Evaluation of the Protection Provided by Hepatitis B Vaccination in India.

Indian J Pediatr. 2018 Jan 10. doi: 10.1007/s12098-017-2601-0. http://rdcu.be/EpEZ

Puliyel J, Naik P, Puliyel A, Agarwal K, Lal V, Kansal N, Nandan D, Tripathi V, Tyagi P, Singh SK, Srivastava R, Sharma U, Sreenivas V.

Evaluation of the Protection Provided by Hepatitis B Vaccination in India
In India Hepatitis B vaccination is recommended at 6 weeks except for hospital-deliveries. We examined protection afforded by the birth dose.
Method A case-control study was done. HBsAg and HBcAb were tested in 2671 children 1 to 5 years and HBsAb was evaluated in a subset of 1413 children. Vaccination history was recorded. Cases were HBsAg carriers. In another analysis children who got infected (HBsAg and/or HBcAb positive) were considered as cases. Exposed were the unvaccinated. In another analysis exposed were those vaccinated without the birth dose.
Results The odds ratio (OR) for HBsAg positivity with birth vaccination was 0.35 (95% CI 0.19 – 0.66); while with vaccination at 6 weeks was 0.29 (95%CI 0.14 – 0.61) both compared to unvaccinated. Birth vaccination has no added protection when compared to the unvaccinated. Unvaccinated children in our study had HBsAg positivity of 4.38%. The NNT to prevent one case of HBsAg positivity was 32.6 (95% CI, 20.9 to 73.6).
The odds of getting HBV infection was 0.42 (CI 0.25-0.68) with birth dose and 0.49 (CI 0.30-0.82) without the birth dose compared to the unvaccinated.
Protective antibody (HBsAb) was present in about 70% of the vaccinated. In the unimmunised, in the first 2 years HBsAb protection was present in 40%. OR for HBsAb in the fully vaccinated between 4 and 5 years was 1.4 (95%CI 0.9-2.18) compared to the unvaccinated.
Conclusions The present study lends support to the pragmatic approach of the Government to vaccinate babies born at home starting at 6 weeks. Word Count 249
Key Words: Immunization, passive immunity, Hepatitis B surface antigen, Hepatitis B core antigen, Antibody to hepatitis B surface antigen, Antibody to hepatitis B core antigen.

Evaluation of the Protection Provided by Hepatitis B Vaccination in India
Hepatitis B virus (HBV) can cause chronic hepatitis, liver cirrhosis and lead to hepato-cellular carcinoma (HCC) in susceptible persons. A systematic review and meta-analysis done by Batham et al. of prevalence of Hepatitis B in India showed that in non-tribal populations the prevalence was 2.4% (95% CI: 2.2%-2.7%) and among tribal populations it was 15.9 % (CI: 11.4%-20.4%) (1). Assuming that 4% are chronic carriers in India and extrapolating trends from Taiwan it was estimated that 250,000 persons in India die of HCC each year (2). However, projections from the ICMR Cancer Registry shows the incidence of HCC due to hepatitis B infection in India is about 5000 cases a year (3) accounting for 0.02% of the annual mortality of approximately 25 million. This is much lower than expected.
The risk of developing chronic HBV infection is inversely related to age of acquisition. The risk of chronic infection is 90% in children younger than 1 year; 30% for those 1-5 years and 2% for adults (4). Mother-to-infant transmission is an important factor responsible for chronic HBV infections (5). This form of perinatal transmission of HBV is believed to be responsible for a third of adult chronic carriers in India (6). Vaccination at birth is done to prevent mother to child transmission and horizontal transmission from infected children to others (7). Vaccination starting at 6 weeks is thought to prevent only horizontal transmission (8).
A large percentage of births in India take place at home away from health-care institutions and so newborns cannot be reached easily, immediately after birth. For this reason the draft National Immunization Policy recommends a pragmatic HBV vaccination schedule wherein the vaccine is given at birth for institutional deliveries and at 6 weeks to children born outside such health-care settings (9).
There is no clear data on how much of the HBV chronic carrier state is prevented by the two schedules. According to a systematic review by the Indian Medical Association there are no studies showing that the ‘pragmatic schedule’ was effective in reducing the chronic carrier rate in any country (10).
This study was undertaken to examine the efficacy of hepatitis B vaccination in those who were vaccinated at birth (within 48 hours of delivery) and those who were vaccinated thereafter, using a case-control model. The secondary objective of the study was to look at hepatitis b surface antibody (HBsAb) levels in the vaccinated for serological response in the two schedules and among the unvaccinated as a marker of natural immunity.
Material and methods
Data collection was done from November 2013 to July 2015. Children between the ages 1 and 5 years were invited to participate in the study if they were having blood taken for other investigations at the hospital or laboratory. Consent was taken from parents to collect an additional 2 ml of blood for Hepatitis B testing. Samples were obtained from participating centres in Delhi, Rajasthan, Uttar Pradesh, Uttarakhand, and Gujarat. The study protocol was approved by the Institute Ethics Committee of St Stephens Hospital, Institutional Ethics Committee of Dr Ram Manohar Lohia Hospital Delhi, and the Emmanuel Health Association Research Committee for centres without their own ethics committees. The study was funded by an ICMR grant which also covered costs for serological testing of the samples (Reference PCR/Virology/20-12/ECD-1)
Immunization status was inferred from patient held immunization cards, supplemented by history when required. Samples were collected in serum gel 4 ml vaccutainers, centrifuged and the serum was stored at minus 20° centigrade till testing. All the samples were tested for hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (HBcAb). A subset was also tested for antibody to surface antigen (HBsAb). All the serological tests were done using ELISA method of immunoassay. GS HBsAg EIA 3.0, MONOLISA™ Anti-HBc EIA and MONOLISA™ Anti-HBs EIA by Bio-Rad© Laboratories was used for serological testing. The titre cut off used for HBsAb was 10 IU/L, and for HBsAg and HBcAb it was 1 IU/l, as specified by the manufacturers of the testing kits. All the tests are being done at Dr Lal PathLabs -Rohini (National Reference Laboratory).

Record collection was though a custom built web-application hosted on large cloud-services vendor.  An append-only data-structure (CouchDB) protected against inadvertent data-loss and allowed us to ensure that individuals involved in reporting serology were blind to the immunization status of the children. Once a child's test results were available, the application parsed and extracted the results of each test directly from the lab's PDF report - avoiding any human-introduced transcription errors. The application, on demand, could produce a table-based, flattened view of the underlying (non-relational) database for Microsoft Excel. This extract was used for analysis at the conclusion of the study. That data, anonymized and garnished with slicers for easy analysis, is made available free online, for further analysis by interested researchers (http://bit.ly/hepbdata).

HBsAg positive children were considered as carriers. Those who got infection and cleared it having developed HBcAb and those who became carriers were considered together (HBcAb and/or HBsAg) as  children who had got infected with hepatitis B. Completely immunized children were those who received at least 3 doses of hepatitis B vaccine, without regard for the birth dose. Birth dose was defined as hepatitis B vaccination within 48 hours of delivery. Unvaccinated were those who received no immunization for hepatitis B. The two groups (children who got infected [HBcAb and/or HBsAg]and children who became carriers [HBsAg]) were each analysed as cases in two different analyses. Controls were the remaining children in the two analyses. Exposure was vaccinated status. Those who had received 3 doses of the vaccine were considered protected (unexposed). In another analysis only those vaccinated from birth were considered protected. For the secondary outcome we examined HBsAb in the fully vaccinated starting at birth, those fully vaccinated but not starting at birth and in the unvaccinated. HBsAb was also analysed by age to look for waning immunity among the vaccinated and in the unvaccinated.
Sample Size Calculations
It was calculated that 94 cases needed to be recruited for this study. Assuming that the prevalence of Hepatitis B virus infection in unvaccinated children was 1% and assuming that it is 0.5% in those vaccinated at 6 weeks, in a two sided test with 80% power, it was calculated that 4700 children each in the vaccinated at birth and vaccinated after 6 weeks groups would need to be recruited. Those vaccinated at birth were assumed to have a 90% chance of being protected against hepatitis B infection.
Statistical Analysis
Statistical analysis was done using MedCalc Statistical Software version 14.8.1 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2014). Fisher’s exact test and Pearson chi square test were used to compare proportion of children in the subgroups of interest. Chi square test for trend was used to assess age-wise positivity of HBsAb. Number needed to treat (NNT) was also calculated.
The prevalence of Hepatitis B infection was higher than projected for sample size calculations. The study was concluded after 2671 children were recruited. In the case control model we had recruited 116 cases of children infected with hepatitis B (defined by presence of either HBcAb and/or HBsAg) and of these 69 were HBsAg positive chronic carriers. Table 1 gives the population characteristics. 1566 (58.63%) children had received complete vaccination whereas 844 (31.60%) were completely unvaccinated. The remaining was partially immunized. Of the study population 880 (32.95%) was fully immunised starting at birth and 686 (25.68%) were fully immunised but without the birth dose. 1413 were also tested for HBsAb out of whom 858 (60.72%) had protective levels.

Table 2 shows results of testing in those completely vaccinated with and without the birth dose. Odds of becoming HBsAg positive was 1.22 (95%CI 0.52-2.83) without birth dose compared to with birth dose. Protection rates (HBsAb) was also very similar (70.3 and 69.5% respectively). The study was however not powered for a head to head comparison between these two groups.
When compared to the unvaccinated the odds of being an HBsAg carrier was 0.35 (95% CI 0.19-0.66) for those vaccinated at birth compared to 0.29 (95% CI 0.14-0.61) without the birth dose. Birth vaccination has no added protection compared to the unvaccinated. The odds of getting infected was 0.42 (95% CI 0. 26-0.68) and 0.49 (95% CI 0.30-0.82) and the odds of being HBsAb positive was also similar (Tables 3 & 4)
Table 5 shows the comparison between those fully vaccinated (3 doses irrespective of whether birth dose was received or not) and the unvaccinated. Protective antibodies (HBsAb ) were present in 69.9% of the fully vaccinated and in 39.5% of the unimmunised. Table 6 shows HBsAb positivity (protective antibodies) increases with increasing doses of vaccination. 52.73% of children who received single dose of Hepatitis B vaccine had protective levels of HBsAb compared to 84% with 4 doses. The trend was statistically significant (p < 0.0001).
Table 7 shows antibody titres in the unvaccinated with age. The highest levels were in the youngest tested and the lowest levels were in the oldest group. 45% of the unvaccinated were naturally immune at 1 year of age. Between 4 and 5 years the odds of being protected with HBsAb was1.4 (95%CI 0.9-2.18) in the vaccinated compared to the unvaccinated.
Prevention of Chronic Carriers
The purpose of this study was to look primarily for the benefits if any, of giving the first dose of Hepatitis B vaccination at birth in India. The findings lend support for the pragmatic approach of the Government of India to vaccinate babies born at home at 6 weeks. The carrier rate was not improved with birth dose.
The OR for HBsAg positivity with birth vaccination was 0.35 (95%CI 0.17 – 0.67); while with vaccination at 6 weeks was 0.29 (95%CI 0.12 – 0.62) both compared to unvaccinated. This empirical evidence contradicts what the principle investigator and others have assumed about the need for the birth dose in India (11-14). Unvaccinated children in our study had HBsAg positivity of 4.38%. The numbers needed to treat to prevent 1 chronic carrier (NNT) was 32.6 (95% CI, 20.9-73.6). The study done by Aggarwal et al. in children 5 to 10 years of age, found HBsAg positivity of 0.15% in immunized children and 0.17% in the unimmunized (p value 0.855) (15). We found HBsAg positivity 1.47% in the vaccinated between 1 and 5 years and 4.38% in the unvaccinated. These differences probably relate to the differences in the populations studied.

Prevention of Hepatitis B Infection
The odds of getting infected (HBsAg and/or HBcAb) was similar with and without birth dose when they were compared to the unvaccinated. 6.28% of those unvaccinated had been infected compared to 2.72% vaccinated with birth dose and 3.21 without birth dose. NNT for preventing Hepatitis B infection was 28.2 (95% CI 18.2 to 62.2) with complete immunized including with birth dose and 32.5 (95% CI, 19.1 to 110.8) without birth dose.
A priori, sample size calculations were not performed to look for differences among the fully vaccinated, with and without the birth dose. The sample we studied had 880 children who were completely vaccinated starting at birth dose. 686 children were fully vaccinated but without the birth dose. Given the very small difference of hepatitis B infection between groups (2.72% and 3.21% respectively) it appears (on retrospective analysis) that the study is not powered to look for such small differences.
However according to INSAL 4.7% of the population in India are carriers (16). This is not very much higher than the HBsAg positive rate of 4.38 we found among unvaccinated children in our study. Nayak et al report that one third of HBV carriers evolve from perinatal transmission (17). This can be prevented by the birth dose. If we assume that 30% of the carrier rate of 4.7% is due to vertical transmission from mother to child (that will be prevented by vaccination starting at birth), then those without birth dose will have 1.7% carrier rate. Assuming 100% efficacy against vertical transmission with the birth dose, the carrier rate due to vertical transmission will be zero with full vaccination starting from birth.  
 For type 1 error of  0.05 and power of 80%  a sample size of 569 in each group (fully vaccinated starting at birth versus no protection against vertical spread at birth) is adequate. The sample size we studied (880 fully vaccinated from birth and 686 fully vaccinated without birth dose) is adequate if assumptions are made based on these previous reports.

This was not a randomised control trial. Unvaccinated children usually belong to a different social class whose parents may be less literate, poorer and therefore more likely to be exposed to unsterile needles and other sources of infection. The lower infection rate seen in this study among the vaccinated could be related both to the fact that immunization is protective and also to the fact that those immunised were less likely to get infected in the first place.

Protective HBsAb
Protective antibody HBsAb was present in about 70% of those fully immunised and was not significantly improved by providing the birth dose. The protective levels waned rapidly from 82% in the first year to 47% by age 5 years.
Protective levels of HBsAb were present in 39.46% of completely unvaccinated. The numbers with protective levels of antibody in the unvaccinated was higher in the early years of life and gradually declined with advancing age. Protection in the second year was 44.9% and at age 5 it was 28.9%. Aggarwal et al who studied children older than 5 years this antibody was 28% of unimmunised at 6 year of age and in 16% at the age of 10 (15). The protective HBsAb in unvaccinated babies could be active immunity developed in these babies after getting infected. However this seems unlikely because most of them were HBcAb negative. Also, if this were active immunity after exposure to natural infections, the numbers are likely to increase with age with more opportunities for exposure. This is not what was seen in this study nor was it the case in the study by Aggarwal 15).

The large numbers of unvaccinated children with protective levels of antibodies (HBsAb) observed in our study and the study by Aggarwal could reflect natural, passively acquired immunity from their mothers which wanes slowly over the years. Even so, the persistence of this passively acquired antibody beyond the age of 9 months is surprising.
There are very few studies that have looked at HBsAb levels in unvaccinated mothers who could transmit it passively to their babies. In a study in Arunachal Pradesh, 40.0% of the unvaccinated had HBsAb above 10 mIU/ml (18). Asim studied HBsAg negative, first-time blood-donors and found 20% had previous natural infection and had developed HBcAb (19). Of these 62% had protective levels of HBsAb levels above 10 IU/L and of them 68% had very high HBsAb levels between 100 and 500IU/L. Studies by Waaijenborgand colleagues looking at measles immunity have shown that the unimmunised population of mothers have higher titres of antibodies and they pass these higher titres to their babies and the duration of the passive protection to the babies is dependent on the antibody titres in the mothers (20). Mothers in highly immunized communities have lower antibody levels as vaccine induces lower antibody levels than does natural infection and the antibody levels of vaccinated cohorts are no longer boosted by exposure to wild-type infection.
The fact that a good number of unvaccinated babies had high levels of antibody, suggests it could be protecting some babies early in life, at the time when they are vulnerable to developing chronic hepatitis (4)
Among the vaccinated to protection decreased with age and the odds of having protective antibodies in children over 4 years was 1.4 (95%CI 0.9-2.18) compared to the unvaccinated children.

This study lends support for the Government of India programme of vaccinating babies born outside of institutional health care facilities at 6 weeks. Those vaccinated starting at 6 weeks had the same benefits as those vaccinated from birth, when both were compared to the unvaccinated.

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