Hepatitis B vaccine in the EPI schedule
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《美国医学杂志》
1 Department of Pediatrics, Maulana Azad Medical College, New Delhi, India
2 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
Abstract
Objective: This study was carried out to evaluate the adequacy of seroconversion when Hepatitis B vaccine is given along with other vaccines at 0, 6 weeks (along with DPT and OPV) and at 9 months (along with measles). Methods: 725 infants born to apparently healthy mothers were enrolled to receive the hepatitis B vaccine at 0, 6 weeks and 9 months (Group A) or at 0, 1 and 6 months as per WHO schedule - (Group B). Baseline HbsAg testing was carried out and the babies were immunized with the first dose of hepatitis B vaccine within 48 hours of birth. BCG and the other EPI vaccines were given as per schedule. Serum samples were collected 4 weeks after the second and the third immunizations. 604 infants (83.3%) completed the study. The testing for HbsAg and Anti Hbs titers were conducted in the Department of Microbiology, Maulana Azad Medical College, New Delhi utilizing standard ELISA kits. Results: The seroconversion rates 4 weeks after the second dose of the vaccine were 90.89% (GMT = 48.23) and 91.82% (GMT = 43.95) (P=0.8) in Group A and Group B respectively. After 4 weeks of the third dose the seroconversion rates were 98.99 (GMT = 161.12) and 98.45 (GMT = 150.12) (p=0.17) in Group A and Group B respectively. The two schedules were comparable on using the Kruskal-Wallis H method for analysis. Conclusion: The schedule of hepatitis B vaccination at 0, 6 weeks and 9 months has the same seroefficacy as the currently recommended schedule of 0, 1 and 6 months.
Keywords: Hepatitis B vaccines; HbsAg mothers; EPI Vaccines; Seroconversion
Hepatitis B is endemic throughout the world, especially in tropical and developing countries. The hepatitis B carriage rates in India range from 3 to 5 percent. In endemic populations the carrier rate is high and perinatal transmission is a common event.[1],[2],[3],[4],[5] The World Health Organization recommends immunization of all children with three doses of 10 micrograms each of hepatitis B vaccine given intramuscularly at 0, 1, 6 months of age.[6],[7],[8],[9],[10] This schedule does not coincide with the Expanded Program of Immunization (EPI) schedule for other vaccines.[11],[12],[13] As a consequence, a greater number of visits are required by the children for immunization, causing a burden on patients and resulting in poor compliance,[13],[14],[15] greater drop out rates, incomplete dosing and non adherence to immunization program.
It will be extremely helpful if an immunization schedule can be designed which can be given simultaneously with the other EPI vaccines, thus decreasing the number of patient visits (40% more visits in the WHO schedule) and increasing compliance. The present study has compared the seroprotection (with the WHO schedule) when the Hepatitis B vaccine is given at birth (along with BCG and OPV), at 6 weeks (along with DPT and OPV), and at 9 months (along with measles), which coincides with the visits in the Expanded Program of Immunization (EPI).
Material and Methods
Infants were selected from among the newborns delivered at LNJP Hospital. Only term, AGA (appropriate for gestational age) babies with uneventful antenatal periods (no history of maternal jaundice or evidence suggestive of a hepatic pathology including previous clinical and lab reports) were selected for the study excluding infants with major congenital anomaly or immunodeficiency excluding infants were randomized by using a computer generated random number into two groups: Group A and Group B. The grouping was based on two different immunization schedules to be followed (vide infra).
Baseline Immunization and Testing
Baseline samples for HbsAg (Hepatitis B surface antigen) detection were taken from the mother and from cord blood. The babies were given the 0 dose hepatitis B vaccine as soon as possible after birth, no later than 48 hours. The status of hepatitis B surface antigen (HBsAg) was evaluated by the ELISA method as per the manufacturers instructions. Vaccine Administration: Details of each child were recorded on a separate Study Performa after a serial number was given to each patient selected into either group A or group B table1 and table2. An informed consent, for vaccination, was obtained from the parents or the guardian of the children. Each child as per the group was administered 10 micrograms of recombinant DNA hepatitis B vaccine intramuscularly into the anteo-lateral part of the thigh. The EPI vaccines when given were injected at separate site with the help of a separate syringe and needle.
Bood Sample Collection and Testing
An informed consent for blood sample collection was obtained from the parents or the guardian of the children. Blood samples were drawn by venepuncture after appropriate aseptic precautions at 10 weeks [Plus / minus 3 days - sample 1] and at 10 months age [Plus / minus 1 week - sample 2] in Group A infants and at 2 months [Plus / minus 3 days - sample 1] and at 7 months age [Plus / minus 1 week - sample 2] in group B infants. The sera was separated and stored at -20°C until tested. The samples were tested using the Enzyme Immunoassay based on the ELISA method for the determination of antibodies to Hepatitis B surface antigen as per the manufacturers instructions. Follow up: Babies selected into the study were followed up and received other vaccinations as per schedule in the well baby clinic of our hospital. The number of patients recruited and available for follow up at each visit is given in table3. The babies who were born to HbsAg positive mothers in either group were also tested for HbsAg status in their follow up sample at 1 year of age.
Results
HbsAg was positive in 2.34% of the all the mothers (17 of 725) with 2.21% (8) in Group A and 2.47% (9) in Group B. 90.89% and 91.82% infants in Group A and Group B respectively had seroprotective titers (3 10 IU/ml), four weeks after the 2nd dose and 98.99% and 98.45% in Group A and Group B respectively had seroprotective titers (3 10 IU/ml), four weeks after the 3rd dose. The results in the two groups were comparable. The GMTs of the serum sample for both groups; four weeks after the second immunization were comparable. The GMTs of the serum sample for both groups; four weeks after the third immunization were also comparable. table4 and table5. All infants born to HbsAg positive mothers were negative for HbsAg at 1 year of age.
Discussion
Hepatitis B is one of the major diseases of mankind and is a serious global public health problem. Of the 2 billion people who have been infected with the hepatitis B virus (HBV), more than 350 million have chronic infections. These chronically infected persons are at high risk of death from cirrhosis of the liver and liver cancer, diseases that kill about one million persons each year. Although safe and effective hepatitis B vaccines have been available for over 20 years, universal vaccination has still not been implemented in many countries. WHO has recommended incorporation of hepatitis B Immunization in all intermediate and high risk countries. India falls in the intermediate risk group of countries, but universal immunization with hepatitis B vaccine has still not been incorporated in the National EPI programme. One of the reasons for its non-incorporation is that the recommended schedule (0,1 and 6 months) calls for separate visits other than the scheduled EPI vaccines. Increased visits in the EPI lead to the possibility of increased dropouts. This study was carried out to see if a modified schedule of hepatitis B vaccine, which coincides with the timing of the other EPI vaccines was equally effective.
The present study results clearly show that both in terms of percent seroconverted and in terms of GMT of antibodies reached, 0, 6 weeks and 9 months schedule is as effective as 0, 1, 6 months schedule and can be incorporated into the National EPI programme. This schedule is effective in providing equivalent seroprotection as the WHO recommended schedule of 0, 1 and 6 months. This schedule was also effective in preventing perinatal transmission as all the infants born to HbsAg positive mothers were negative for HbsAg at 1 year of age. As the number of infants born to HbsAg positive mothers was small in the current study, the possibility of high rates of breakthrough infection among infants who are given a delayed vaccine schedule (0, 6 weeks and 9 months) compared to the 0, 1 and 6 months schedule cannot be answered from this study. A much larger number of infants born to HbsAg positive mothers would have to be included to reply to that question.
Other studies have shown that vaccination at 6, 10 and 14 weeks is also effective and capable of being incorporated into the EPI schedule. However with this schedule the GMT of hepatitis B antibodies achieved were lower than those reached by the WHO schedule[14] and further as this schedule does not include a 0 dose (birth), its efficacy for prevention of perinatal transmission remains doubtful. As perinatal transmission contributes a significant proportion to chronic carrier state in our country,[2],[4],[5] the 6, 10 and 14 weeks schedule cannot be recommended for universal immunization. The schedule adopted in the present study (0, 6 weeks and 9 months) could take care of both the perinatal transmission as well as prevent horizontal transmission and is as efficacious as the WHO schedule and therefore viable for universal immunization.
Simultaneously administration of Hepatitis B vaccine could interfere with the seroefficacy of other EPI vaccines. The authors have not looked into that aspect, however other studies have shown the efficacy of other EPI vaccines when given simultaneously with hepatitis B vaccine, demonstrating that there were no interactions due to simultaneous administration. [13],[14],[15],[16],[17],[18],[19],[20],[21]
Conclusion
The schedule of hepatitis B vaccination at 0, 6 weeks and 9 months has the same seroefficacy as the currently recommended schedule of 0,1 and 6 months. This schedule has the potential of being incorporated into the National EPI schedule.
Acknowledgement
Dr. Ajay Jain was the recipient of Senior Research Fellowship award of ICMR for this project. University Grants Commission provided financial grant for providing reagents and kits under the Minor Research Grant scheme. The help of both these agencies is greatly acknowledged.
References
1. Kane M. Global Program for control of hepatitis B infection. Vaccine 1995; 13: S47-49.
2. Tandon BN. Dimensions and issues of HBV control in India. In Sarin SK, Singhal AK, eds. Hepatitis B in India: Problems and Prevention. New Delhi, CBS Publishers and Distributors; 1996; 1-4.
3. Tandon BN, Irshad M, Raju M et al. Prevalence of HbsAg and antiHbs in children and stratergy for immunization in India. Ind J Med Res 1991; 93: 337.
4. Govt. of India (1994), Annual Report 1993, DGHS, Ministry of Health and Family Welfare, New Delhi.
5. Nayak NC, Panda SK, Zuckerman AJ, Bhan MK, Guha DK. Dynamics and impact of perinatal transmission of Hepatitis B virus in North India. J Med Virol 1987; 21: 137-145.
6. John D. Snyder and Larry K. Pickering. Viral Hepatitis. In Behrman RE, Kleigman RM, Nelson WE, eds. Nelson Textbook of Pediatrics, 16th edn. Harcourt Asia PTE, Ltd 2000; 771-773.
7. World Health Organization. Immunization Policy WHO/EPI/GEN/95.3, 1995
8. Expanded Programme on Immunization: Framework for evaluating a vaccine for the EPI. WHO Document WHO/EPI/GEN/93.5; 1993g.
9. World Health Organization. Hepatitis B vaccine - making global progress. EPI update , October, 1996.
10. John TJ. Hepatitis B immunization. Indian Pediatr 1995; 32: 609-613.
11. Park K. In K. Park, ed. Principles of Epidemiology and Epidemiologic Methods. Park's Textbook of Preventive and Social Medicine. 15th edn. New Delhi; M/S Banarsidas Bhanot Publishers. 1997; 100.
12. Bassily S, Kotkat A, Gray G, Hyams KC, Brown FM, Imam IZ, Arthur R. U.S. Comparative study of the immunogenicity and safety of two dosing schedules of hepatitis B vaccine in neonates. Am J Trop Med Hyg 1995; 53(4): 419-422.
13. Mittal SK, Rao S, Kumari S, Aggarwal V, Prakash C, Thirupuram S. Simultaneous administration of hepatitis B vaccine with other E.P.I. vaccines. Indian J Pediatr 1994; 61(2): 183-188.
14. Gomber Sunil, Sharma Rajesh, Ramachandran VG, Talwar Vibha, Singh Bharat. Immunogenicity of Hepatitis B Vaccine Incorporated into the Expanded Program of Immunization Schedule. Indian Pediatr 2000; 37: 411-413.
15. Kottenhahn RK, Rosenthal SL, Biro FM. Hepatitis B vaccine completion among adolescents. Del Med J 1996; 68(6): 309-311.
16. Park K. Communicable Diseases. K. Park, ed. Park's Textbook Of Preventive and Social Medicine. 15th edn. New Delhi; M/S Banarsidas Bhanot Publishers. 1997; 158-161.
17. Tsega E, Tafesse B, Nordenfelt E, Wolde-Hawariat G, Hansson BG, Lindberg J. Immunogenicity of hepatitis B vaccine simultaneously administered with the expanded programme on immunisation (EPI). J Med Virol 1990; 32(4): 232-235.
18. Sathish T K, Abraham P, Raghuraman Sukanya and Cherian T. Immunogenicity of Indigenous Recombinant Hepatitis B Vaccine in Infants Following a 0, 1, 2-Month Vaccination Schedule. Indian Pediatrics 2000; 37: 75-80.
19. Papaevangelou G, Karvelis E, Alexiou D et al. Evaluation of combined tetravalent diptheria, tetanus, whole cell pertusis and hepatitis B candidate vaccine administered to healthy infants according to a three dose vaccination schedule. Vaccine 1995; 13: 175
20. Giammanco G, Li Volti S, Mauro L, Bilancia GG, Salemi I, Barone P, Musumeci S. Immune response to simultaneous administration of a recombinant DNA hepatitis B vaccine and multiple compulsory vaccines in infancy. Vaccine 1991; 9(10): 747-750.
21. Bhan M. K. Hepatitis B Immunization for the newborn: Rationale and Regimens. In SK Sarin, AK Singhal, eds. Hepatitis B In India, Problems and Prevention. CBS Publishers And Distributors, 1996: 204.(Jain Ajay Kumar, Mittal S)
2 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
Abstract
Objective: This study was carried out to evaluate the adequacy of seroconversion when Hepatitis B vaccine is given along with other vaccines at 0, 6 weeks (along with DPT and OPV) and at 9 months (along with measles). Methods: 725 infants born to apparently healthy mothers were enrolled to receive the hepatitis B vaccine at 0, 6 weeks and 9 months (Group A) or at 0, 1 and 6 months as per WHO schedule - (Group B). Baseline HbsAg testing was carried out and the babies were immunized with the first dose of hepatitis B vaccine within 48 hours of birth. BCG and the other EPI vaccines were given as per schedule. Serum samples were collected 4 weeks after the second and the third immunizations. 604 infants (83.3%) completed the study. The testing for HbsAg and Anti Hbs titers were conducted in the Department of Microbiology, Maulana Azad Medical College, New Delhi utilizing standard ELISA kits. Results: The seroconversion rates 4 weeks after the second dose of the vaccine were 90.89% (GMT = 48.23) and 91.82% (GMT = 43.95) (P=0.8) in Group A and Group B respectively. After 4 weeks of the third dose the seroconversion rates were 98.99 (GMT = 161.12) and 98.45 (GMT = 150.12) (p=0.17) in Group A and Group B respectively. The two schedules were comparable on using the Kruskal-Wallis H method for analysis. Conclusion: The schedule of hepatitis B vaccination at 0, 6 weeks and 9 months has the same seroefficacy as the currently recommended schedule of 0, 1 and 6 months.
Keywords: Hepatitis B vaccines; HbsAg mothers; EPI Vaccines; Seroconversion
Hepatitis B is endemic throughout the world, especially in tropical and developing countries. The hepatitis B carriage rates in India range from 3 to 5 percent. In endemic populations the carrier rate is high and perinatal transmission is a common event.[1],[2],[3],[4],[5] The World Health Organization recommends immunization of all children with three doses of 10 micrograms each of hepatitis B vaccine given intramuscularly at 0, 1, 6 months of age.[6],[7],[8],[9],[10] This schedule does not coincide with the Expanded Program of Immunization (EPI) schedule for other vaccines.[11],[12],[13] As a consequence, a greater number of visits are required by the children for immunization, causing a burden on patients and resulting in poor compliance,[13],[14],[15] greater drop out rates, incomplete dosing and non adherence to immunization program.
It will be extremely helpful if an immunization schedule can be designed which can be given simultaneously with the other EPI vaccines, thus decreasing the number of patient visits (40% more visits in the WHO schedule) and increasing compliance. The present study has compared the seroprotection (with the WHO schedule) when the Hepatitis B vaccine is given at birth (along with BCG and OPV), at 6 weeks (along with DPT and OPV), and at 9 months (along with measles), which coincides with the visits in the Expanded Program of Immunization (EPI).
Material and Methods
Infants were selected from among the newborns delivered at LNJP Hospital. Only term, AGA (appropriate for gestational age) babies with uneventful antenatal periods (no history of maternal jaundice or evidence suggestive of a hepatic pathology including previous clinical and lab reports) were selected for the study excluding infants with major congenital anomaly or immunodeficiency excluding infants were randomized by using a computer generated random number into two groups: Group A and Group B. The grouping was based on two different immunization schedules to be followed (vide infra).
Baseline Immunization and Testing
Baseline samples for HbsAg (Hepatitis B surface antigen) detection were taken from the mother and from cord blood. The babies were given the 0 dose hepatitis B vaccine as soon as possible after birth, no later than 48 hours. The status of hepatitis B surface antigen (HBsAg) was evaluated by the ELISA method as per the manufacturers instructions. Vaccine Administration: Details of each child were recorded on a separate Study Performa after a serial number was given to each patient selected into either group A or group B table1 and table2. An informed consent, for vaccination, was obtained from the parents or the guardian of the children. Each child as per the group was administered 10 micrograms of recombinant DNA hepatitis B vaccine intramuscularly into the anteo-lateral part of the thigh. The EPI vaccines when given were injected at separate site with the help of a separate syringe and needle.
Bood Sample Collection and Testing
An informed consent for blood sample collection was obtained from the parents or the guardian of the children. Blood samples were drawn by venepuncture after appropriate aseptic precautions at 10 weeks [Plus / minus 3 days - sample 1] and at 10 months age [Plus / minus 1 week - sample 2] in Group A infants and at 2 months [Plus / minus 3 days - sample 1] and at 7 months age [Plus / minus 1 week - sample 2] in group B infants. The sera was separated and stored at -20°C until tested. The samples were tested using the Enzyme Immunoassay based on the ELISA method for the determination of antibodies to Hepatitis B surface antigen as per the manufacturers instructions. Follow up: Babies selected into the study were followed up and received other vaccinations as per schedule in the well baby clinic of our hospital. The number of patients recruited and available for follow up at each visit is given in table3. The babies who were born to HbsAg positive mothers in either group were also tested for HbsAg status in their follow up sample at 1 year of age.
Results
HbsAg was positive in 2.34% of the all the mothers (17 of 725) with 2.21% (8) in Group A and 2.47% (9) in Group B. 90.89% and 91.82% infants in Group A and Group B respectively had seroprotective titers (3 10 IU/ml), four weeks after the 2nd dose and 98.99% and 98.45% in Group A and Group B respectively had seroprotective titers (3 10 IU/ml), four weeks after the 3rd dose. The results in the two groups were comparable. The GMTs of the serum sample for both groups; four weeks after the second immunization were comparable. The GMTs of the serum sample for both groups; four weeks after the third immunization were also comparable. table4 and table5. All infants born to HbsAg positive mothers were negative for HbsAg at 1 year of age.
Discussion
Hepatitis B is one of the major diseases of mankind and is a serious global public health problem. Of the 2 billion people who have been infected with the hepatitis B virus (HBV), more than 350 million have chronic infections. These chronically infected persons are at high risk of death from cirrhosis of the liver and liver cancer, diseases that kill about one million persons each year. Although safe and effective hepatitis B vaccines have been available for over 20 years, universal vaccination has still not been implemented in many countries. WHO has recommended incorporation of hepatitis B Immunization in all intermediate and high risk countries. India falls in the intermediate risk group of countries, but universal immunization with hepatitis B vaccine has still not been incorporated in the National EPI programme. One of the reasons for its non-incorporation is that the recommended schedule (0,1 and 6 months) calls for separate visits other than the scheduled EPI vaccines. Increased visits in the EPI lead to the possibility of increased dropouts. This study was carried out to see if a modified schedule of hepatitis B vaccine, which coincides with the timing of the other EPI vaccines was equally effective.
The present study results clearly show that both in terms of percent seroconverted and in terms of GMT of antibodies reached, 0, 6 weeks and 9 months schedule is as effective as 0, 1, 6 months schedule and can be incorporated into the National EPI programme. This schedule is effective in providing equivalent seroprotection as the WHO recommended schedule of 0, 1 and 6 months. This schedule was also effective in preventing perinatal transmission as all the infants born to HbsAg positive mothers were negative for HbsAg at 1 year of age. As the number of infants born to HbsAg positive mothers was small in the current study, the possibility of high rates of breakthrough infection among infants who are given a delayed vaccine schedule (0, 6 weeks and 9 months) compared to the 0, 1 and 6 months schedule cannot be answered from this study. A much larger number of infants born to HbsAg positive mothers would have to be included to reply to that question.
Other studies have shown that vaccination at 6, 10 and 14 weeks is also effective and capable of being incorporated into the EPI schedule. However with this schedule the GMT of hepatitis B antibodies achieved were lower than those reached by the WHO schedule[14] and further as this schedule does not include a 0 dose (birth), its efficacy for prevention of perinatal transmission remains doubtful. As perinatal transmission contributes a significant proportion to chronic carrier state in our country,[2],[4],[5] the 6, 10 and 14 weeks schedule cannot be recommended for universal immunization. The schedule adopted in the present study (0, 6 weeks and 9 months) could take care of both the perinatal transmission as well as prevent horizontal transmission and is as efficacious as the WHO schedule and therefore viable for universal immunization.
Simultaneously administration of Hepatitis B vaccine could interfere with the seroefficacy of other EPI vaccines. The authors have not looked into that aspect, however other studies have shown the efficacy of other EPI vaccines when given simultaneously with hepatitis B vaccine, demonstrating that there were no interactions due to simultaneous administration. [13],[14],[15],[16],[17],[18],[19],[20],[21]
Conclusion
The schedule of hepatitis B vaccination at 0, 6 weeks and 9 months has the same seroefficacy as the currently recommended schedule of 0,1 and 6 months. This schedule has the potential of being incorporated into the National EPI schedule.
Acknowledgement
Dr. Ajay Jain was the recipient of Senior Research Fellowship award of ICMR for this project. University Grants Commission provided financial grant for providing reagents and kits under the Minor Research Grant scheme. The help of both these agencies is greatly acknowledged.
References
1. Kane M. Global Program for control of hepatitis B infection. Vaccine 1995; 13: S47-49.
2. Tandon BN. Dimensions and issues of HBV control in India. In Sarin SK, Singhal AK, eds. Hepatitis B in India: Problems and Prevention. New Delhi, CBS Publishers and Distributors; 1996; 1-4.
3. Tandon BN, Irshad M, Raju M et al. Prevalence of HbsAg and antiHbs in children and stratergy for immunization in India. Ind J Med Res 1991; 93: 337.
4. Govt. of India (1994), Annual Report 1993, DGHS, Ministry of Health and Family Welfare, New Delhi.
5. Nayak NC, Panda SK, Zuckerman AJ, Bhan MK, Guha DK. Dynamics and impact of perinatal transmission of Hepatitis B virus in North India. J Med Virol 1987; 21: 137-145.
6. John D. Snyder and Larry K. Pickering. Viral Hepatitis. In Behrman RE, Kleigman RM, Nelson WE, eds. Nelson Textbook of Pediatrics, 16th edn. Harcourt Asia PTE, Ltd 2000; 771-773.
7. World Health Organization. Immunization Policy WHO/EPI/GEN/95.3, 1995
8. Expanded Programme on Immunization: Framework for evaluating a vaccine for the EPI. WHO Document WHO/EPI/GEN/93.5; 1993g.
9. World Health Organization. Hepatitis B vaccine - making global progress. EPI update , October, 1996.
10. John TJ. Hepatitis B immunization. Indian Pediatr 1995; 32: 609-613.
11. Park K. In K. Park, ed. Principles of Epidemiology and Epidemiologic Methods. Park's Textbook of Preventive and Social Medicine. 15th edn. New Delhi; M/S Banarsidas Bhanot Publishers. 1997; 100.
12. Bassily S, Kotkat A, Gray G, Hyams KC, Brown FM, Imam IZ, Arthur R. U.S. Comparative study of the immunogenicity and safety of two dosing schedules of hepatitis B vaccine in neonates. Am J Trop Med Hyg 1995; 53(4): 419-422.
13. Mittal SK, Rao S, Kumari S, Aggarwal V, Prakash C, Thirupuram S. Simultaneous administration of hepatitis B vaccine with other E.P.I. vaccines. Indian J Pediatr 1994; 61(2): 183-188.
14. Gomber Sunil, Sharma Rajesh, Ramachandran VG, Talwar Vibha, Singh Bharat. Immunogenicity of Hepatitis B Vaccine Incorporated into the Expanded Program of Immunization Schedule. Indian Pediatr 2000; 37: 411-413.
15. Kottenhahn RK, Rosenthal SL, Biro FM. Hepatitis B vaccine completion among adolescents. Del Med J 1996; 68(6): 309-311.
16. Park K. Communicable Diseases. K. Park, ed. Park's Textbook Of Preventive and Social Medicine. 15th edn. New Delhi; M/S Banarsidas Bhanot Publishers. 1997; 158-161.
17. Tsega E, Tafesse B, Nordenfelt E, Wolde-Hawariat G, Hansson BG, Lindberg J. Immunogenicity of hepatitis B vaccine simultaneously administered with the expanded programme on immunisation (EPI). J Med Virol 1990; 32(4): 232-235.
18. Sathish T K, Abraham P, Raghuraman Sukanya and Cherian T. Immunogenicity of Indigenous Recombinant Hepatitis B Vaccine in Infants Following a 0, 1, 2-Month Vaccination Schedule. Indian Pediatrics 2000; 37: 75-80.
19. Papaevangelou G, Karvelis E, Alexiou D et al. Evaluation of combined tetravalent diptheria, tetanus, whole cell pertusis and hepatitis B candidate vaccine administered to healthy infants according to a three dose vaccination schedule. Vaccine 1995; 13: 175
20. Giammanco G, Li Volti S, Mauro L, Bilancia GG, Salemi I, Barone P, Musumeci S. Immune response to simultaneous administration of a recombinant DNA hepatitis B vaccine and multiple compulsory vaccines in infancy. Vaccine 1991; 9(10): 747-750.
21. Bhan M. K. Hepatitis B Immunization for the newborn: Rationale and Regimens. In SK Sarin, AK Singhal, eds. Hepatitis B In India, Problems and Prevention. CBS Publishers And Distributors, 1996: 204.(Jain Ajay Kumar, Mittal S)