapy) and children with any chronic disease that might affect the immune response (e.g. malnutrition, malignancies, etc) were not included in the study sample.

Parents and/or caregivers were informed about the aim of the study and their consent was obtained for the inclusion of their children in the study. The sample studied included 189 children 2–16 years of age (109 females and 80 males). The sample was divided into preschool (2 to ≤6 years) and school-age children (6–16 years of age). The school-age group was further divided into five age subgroups (from 6 to <8, 8 to <10, 10 to <12, 12 to <14, and 14–16 years of age). All children in the study sample were subjected to a full assessment of medical history and a thorough medical examination. A 5 ml blood sample was drawn aseptically by venepuncture and serum was separated by centrifugation and stored at −70°C until needed for analysis. The samples were thawed for the quantitative determination of antibody to hepatitis b surface antigen (HBsAg) by a competitive enzyme-linked immune sorbent assay (DiaPro, Milano, Italy).

Back to Top | Article Outline

Principle of the test

The method for the quantitative HBsAb determination is a direct, noncompetitive sandwich assay. Microplates are coated with a preparation of highly purified HBsAg that, in the first incubation with the sample, specifically captures anti-HBsAg antibodies to the solid phase. After washing, captured antibodies are detected by an HBsAg labeled with peroxidase (HRP) that specifically binds the second available binding site of these antibodies. The enzyme specifically binds to wells by acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of HBsAb in the sample, and can be detected by an enzyme-linked immune sorbent assay reader. Antibody levels were determined quantitatively by a standard curve calibrated against the WHO reference preparation.

Back to Top | Article Outline

Statistical analysis

Statistical analysis was carried out using SPSS version 14.0 (SPSS Inc., Chicago, Illinois, USA). Qualitative data were presented as number and %, and quantitative data as mean±SD. χ 2-testwas used for analysis of qualitative data and the likelihood ratio was used if more than 25% of the cells had an expected count less than 5. Least significant difference was used in the analysis to compare the HBV antibodies’ titer between the different age groups. Correlation coefficient (r) was used to study the relationship between the titer and ages of the children. P value less than 0.05 was considered significant.

Back to Top | Article Outline

Results

The sample studied included 189 children 2–16 years of age divided into a preschool-age group (2 to <6 years, 21 children) and school-age group (6–16 years of age, 168 children). Both groups were analyzed according to the level of HBsAb.

The percentages of children with protective HBsAb titer against those with nonprotective titer in both age groups (<6 and >6 years of age) are presented and compared in Fig. 1. The results showed that among the children younger than 6 years of age, 4.8% had a nonprotective level, whereas 95.2% had a protective level of HBV antibodies (≥10 mIU/ml). However, 58.3% of the study sample had a nonprotective level, whereas 41.7% had a protective level in children older than 6 years of age. In other words, the protective level of HBsAb was 95.2% in children younger than 6 years of age in contrast to 41.7% in children older than 6 years of age. The χ 2-test was used to test the significance of these differences, which indicated a result of 21.5 and a P value of less than 0.0001. These findings indicated that the HBsAb level was significantly higher in younger children than in older ones.

Fig. 1

Fig. 1

Both study groups (<6 and >6 years of age) were further classified according to the level of HBV antibodies (Table 1). The likelihood ratio was applied, which was 51.9 with a P value less than 0.0001. This result indicated a highly significant difference between the levels of HBsAb in children younger than 6 years when compared with those older than 6 years of age.

Table 1

Table 1

The study sample older than 6 years of age was further classified according into five subgroups (6 to <8 years, 31 children; second group: 8 to <10 years, 31 children; third group, 10 to <12 years, 36 children; fourth group, 12 to <14 years, 33 children; and fifth group, 14–16 years, 37 children). Age subgroups were analyzed according to the level of HBsAb and were compared with the level of children younger than 6 years of age using the χ 2-test (Fig. 2). The results showed a highly significant difference by age (χ 2=40.3, P<0.0001). There was a significantly higher percentage of protective HBsAb titer in the children younger than 6 years of age compared with the other age groups. In addition, the decreasing protective percentage was evident among children older than 6 years of age, which means that as the child grows, the lower the protective ability of the vaccine.

Fig. 2

Fig. 2

The different groups were compared according to the level of HBsAb and the likelihood ratio was calculated with a result of 87.26 and P less than 0.0001 (Table 2). The titer of HBsAb was significantly higher in children younger than 8 years compared with all the other children and in children younger than 6 years compared with those 6–8 years of age.

Table 2

Table 2

The correlation test (r) between the HBsAb titer and age of children younger than 6 years of age (Fig. 3) showed almost no correlation (r=0.1, P>0.05), whereas in children older than 6 years of age (Fig. 4), there was a significant negative correlation (r=−0.2, P<0.005).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Back to Top | Article Outline

Discussion

HBV infection and its complications are currently a major issue in medicine worldwide. Children are one of the at-risk populations for HBV infection. The inclusion of hepatitis B vaccine in national infant immunization programs could prevent more than 80% of HBV-related deaths 15. Neonatal HBV vaccination is the best effective measure for the prevention of HBV infection in countries with an intermediate to high level of HBV endemicity 16. As recommended by the WHO, all infants should receive the hepatitis B vaccine. The program of universal immunization of infants against hepatitis B infection started in Egypt in 1992. The schedule adopted by the Egyptian Ministry of Health was three doses of yeast-recombinant hepatitis B vaccine administered to all infants at 2, 4, and 6 months of age to coincide with other compulsory vaccines 17. The protective cutoff level was set at at least 10 mIU/ml anti-HBs on the basis of vaccine efficacy studies 18.

Vaccinated children against hepatitis B may show serological evidence of breakthrough infections particularly if the level of HBsAb induced by the vaccine is low 8,19,20 or because of waning of immunity to the HBV vaccine 20. A study at Ismalia reported that more than 17% of vaccinated children were unprotected serologically 12–18 months after the vaccination cycle 21. A small proportion of healthy children fail to develop antibodies against hepatitis B after three doses of vaccine 22, whereas another proportion of children are at a risk of a poor response 23. A distinction between true nonresponders (after adequate immunization) and waning of the HBsAb level is important, the latter of which is not uncommon among populations in areas of the world with low endemicity of HBV infection 24. It has been reported that many host and immunization factors affect the immune response and duration of immunity. Some of these host factors include age, weight, immunocompetence of host, genetics, and socioeconomic state 25. Among a variety of genetic and environmental factors implicated in immune responsiveness to a nominal antigen, the human leukocyte antigen complex deserves special consideration. Human leukocyte antigen may regulate the immune response to HBsAg by at least two different mechanisms. The lack of immune response could either be a defect in antigen presentation or a defect in the T-cell repertoire. Evidence for both propositions has been reported to explain the unresponsiveness to HBsAg vaccination in humans 26.

In the current study, 189 healthy children were included over a period of 1 year. The sample studied was classified into a preschool-age group 1.5–5.5 years after the end of the vaccination cycle (2 to <6 years), 21 children, and a school-age group 5–15.5 years after the end of the vaccination cycle (6–16 years of age), 168 children, who were further classified into five age subgroups (6 to <8, 8 to <10, 10 to <12, 12 to <14, and 14–16 years of age). In the preschool group, 4.8% (1/21, whose age was 4 years) had a 0 titer, who were either nonresponders or hyporesponders, whereas 95.2% (20/21) had a protective titer of HBsAb (Fig. 1), two of whom were around 2 years of age with a weak protective titer 50 mIU/ml, so-called hyporesponders, and the remaining (17/21) had an adequate protective titer of at least 100 mIU/ml (Table 1). This was very similar to an Egyptian study carried out to detect the efficacy of vaccination through the presence of HBsAb among 180 infants and children for whom time lapse since the last vaccination varied between 1 month and 5 years; it was found that although a high seroprotection rate (93.3%) was elicited 1 month after vaccination, there were low initial HBsAb concentrations and both decreased rapidly over time 27. In the school-age group, 58.3% lacked a protective titer, and almost half of them (24.4%) had a 0 titer (Table 1); thus, only 41.7% (70/168) had a protective titer of HBsAb. Our results were almost in agreement with other studies carried out in Egypt in the last few years. Two studies obtained very similar results; in one study carried out in 2003/2004 on children between the age of 6 and 12 years, 39.4% of the children included had a protective HBsAb titer 7 and in the other study carried out on children between 6 and 11 years of age, 39.3% of the children studied had a protective HBsAb titer 11. The previous studies, in addition to other studies in Alaska 28, Yemen 29, and Iran 30,31, recommended a booster vaccination dose for HBV to enhance the immune response.

The present study showed a significant negative correlation between the HBsAb titer and ages of the school-age subgroups studied as shown in Fig. 4. The school-age group (168 children) was classified into five subgroups as a trial to pinpoint the age at which the severe decrease in protective HBsAb titer could occur, thus leading to an increased risk of acquiring a hepatitis B infection with all its health risks, besides being a source of infection in the community. This was not attempted in any previous study on Egyptian children. The five subgroups included (as shown in Fig. 2) children (6 to <8, 8 to <10, 10 to <12, 12 to <14, and 14–16 years of age), who showed a marked decrease in the protective antibody titer (61.3, 58.1, 44.4, 15.2, and 32.4%, respectively), with a significant P value less than 0.001, and in those who maintained an adequate protective titer as shown in Table 2 (32.2, 16.1, 11.1, 6.1, and 13.5%, respectively). The result of our study was in agreement with other studies 28; Dentinger et al. 28 detected a reduction in HBsAb from 6% after 5 years of vaccination to 3% after 10 years. Another more recent study found that 81% of children had a protective titer among 100 children at 6 years of age – which seems higher than the percentage reported by other Egyptian studies – whereas among 100 children 11 years of age, only 48% had a protective titer 14. A negative correlation did not exist between HBsAb titer and ages in the preschool-age group as shown in Fig. 3 by an insignificant P value (>0.005).

The decrease in the percentage of children with a protective HBsAb titer from 95.2% in the preschool-age group to 41.7% in the school-age group (Fig. 1) enabled us to support the recommendation of other studies to enhance the immune response by a booster dose 7,13,21,32. This finding supports other studies which recommend administration of a booster dose at school-age timing, coinciding with other compulsory vaccines at that age 11,14,20,31,33. Yet, it needs to be emphasized that 8 years is a critical age as only 16.1% had an adequate protective titer whereas 42% had a weak protective titer, indicating the waning effect of the latter; in addition, 41.9% lacked the protective antibody titer at that age. Thus, a large percentage of children are at risk of developing an infection, with its attendant health risks, and its impact on the community. The only explanation for the increase in the percentage of children with a protective HBsAb titer in the 14–16 years subgroup is that they might have acquired the infection subclinically, which enhanced their immune response. This could have been proved by detection of hepatitis b core IgG (HBcIgG) as it is the only way to distinguish between HBsAb as a result of vaccination or acquisition of an infection, whether clinically or subclinically.

The failure to achieve satisfactory seroprotection levels reflects the need to carry out a larger scale study with narrow age subgroups in different Egyptian governorates and in different communities taking into consideration the different socioeconomic levels in order to better evaluate the need for changes in the HBV vaccination strategy in Egypt in view of the current findings. It should also be determined whether this low seroprotection level is because of waning immunity with time or because of an initial low response. Detection of HBsAb titer should also be performed along with detection of HBsAg in all children with 0 titer to differentiate whether they were infected earlier and became chronic patients or whether they are just hyporesponders or nonresponders to the vaccination cycle and may thus benefit from a booster dose. The efficiency of the hepatitis b vaccination can also be evaluated by detecting the HBcIgG marker in all children who show a protective HBsAb titer as it is the only marker to distinguish between those who have an HBsAb titer because of a compulsory vaccination schedule and those who might have acquired the infection by any means earlier in life because of lack of a protective titer of HBsAb.

Table

Table

Back to Top | Article Outline

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Jabber SM.Prevalence of anti-hepatitis B and anti-hepatitis A among school aged children in Western Saudi Arabia.Saudi Med J2006;27:1515–1522.
2. Poovorawan Y, Chatchatee P, Chongsrisawat V.Epidemiology and prophylaxis of viral hepatitis: a global perspective.J Gastroentrol Hepatol2002;17:S155–S166.
3. Poland GA, Jacobson RM.Prevention of hepatitis B with the hepatitis B vaccine.N Engl J Med2004;351:2832–2838.
4. Reda AA, Arafa MA, Youssry AA, Wandan EH, Ab de Ati M, Daebees H.Epidemiologic evaluation of the immunity against hepatitis B in Alexandria, Egypt.Eur J Epidemiol2003;18:1007–1011.
5. Lu CY, Chiang BL, Chi WK, Chang MH, Ni YH, Hsu HM, et al..Waning immunity to plasma-derived hepatitis B vaccine and the need for boosters 15 years after neonatal vaccination.Hepatology2004;40:1415–1420.
6. Puvacić S, Curić I, Puvacić Z, Ravlija J, Mulaomerović M.Strategy for administering hepatitis B vaccine to newborns in Bosnia Herzegovina.Med Arh2004;58:7–10.
7. Shaaban FA, Hassanin AI, Samy SM, Salama SI, Said ZN.Long term immunity to hepatitis B among a sample of fully vaccinated children in Cairo Egypt.East Mediterr Health J2007;13:750–757.
8. Yu AS, Cheung RC, Keeffe EB.Hepatitis B vaccines.Clin Liver Dis2004;8:283–300.
9. Simo MJ, Gaztambide GM, Femandez MP, Pena FM.Hepatitis B vaccine immunoresponsiveness in adolescents: a revaccination proposal after primary vaccination.Vaccine1996;14:103–106.
10. Abdul Ahad Md, Abdul Alim Md, Guho A, Islam QT, Azad KA.Role of booster dose on antibody titre after recombinant hepatitis B vaccination.J Med2009;10:67–76.
11. Afifi SS, Mahran MH, Said ZN, Salama II, El-Khayat H.Serum level of anti-hepatitis B surface antigen among newborns and fully vaccinated infants and children aged 6 to 11 years.Aust J Basic Appl Sci2009;3:3239–3245.
12. Mokhles M, El Ashry R, Sedky M, Emara N, Rasheed M, El-Wassef M, et al..Long term efficacy of hepatitis B vaccine among high risk multiple blood transfusion children in Egypt.J Appl Sci Res2009;5:2504–2510.
13. Khashba A, El-Defrawy M, Abdel Motaleb G, El-Shafaey M, Rashad G.Study of the immune response of hepatitis B vaccination in Egyptian pre-school children.Benha Med J2005;22:515.
14. El-Sayed B, El-Guindi M, El-Shaarawy A, Salama EI, Sobhy GA.Long-term protection of hepatitis B vaccination among Egyptian children.Egypt J Pediatr Allergy Immunol2011;9:35–40.
15. Goldstein ST, Zhou F, Hadler SC, Bell BP, Mast EE, Margolis HS.A mathematical model to estimate global hepatitis B disease burden and vaccination impact.Int J Epidemiol2005;34:1329–1339.
16. Saffar MJ, Rezai MS.Long-term antibody response and immunologic memory in children immunized with hepatitis B vaccine at birth.Indian Pediatr2004;41:1232–1237.
17. Mansour E, Abdul-Rahim S, Batouty G, Zaghloul I, Abdel-Hadi S.Integration of hepatitis B immunization in the Expanded Program on Immunization of the Child Survival Project.J Egypt Public Health Assoc1993;68:487–494.
18. Jack AD, Hall AJ, Maine N, Mendy M, Whittle HC.What level of hepatitis B antibody is protective?J Infect Dis1999;179:489–492.
19. Banatvala J, Van Damme P, Oehen S.Lifelong protection against hepatitis B: the role of vaccine immunogenicity in immune memory.Vaccine2001;19:877–885.
20. Abu shady EA, Fahmy GM, Abdel-Ghany SA, Abdel-Azim EM.Evaluation of hepatitis B vaccination among Egyptian children and genotyping of hepatitis-B by multiplex polymerase chain reaction.Int J Virol2005;1:55.
21. El-Ghandour S, El-Sayed H, Abel-hamid A, Gad S.Effectiveness of hepatitis B vaccination in Egyptian infants in Ismalia Governorate.Suez Canal Univ Med J1998;1:123–130.
22. Duval B, boulianne N, De Serres G, De Wals P, Massé R, Trudeau G.preadolescent non-and hyporesponders following three doses of hepatitis B vaccine need only one more dose.Vaccine2002;20:3632–3634.
23. Adwan k, Abu-Hassan N, Adwan G, Abu-Khater K.Hepatitis B surface antibody response of household contacts of hepatitis B virus carriers in Palestine.East Mediterr Health J2005;11:494–498.
24. Sjogren MH.Prevention of hepatitis B in non-responders to initial hepatitis B virus vaccination.Am J Med2005;118:34S–39S.
25. Tsega E, Horton J, Nordenfelt E, Hansson BJ, Tafesse B, Wolde-Hawariat G, et al..Antibody levels in Ethiopian children five years after vaccination with two different doses of hepatitis B vaccine: is there a need for booster vaccination?Can J Gastroenterol1998;12:57–60.
26. Amirzargar AA, Mohseni N, Shokrgozar MA, Arjang Z, Ahmadi N, Behzadi MY, et al..HLA DRB1, DQA1 and DQB1 alleles and haplotypes frequencies in Iranian healthy adult responders and non-responders to recombinant hepatitis B vaccine.Iran J Immunol2008;5:92–99.
27. El-Sawy IH, Mohamed ON.Long term immunogenicity and efficacy of recombinant hepatitis B vaccine in Egyptian children.East Mediterr Health J1999;5:922–932.
28. Dentinger CM, McMahon BJ, Butler JC, Dunaway CE, Zanis CL, Bulkow LR, et al..Persistence of antibody to hepatitis B and protection from disease among Alaska natives immunized at birth.Pediatr Infect Dis J2005;24:786–792.
29. Sallam TA, Alghshm HM, Ablohom AA, Alarosi MS, Almotawakel RE, Farea NH, Mosleh AA.Immune response to hepatitis B vaccine among children in Yemen.Saudi Med J2005;26:281–284.
30. Jafarzadeh A, Montazerifar SJ.Persistence of anti-HBs antibody and immunological memory in children vaccinated with hepatitis B vaccine at birth.J Ayub Med Coll Abbottabad2006;18:4–9.
31. Kazemi A, Koosha A, Rafizadeh B, Mousavinasab N, Mahram M.Serum level of anti-hepatitis B surface antigen 6-8 years after vaccination at birth.East Mediterr Health J2008;14:960–965.
32. Poovorawan Y, Chongsrisawat V, Theamboonlers A, Bock HL, Leyssen M, Jacquet JM.Persistance of antibodies and immune memory to hepatitis B vaccine 20 years after vaccination in Thailand.Vaccine2010;28:730–736.
33. Petersen KM, Bulkow LR, McMahon BJ, Zanis C, Getty M, Peters H, Parkinson AJ.Duration of hepatitis B immunity in low risk children receiving hepatitis B vaccinations from Birth.Pediatr Infect Dis J2004;23:650–655.
© 2013 Medical Research Journal