Chronic hepatitis B (HB) is a major global healthcare problem 1. Egypt is considered an area of intermediate endemicity of hepatitis B virus (HBV) (2–7%) as reported by the WHO 2. HB vaccine was first introduced in Egypt as a part of the Expanded Program of Immunization (EPI) for infants in 1993 3,4. The WHO 2 recommended that the vaccine be given as either three or four separate doses, as part of the existing routine immunization schedules. Seroprotection after vaccination, defined as HBsAb of at least 10 IU/l, is achieved in over 95% of all vaccinated children 5.
The duration of protection provided by HB vaccination is unknown and the need for a challenge dose after complete series of vaccination is an issue of debate. The presence of immune memory can be evaluated by measuring the anamnestic response to a challenge dose of vaccine 6. Therefore, one way of indirectly measuring the long-term immunity is measuring the response to a challenge dose in individuals with reduced or absent anti-HBs over time 7–9.
The aim of the current study was to assess the long-term effectiveness of HBV vaccine and the need for a booster dose among fully vaccinated children during infancy in Gharbeya Governorate.
Patients and methods
The present study is a part of a national community-based multistage cluster sampling design conducted during the period from July 2010 to June 2013 in six governorates, representing all geographic regions of Egypt. For the sampling process and selecting the clusters, probability proportional to size sampling was used. The sample frame for the current survey was on the basis of the most recent population census 2006. According to the population size of each governorate, number of participating clusters was identified in each governorate. First, implicit stratification was carried out by geographic location in each governorate and lists of cities and villages were arranged in serpentine order geographically. This stratification was carried out independently for urban and rural areas. A sampling interval was calculated and accordingly a random number was selected using a table of random numbers. From these lists, areas such as villages or city blocks were selected. In each selected area, lists of maternal and child health centers (MCH), kindergarten, and school facilities were identified and five facility clusters were randomly selected.
The current study presents the part of the project results concerning Gharbeya Governorate located in the middle region of the Nile Delta in Lower Egypt. Project results concerning a total of 762 children aged 9 months to 16 years who participated in Gharbeya were analyzed and presented. From this governorate, four cluster areas were randomly chosen: one urban area represented by Tanta city and three rural areas – Meet-Habeeb village in Samanood, Damatt village in Kotour, and Qurasheya village in Elsanta. From each cluster area, five facilities were randomly selected: one MCH or health unit, one kindergarten, and three schools (primary, preparatory, and secondary schools).
Approval was taken from the ethical committee of Ministry of Health and National Research Center and from the Ministry of Education. A closed-end questionnaire was designed and tested. For quality assurance, training sessions for supervisors, interviewers, and MOH staff in each governorate were given and peel-off barcode sheets were used. All participating children had received the three compulsory doses of HBV vaccine during infancy. Parents signed a written consent form before the interview. Face-to-face interview was carried out with the parents or caretakers of the children. Adolescents above 10 years were also interviewed after their verbal consent. The questionnaire contained demographic data about child age, sex, date of birth, and socioeconomic variables as well. Data were also collected concerning the child HBV vaccination status, and the available vaccination card was revised for date and dose intervals of HBV vaccine. Socioeconomic status was determined according to Fahmy and El-Sherbiny 10. It depends on education of parents, maternal working status, water source, sewage disposable, electricity, and some modification of family income.
Blood sampling and laboratory analysis
About 3–5 ml blood sample was withdrawn aseptically from each child. Serum samples were emptied into two aliquot and stored at –20°C. HBV markers detection was carried out in the Virology Lab of the Microbiology Department at the Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt. Qualitative determination of serum total HBV core antibody (anti-HBc) and hepatitis B surface antigen (HBsAg) and quantitative detection of serum anti-HBs were carried out using commercially available enzyme-linked immunosorbent assays (ELISA; DiaSorin, Saluggia, Italy) and according to the manufacturer’s instructions. For quality assurance, individuals found to be positive for anti-HBc or HBsAg were reassessed. According to the international standards, anti-HBs level of at least 10 IU/l was considered to be protective against HBV infection 2,11. Undetectable level was defined as anti-HBs level less than 1 IU/l and detectable level when it was 1–9 IU/l 12.
Challenging booster dose
Children with nonprotective levels of residual anti-HBs titers received a challenging booster dose of HBV vaccine (10 μg of monovalent Euvax HB vaccine, intramuscularly in the deltoid muscle). After 3 weeks, a blood sample was withdrawn and postchallenging dose of anti-HBs was quantitatively evaluated to assess early anamnestic response. An anamnestic response was defined as a rise in anti-HBs level to at least 10 IU/l 11. Individuals still showing antibody concentrations less than 10 IU/l were offered an additional complete course of vaccination.
Data analysis and presentation
Data entry was carried out on excel sheet and statistical analysis was performed using SPSS software program version 18.0 (SPSS Inc., Chicago). The geometric mean titer (GMT) was calculated to indicate the central tendency of anti-HBs titers in consideration of the skewed distribution of anti-HBs level. For the calculation of the GMT, children who had an undetectable anti-HBs titer were assigned a nominal serum anti-HBs titer value of 0.05 IU/l 13. The χ2-test was performed for qualitative data and was presented by numbers and percentages. The t-test was performed for comparison between two means and one-way analysis of variance for more than two means. Multivariate logistic analysis was performed to predict the risk factors significantly associated with nonseroprotection and postchallenging dose nonresponders. P value was considered statistically significant when its value was less than 0.05 and considered statistically highly significant when its value was less than 0.01.
The total number of studied children was 762 (327 boys and 435 girls). Their mean age was 9.2±5.4 years. None of the participants had an evidence of chronic HBV or breakthrough infection. Characteristics of the studied children are shown in Table 1. However, 57% of children retained protective levels of anti-HBs; only 1.4% of them had anti-HBs levels of at least 1000 IU/l.
There was a significant negative correlation between the age of the studied children and anti-HBs titer among both girls and boys (correlation coefficient 0.431 and 0.434, respectively, P<0.01). Table 2 shows that the nonseroprotection rate significantly increased with age (it increased from 11.8% at age group <5 years to 72.8% at age >15 years, P<0.0005). The rate was also significantly higher among girls (50.1%) compared with boys (33.6%) (P<0.0005) and in rural areas (45.0%) compared with urban areas (37.0%) (P<0.05).
As shown in Table 3, multivariate logistic analysis revealed that the risk of nonseroprotection increased significantly with increasing age from 5 to less than 10, 10 to less than 15, and at least 15 years compared with younger children less than 5 years, with adjusted odds ratio (AOR) of 3.4, 10.2, and 18.5, respectively. Other significant predictor risk factors were high in girls compared with boys (AOR=1.5) and in children living in Kotour and Elsanta villages compared with those in Tanta city, with AOR of 1.8.
Three weeks after receiving the challenging dose, 243/265 (91.7%) children with nonseroprotective level developed anamnestic response (anti-HBs level≥10 IU/l). Figure 1 shows that the prechallenging dose seroprotection rates decreased significantly with increasing age (from 90.5% at age 1 year to 26.7% at age 16 years) (P<0.05). However, the postchallenging dose anamnestic response rate was 100% at age 1 year and it reached 89% at age 16 years, with no significant difference (P>0.05).
Children with nonseroprotective levels (265 children) were grouped according to their prechallenging dose anti-HBs titer into undetectable (<1 IU/l) and detectable (1.0 to <10.0 IU/l). Table 4 shows that the majority of studied participants (77.7%) developed a good anamnestic response to the challenging dose (anti-HBs>100 IU/l). Good anamnestic response rate was significantly higher (85.0%) among children with detectable initial level of anti-HBs compared with 70.2% among those with undetectable level (P<0.01). Although anamnestic response was much better among younger children, there was no significant difference in the distribution of postchallenging dose anti-HBs levels with respect to age and sex (P>0.05).
Table 5 shows the GMT of prechallenging and postchallenging dose anti-HBs with respect to the level of prechallenging dose anti-HBs among nonseroprotected boys and girls. The differences between prechallenging and postchallenging dose anti-HBs levels were all significant (P<0.001) irrespective of sex and prechallenging dose anti-HBs levels. In addition, girls who had detectable prechallenging dose anti-HBs developed significantly higher postchallenging dose GMT compared with those with undetectable level (P<0.01). Of the children with undetectable prechallenging dose level, boys developed a greater increase in postchallenging dose GMT compared with girls and vice versa with detectable level, with no significant difference between both sexes (P>0.05).
The current study is considered the first study to analyze the status of anti-HBs level among children in Gharbeya Governorate in Lower Egypt. A total of 762 children who had received three doses of HB vaccine during infancy were recruited from urban and rural areas belonging to different socioeconomic backgrounds on a community basis.
None of the participants had an evidence of chronic or breakthrough HBV infection. Similar results were reported among Alaskan children and adolescents 14 and in Iran among children aged 5–7 years 15.
In the current study, 90.5% of participants aged less than or equal to 1 year were HBV seroprotected, and the overall seroprotection rate among the studied children was 57%. This is in agreement with many studies stating that, although about 90–99% of all vaccinated individuals acquire seroprotective anti-HBs concentrations 1–3 months following full HB immunization at infancy, a considerable segment show a decline to low or virtually undetectable titers over time 16–18.
Similarly, in Cairo Governorate, El Sherbini et al.19 found that the seroprotection rate was 54% among vaccinated children aged 6–10 years. These results were also in agreement with many other studies stating that, by 10–15 years after the first immunization, the seroprotection rate decreased by 10–50% 13,17,20,21. Furthermore, other follow-up studies reported even higher nonseroprotection rates (15–90%) 5–15 years after vaccination 22–25.
In the present study, the nonseroprotection rate was significantly higher among girls (50.1%) compared with boys (33.6%) (P<0.0005). Another Egyptian study found that the nonprotective rate of anti-HBs was significantly higher among girls than boys aged more than 6 years, and no statistical difference was found among children less than 6 years 1. However, Yazdanpanah et al.15 found that there were no sex differences among boys and girls aged 5–7 years, 83.8 and 85.2%, respectively. This variability in results may be due to other confounding factors such as age and socioeconomic or nutritional status.
The current study revealed that the nonseroprotection rate increases significantly with age, from 11.8% among children less than 5 years to 33.3, 57.5, and 72.8% among those aged from 5 to less than 10 years, 10 to less than 15 years, and at 15 years or above, respectively (P<0.001). This result corroborates with the findings of previous studies that indicate that anti-HBs concentrations decline over time 15,25,26. Nearly similar results were reported by Garcia et al.7 and Behjati et al.27, who reported 29.4 and 31% nonseroprotection rates among children aged 6–7 years after primary vaccination. With respect to older children, Tosun et al.28 reported lower nonseroprotection rate (50%) among children 9 years after vaccination.
The present study showed that children in rural areas had higher nonseroprotection rate (45.1%) compared with those living in urban areas (37.0%) (P=0.05). In Turkey, Tosun et al.28 found that 48.5% of children in semiurban areas and 42.3% in urban areas had antibody titers below the protective levels.
Concerning the seroprotection rate, the current study found it to be higher among high socioeconomic level individuals (61.2%) compared with very low class individuals (49.6%) (P<0.05). The same direct relationship was also reported by Guho and colleagues 29,30. This may be correlated with malnutrition prevailing among individuals of low socioeconomic class, with consequent lower immunity 30.
However, the current study revealed that 51.4% of children aged less than 5 years retained good seroprotection rate (anti-HBs≥100 IU/l), whereas Mahyar 31 found that only 29.2% Iranian children at 6 years of age retained this high levels of antibody. Mendy et al.32 reported that, in Gambia, the nonseroprotection rate at age 5, 10, and 15 years was 93.8, 51.2, and 27.0%, respectively, among vaccinated children.
The present study showed that three weeks after receiving the challenging booster dose 91.7% of nonseroprotected children developed anamnestic response (anti-HBs≥10 IU/l) and 77.8% of them reported good response (anti-HBs≥100 IU/l).
Lower rate of anamnestic response was reported by Yazdanpanah et al.15 where only 78.1% of children responded to the booster dose and nearly half of them only had good antibody levels.
However, the current study found that the nonresponder rate to the challenging dose was 8.3%. Samandari et al.14 reported that, in Alaska, only 1% of children aged 5–7 years and 17% of adolescents from 10 to 15 years were nonresponders to the given booster. They attributed this to waning of the immune memory, especially among older age individuals. Jan et al. 9 reported that 20 years after vaccination, the immune system memory is lost in up to 80% of vaccinated children.
The current study showed that the postchallenging dose anamnestic response was significantly higher (94%) among children who had detectable prechallenging dose anti-HBs level (1–9 IU/l) than among those who had undetectable level (89.3%) (P<0.05). Similarly, in Taiwan, Wang and Lin 33 stated that poor response to a booster dose of HB vaccine was significantly higher (47.5%) among adolescents aged 16 years, with undetectable prebooster dose anti-HBs level compared with 97.2% among those with detectable level.
The anamnestic response rate to the booster dose did not vary between boys and girls in the current study (90.1 and 92.5%, respectively) (P>0.05). Of the boys and girls with undetectable prebooster anti-HBs level, boys developed a greater increase in post-GMT compared with girls and vice versa with detectable level, with no significant difference between both sexes (P>0.05). This is in agreement with the study by Wang and Lin 33 who found no significant difference between boys and girls. However, Su et al.13 found that, among adolescents with the same prebooster anti-HBs level, female individuals always had a greater anamnestic response (P<0.001).
The current study found that the strength of the immune response to the challenging booster dose decreased with longer time interval since vaccination, that is younger ages respond better (100% among those younger than 5 years, 83.3% in those from 5 to 10 years, and 74% in those older than 10 years). High response observed after the booster dose even among older age groups suggests the persistence of an excellent immune memory. Similarly, Samandari et al.14 reported that almost all children and 80% adolescents responded to a booster dose. Chaves et al.25 stated that nearly 20–30% of adolescent participants are unable to mount an immune response after booster dose. However, Gilca et al.34, in Canada, reported higher anamnestic response rate (99.1–100%) when the booster was administered 5–15 years after vaccination.
From the current study, it can be concluded that long-term immunity exists among children who had complete series of HB vaccination in infancy even in case of reduced or absent anti-HBs over time. HBV vaccine is effective in preventing HBV infection in the studied areas in Gharbeya Governorate. The developed high postchallenging dose anamnestic response rates signify the presence of immune memory and indicate that there is no need to offer a booster dose of HBV vaccine at least for the studied age.
This study was funded by the Science and Technology Development Fund (STDF), Egypt, Grant No. 1611. The authors thank Ministry of Health represented by Dr Nasr El-Sayed, Dr Amr Kandil, Dr Sahar El Shorbagi and other facility members in Gharbeya Governorate for their great cooperation through facilitating and preparing the field work. They also thank all members of the research team for their efforts to conduct the project as well as the children and their parents.
Conflicts of interest
There are no conflicts of interest.
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