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Oral polio immunization of the newborn infant; a possible method for overcoming interference by ingested antibodies
TROPICAL PEDIATRICS
Oral polio immunization of the newborn infant; a possible method for overcoming in terference by ingested a ntibodies Thirty-eight in[ants weighing 1,500 grams or more were given oral poliovirus type 1 vaccine and were led [ormula containing neutralizing antibody against poliovirus type 1 in a concentration o[ 1:256 per milliliter, at various time intervals in relation to the administration o[ the vaccine. Inter[erence against injection with the vaccine virus was observed when the ingestion o[ antibody preceded or [ol[owed vaccine administration by 2 hours. There was no inter[erence i[ ingestion o[ antibody took place 6 hours be[ore and 6 hours alter vaccine administration. This model system suggests a method [or overcoming the inhibitory effect o[ breast-feeding on oral polio vaccination. Application of this method to polio immunization of in[ants in the developing countries is discussed.
Michael K a t z , M . D . , and Stanley A . P l o t k i n , M . D . PHILADELPHIA~
PA.
B R E A s T - F E ~, D I N O
has an inhibitory effect on vaccination with live poliovirus. This was shown in a study by Lepow and co-workers~ in Cleveland. Our study in Uganda 2 confirmed this finding: polio vaccination of breast-fed 3-day-old neonates resulted in a rate of intestinal infection with vaccine virus in only 57 per cent of the infants. This interference was directly re-
From The Wistar Institute o[ Anatomy and Biology and the Department o[ Pediatrics, University o[ Pennsylvania. Supported in part by United States Public Health Service Grant No. 5-ROI-AIO1799 from The National Institute o[ Allergy and In[ectious Diseases.
lated to the quantity of antibody present in the breast milk. In a subsequent study 3 we attempted to circumvent this interference by vaccinating neonates immediately after birth, approximately 12 hours before the initial breast-feeding. However, because of the relative insusceptibility of the neonatal gut during the first 3 days of life to this virus, 4 we experienced a failure rate of approximately 40 per cent. It is clear that for vaccination to be successful the infants should be at least 3 days old and that a method be developed to overcome interference by the ingested antibody. The present study was designed to test such a method. Vol. 73, No. 2, pp. 267-270
268
The .[ournal o/~Pediatrics August 1968
t(atz and Plotkin
MATERIALS
AND
METHODS
Subjects. Thirty-eight premature infants in the nurseries of Philadelphia General Hospital served as subjects. Each weighed 1,500 grams or more and was in good health. Informed consent was obtained from each mother, and arrangements were made for a complete polio vaccination subsequent to this study. T h e infants were divided into 4 groups, as follows: Group I. ingested antibody against the vaccine virus 2 hours before and 2 hours after the vaccine administration; Group II. ingested antibody 6 hours before and 2 hours after the vaccine; Group I I I . ingested antibody 2 hours before and 6 hours after the vaccine; Group IV. ingested antibody 6 hours before and 6 hours after the vaccine. Antibody. Antiserum against the Mahoney strain of wild type 1 poliovirus was prepared in a rabbit and titrated in primary green monkey kidney ( G M K ) tissue cultures, according to the method of Gard. 5 T h e antiserum was stored at -20 ~ C. in individual tubes and added to the formulas immediately before feeding. When mixed with the formula it produced a titer of 1:256 per milliliter against the type 1 virus. Formulas. Standard formulas of cow's milk simulating the composition of human milk, prepared by different manufacturers, were used; a regular feeding schedule was maintained. Vaccine. Commercially prepared Sabin type 1 attenuated poliovirus was used. It was divided into 1 ml. lots containing 10~TTCDso of the virus and stored at -20 ~ C. Administration of the vaccine. Virus was administered through an orogastric tube, followed by 5 c.c. of saline solution. The infant was then observed for I5 minutes to ascertain that there was no regurgitation of the vaccine. Collection of stools. Stool specimens were obtained before the administration of the vaccine and at subsequent 3 day intervals following vaccination for a total of 3 postvaccination specimens. They were stored a t - 2 0 ~ C.
Virus isolation. Ten per cent fecal suspensions were prepared, and 0. I ml. of each was inoculated into sets of 4 primary G M K tissue culture tubes, from which the medium had been removed. After incubation at 37 ~ C. for one hour the tissue cultures were washed twice with buffered saline and covered with medium containing 2 per cent fetal calf serum. Cytopathic effect was taken as presumptive evidence of poliovirus, and the cytopathogenic agent was then identified as poliovirus type 1 by neutralization with an appropriate antiserum prepared in a rabbit. All specimens negative for cytopathic effect were blind-passed once to another set of G M K tissue culture tubes. RESULTS
As shown in Table I, administration of the antibody 2 hours before and 2 hours after the vaccine (Group I) completely prevented intestinal infection. In Groups I I (antibody ingested 6 hours before and 2 hours after vaccine administration) and I I I (antibody ingested 2 hours before and 6 hours after vaccine administration) 6 out of the 10 infants in each group became infected with the vaccine virus. In Group I V (antibody ingested 6 hours before and 6 hours after vaccine administration) 8 out of 8 infants became infected. Virus was re-, covered from all infants who became infected in each of the 3 stool specimens. These differences are significant at the 0.0i level by the ehi square method. DISCUSSION Breast-feeding, which is an almost universal practice in the developing countries,
Table I. Influence of the time of antibody ingestion and infection with attenuated poliovirus
Group*
No. infected in/ants
[ No. infants I given vaccine
I II
0 6
10 10
III IV
6 8
10 8
*See text for definition of the groups.
Volume 73 Number 2
should be encouraged as a preventive measure against infant malnutrition. I t is also desirable, in these countries, to immunize against poliomyelitis at birth or immediately after, because newborn infants are most accessible to health workers. I n the postnatal period, only a small proportion of infants can be reached for the purpose of vaccination; yet poliomyelitis has a high prevalence in infancy. 6, 7 O u r results show that interference by the ingested antibody against infection with attenuated poliovirus in neonates depends on the time interval separating antibody ingestion and vaccine administration. If the feedings within 2 hours before and after the administration of the vaccine contained antibodies against the virus, complete prevention of the infection resulted. If only one of those feedings contained the antibody, interference was partial; only 6 out of 10 infants became infected. W h e n the antibody was administered 6 hours before and 6 hours after the vaccine virus, no interference was observed since all infants became infected. Although no antibody was ingested by the infants in our study beyond 6 hours, it is unlikely that any later, or repetitive administration of antibody would result in interference. As was shown by Gonzaga and associates, s feeding of bovine colostrum for 48 hours after intestinal infection has taken place did not prevent virus excretion. Serum antibody levels were not measured because the objective of this study was the determination of infectibility of the intestine in the presence of ingested antibody. Once infection in the intestine takes place, the rate of seroconversion in infants is 90 per cent2 I n the premature iqfant, the antibody response has been variable. It was relatively poor in type 1 poliovirus, 1~ since only 59 per cent of such infants converted, but it was m u c h better in case of the type 3 vaccine, which produced a 90 per cent rate of seroconversion? 1 I n any mass immunization p r o g r a m directed at the newborn infants, premature infants would constitute only a small fraction of the whole group. I n view of our data, it is likely that in a developing country, in which newborn in-
Polio immunization o/ newborn
2 69
rants should be vaccinated before leaving the community health center or before an attendant midwife leaves the area of the infant's residence, the interference of breast milk to vaccination can be overcome. This can be accomplished by the omission of one breast-feeding before and one after the administration of the vaccine and substituting boiled water for breast milk at these times. This study, which was limited to type 1 vaccine, should be extended to include a trivalent vaccine. Moreover, it should be supported by a field trial before this method can be definitely recommended. Such a trial seems warranted in view of the data we have presented. The authors thank Dr. Margaret L. Williams and her staff of the Department of Pediatrics, Philadelphia General Hospital, for their cooperation in this study.
REFERENCES
1. Lepow, M. L., Warren, R. J., Gray, N., Ingrain, V. G., and Robbins, F. C.: Effect of Sabin Type I poliomyelitis vaccine administered by mouth to newborn infants, New England J. Med. 264: 1071, 1961. 2. Plotkin, S. A., Katz, M., Brown, R. E., and Pagano, J. S.: Oral poliovirus vaccination in newborn African infants: The inhibitory effect of breast feeding, Am. J. Dis. Child. 111: 27, 1966. 3. Katz, M., Brown, R. E,, and Plotkin, S. A.: Oral poliovirus vaccination in newborn African infants; relative ineffectiveness of early feeding of vaccine, Trop. & Geograph. Med. In press. 4. Warren, R. J., Lepow, M. L., Bartsch, G. E., and Robbins, F. C.: The relationship of maternal antibody, breast feeding, and age to the susceptibility of newborn infants to infection with attenuated polioviruses, Pediatrics 34: 4, 1964. 5. Gard, S.: Immuno-inactivation of poliovirus, Arch. ges. Virusforsch. 7: 449, 1957. 6. Paul, J. R., Melnick, J, L., Barnett, V. H., and Goldblum, N.: Survey of neutralizing antibodies to poliomyelitis in Cairo, Egypt, Am. J. Hygiene 55: 402, 1952. 7. Lebrun, A., Cerf, J., Gelfand, t-I. M., Courtois, G., Plotkin, S. A., and Koprowski, H.: Vaccination with the CHAT strain of Type I attenuated poliomyelitis virus in Leopoldville, Belgian Congo. I. Description of the city, its history of poliomyelitis, and the plan of vaccination campaign, Bull. World Health Organ. 22: 203, 1960. 8. Gonzaga, A. J., Warren, R. J., and Robbins, F. C.: Attenuated pollovirus infection in in-
270
Cook and Jelliffe
fants fed colostrum from poliomyelitis immune cows, Pediatrics 32: 1039, 1963. 9. Plotkin, S. A.: Proceedings of the 6th International Congress of Microbiological Standardization, Berlin, 1961, Hoffman Verlag, pp. 48-73. 10. Pagano, J. S., PIotkin, S. A., Comely, D., Leuterer, W., and Koprowski, H.: The re-
Immunization in developing regions
The Journal of Pediatrics August 1968
sponses of premature infants to infection with attenuated poliovirus, Pediatrics 29: 794, 1962. 11. Pagano, J. S., Plotkin, S. A., and CorneIy, D.: The response of premature infants to infection with type 3 attenuated poliovirus, J. PEmAT. 65: 165, 1964.
e. f. g. h. i. j. k.
Cost of immunization Simplicity of the immunization procedure Cost of rehabilitation Economic burden of disabled Availability of the antigen Popular demand Cost of alternative methods of prophylaxis
Editorial comment The observations presented here by Katz and Plotkin appear to be a practical contribution to the prevention of poliomyelitis in developing countries. This article points up the question of the most suitable immunization schedule for such countries, where geographical and cultural difficulties in making contact with young children assume considerable importance, where staff and finance are in short supply, and where one must take every opportunity that offers of immunizing children, provided only that the vaccine will be effective at that age. The schedule recently suggested by Stanfield1 is a feasible one which we can now modify to include the first dose of oral poliomyelitis vaccine at birth. By adopting the plan of Katz and Plotldn, the infant may leave the maternity ward immunized with poliomyelitis vaccine as well as BCG and possibly also smallpox vaccine. Even if no further opportunity for immunization arises, at least something will have been achieved. However, one should seek to add an additional dose of poliomyelitis vaccine at a Iater date in infancy, and before 6 months of age, triple vaccine, and as soon as possible after 9 months of age, measles vaccine. Cook 2, s has described a plan by which the majority of young children in areas with only a limited health service can be cheaply immunized. In determining schedules of immunization for developing regions it is of value to bear in mind the following considerations4: 1. D I S E A S E
a. b. c. d.
PRIORITIES
Incidence of disease Mortality rate from disease "Conditioning" infection or not Cost of treatment
2. A G E - G R O U P
PRIORITIES
a. Above considerations by age group b. Accessibility of the group (e.g., newborn, schools) c. Ability of group to respond to antigen (e.g., BCG compared with measles in newborn) 3. T E C H N I Q U E S a. Number of doses required, minimum and optimum b. Dosage c. Methods of administration (oral, hypodermic, intradermal) d. Choice of preparations available e. Refrigeration requirements at base and in field 4. R E S U L T S a. Intermediate results of successful immunization b. Long-term results of successful immunization c. Number of adverse individual reactions to be expected R.
COOK~
IVf.D.
D. B. JELLIFFE~ M.D.
REFERENCES
1. Stanfield, J. P.: Organization of MCH services in devoloping regions. VI. Special programmes in immunization, J. Trop. Pediat. 13: 102, 1967. 2. Cook, R.: Comprehensive immunization in rural areas, J. Trop. Pediat. 2: 13, 1966. 3. Cook, R.: The Ankole pre-school protection programme 1964-67, Mimeographed document, Department of Paediatrics, Makerere Medical School. 4. JelIiffe, D. B.: Recent experience in maternal and child health in East Africa, J. Trop. Pediat. 2: 15, 1966.
Oral polio immunization of the newborn infant; a possible method for overcoming in terference by ingested a ntibodies Thirty-eight in[ants weighing 1,500 grams or more were given oral poliovirus type 1 vaccine and were led [ormula containing neutralizing antibody against poliovirus type 1 in a concentration o[ 1:256 per milliliter, at various time intervals in relation to the administration o[ the vaccine. Inter[erence against injection with the vaccine virus was observed when the ingestion o[ antibody preceded or [ol[owed vaccine administration by 2 hours. There was no inter[erence i[ ingestion o[ antibody took place 6 hours be[ore and 6 hours alter vaccine administration. This model system suggests a method [or overcoming the inhibitory effect o[ breast-feeding on oral polio vaccination. Application of this method to polio immunization of in[ants in the developing countries is discussed.
Michael K a t z , M . D . , and Stanley A . P l o t k i n , M . D . PHILADELPHIA~
PA.
B R E A s T - F E ~, D I N O
has an inhibitory effect on vaccination with live poliovirus. This was shown in a study by Lepow and co-workers~ in Cleveland. Our study in Uganda 2 confirmed this finding: polio vaccination of breast-fed 3-day-old neonates resulted in a rate of intestinal infection with vaccine virus in only 57 per cent of the infants. This interference was directly re-
From The Wistar Institute o[ Anatomy and Biology and the Department o[ Pediatrics, University o[ Pennsylvania. Supported in part by United States Public Health Service Grant No. 5-ROI-AIO1799 from The National Institute o[ Allergy and In[ectious Diseases.
lated to the quantity of antibody present in the breast milk. In a subsequent study 3 we attempted to circumvent this interference by vaccinating neonates immediately after birth, approximately 12 hours before the initial breast-feeding. However, because of the relative insusceptibility of the neonatal gut during the first 3 days of life to this virus, 4 we experienced a failure rate of approximately 40 per cent. It is clear that for vaccination to be successful the infants should be at least 3 days old and that a method be developed to overcome interference by the ingested antibody. The present study was designed to test such a method. Vol. 73, No. 2, pp. 267-270
268
The .[ournal o/~Pediatrics August 1968
t(atz and Plotkin
MATERIALS
AND
METHODS
Subjects. Thirty-eight premature infants in the nurseries of Philadelphia General Hospital served as subjects. Each weighed 1,500 grams or more and was in good health. Informed consent was obtained from each mother, and arrangements were made for a complete polio vaccination subsequent to this study. T h e infants were divided into 4 groups, as follows: Group I. ingested antibody against the vaccine virus 2 hours before and 2 hours after the vaccine administration; Group II. ingested antibody 6 hours before and 2 hours after the vaccine; Group I I I . ingested antibody 2 hours before and 6 hours after the vaccine; Group IV. ingested antibody 6 hours before and 6 hours after the vaccine. Antibody. Antiserum against the Mahoney strain of wild type 1 poliovirus was prepared in a rabbit and titrated in primary green monkey kidney ( G M K ) tissue cultures, according to the method of Gard. 5 T h e antiserum was stored at -20 ~ C. in individual tubes and added to the formulas immediately before feeding. When mixed with the formula it produced a titer of 1:256 per milliliter against the type 1 virus. Formulas. Standard formulas of cow's milk simulating the composition of human milk, prepared by different manufacturers, were used; a regular feeding schedule was maintained. Vaccine. Commercially prepared Sabin type 1 attenuated poliovirus was used. It was divided into 1 ml. lots containing 10~TTCDso of the virus and stored at -20 ~ C. Administration of the vaccine. Virus was administered through an orogastric tube, followed by 5 c.c. of saline solution. The infant was then observed for I5 minutes to ascertain that there was no regurgitation of the vaccine. Collection of stools. Stool specimens were obtained before the administration of the vaccine and at subsequent 3 day intervals following vaccination for a total of 3 postvaccination specimens. They were stored a t - 2 0 ~ C.
Virus isolation. Ten per cent fecal suspensions were prepared, and 0. I ml. of each was inoculated into sets of 4 primary G M K tissue culture tubes, from which the medium had been removed. After incubation at 37 ~ C. for one hour the tissue cultures were washed twice with buffered saline and covered with medium containing 2 per cent fetal calf serum. Cytopathic effect was taken as presumptive evidence of poliovirus, and the cytopathogenic agent was then identified as poliovirus type 1 by neutralization with an appropriate antiserum prepared in a rabbit. All specimens negative for cytopathic effect were blind-passed once to another set of G M K tissue culture tubes. RESULTS
As shown in Table I, administration of the antibody 2 hours before and 2 hours after the vaccine (Group I) completely prevented intestinal infection. In Groups I I (antibody ingested 6 hours before and 2 hours after vaccine administration) and I I I (antibody ingested 2 hours before and 6 hours after vaccine administration) 6 out of the 10 infants in each group became infected with the vaccine virus. In Group I V (antibody ingested 6 hours before and 6 hours after vaccine administration) 8 out of 8 infants became infected. Virus was re-, covered from all infants who became infected in each of the 3 stool specimens. These differences are significant at the 0.0i level by the ehi square method. DISCUSSION Breast-feeding, which is an almost universal practice in the developing countries,
Table I. Influence of the time of antibody ingestion and infection with attenuated poliovirus
Group*
No. infected in/ants
[ No. infants I given vaccine
I II
0 6
10 10
III IV
6 8
10 8
*See text for definition of the groups.
Volume 73 Number 2
should be encouraged as a preventive measure against infant malnutrition. I t is also desirable, in these countries, to immunize against poliomyelitis at birth or immediately after, because newborn infants are most accessible to health workers. I n the postnatal period, only a small proportion of infants can be reached for the purpose of vaccination; yet poliomyelitis has a high prevalence in infancy. 6, 7 O u r results show that interference by the ingested antibody against infection with attenuated poliovirus in neonates depends on the time interval separating antibody ingestion and vaccine administration. If the feedings within 2 hours before and after the administration of the vaccine contained antibodies against the virus, complete prevention of the infection resulted. If only one of those feedings contained the antibody, interference was partial; only 6 out of 10 infants became infected. W h e n the antibody was administered 6 hours before and 6 hours after the vaccine virus, no interference was observed since all infants became infected. Although no antibody was ingested by the infants in our study beyond 6 hours, it is unlikely that any later, or repetitive administration of antibody would result in interference. As was shown by Gonzaga and associates, s feeding of bovine colostrum for 48 hours after intestinal infection has taken place did not prevent virus excretion. Serum antibody levels were not measured because the objective of this study was the determination of infectibility of the intestine in the presence of ingested antibody. Once infection in the intestine takes place, the rate of seroconversion in infants is 90 per cent2 I n the premature iqfant, the antibody response has been variable. It was relatively poor in type 1 poliovirus, 1~ since only 59 per cent of such infants converted, but it was m u c h better in case of the type 3 vaccine, which produced a 90 per cent rate of seroconversion? 1 I n any mass immunization p r o g r a m directed at the newborn infants, premature infants would constitute only a small fraction of the whole group. I n view of our data, it is likely that in a developing country, in which newborn in-
Polio immunization o/ newborn
2 69
rants should be vaccinated before leaving the community health center or before an attendant midwife leaves the area of the infant's residence, the interference of breast milk to vaccination can be overcome. This can be accomplished by the omission of one breast-feeding before and one after the administration of the vaccine and substituting boiled water for breast milk at these times. This study, which was limited to type 1 vaccine, should be extended to include a trivalent vaccine. Moreover, it should be supported by a field trial before this method can be definitely recommended. Such a trial seems warranted in view of the data we have presented. The authors thank Dr. Margaret L. Williams and her staff of the Department of Pediatrics, Philadelphia General Hospital, for their cooperation in this study.
REFERENCES
1. Lepow, M. L., Warren, R. J., Gray, N., Ingrain, V. G., and Robbins, F. C.: Effect of Sabin Type I poliomyelitis vaccine administered by mouth to newborn infants, New England J. Med. 264: 1071, 1961. 2. Plotkin, S. A., Katz, M., Brown, R. E., and Pagano, J. S.: Oral poliovirus vaccination in newborn African infants: The inhibitory effect of breast feeding, Am. J. Dis. Child. 111: 27, 1966. 3. Katz, M., Brown, R. E,, and Plotkin, S. A.: Oral poliovirus vaccination in newborn African infants; relative ineffectiveness of early feeding of vaccine, Trop. & Geograph. Med. In press. 4. Warren, R. J., Lepow, M. L., Bartsch, G. E., and Robbins, F. C.: The relationship of maternal antibody, breast feeding, and age to the susceptibility of newborn infants to infection with attenuated polioviruses, Pediatrics 34: 4, 1964. 5. Gard, S.: Immuno-inactivation of poliovirus, Arch. ges. Virusforsch. 7: 449, 1957. 6. Paul, J. R., Melnick, J, L., Barnett, V. H., and Goldblum, N.: Survey of neutralizing antibodies to poliomyelitis in Cairo, Egypt, Am. J. Hygiene 55: 402, 1952. 7. Lebrun, A., Cerf, J., Gelfand, t-I. M., Courtois, G., Plotkin, S. A., and Koprowski, H.: Vaccination with the CHAT strain of Type I attenuated poliomyelitis virus in Leopoldville, Belgian Congo. I. Description of the city, its history of poliomyelitis, and the plan of vaccination campaign, Bull. World Health Organ. 22: 203, 1960. 8. Gonzaga, A. J., Warren, R. J., and Robbins, F. C.: Attenuated pollovirus infection in in-
270
Cook and Jelliffe
fants fed colostrum from poliomyelitis immune cows, Pediatrics 32: 1039, 1963. 9. Plotkin, S. A.: Proceedings of the 6th International Congress of Microbiological Standardization, Berlin, 1961, Hoffman Verlag, pp. 48-73. 10. Pagano, J. S., PIotkin, S. A., Comely, D., Leuterer, W., and Koprowski, H.: The re-
Immunization in developing regions
The Journal of Pediatrics August 1968
sponses of premature infants to infection with attenuated poliovirus, Pediatrics 29: 794, 1962. 11. Pagano, J. S., Plotkin, S. A., and CorneIy, D.: The response of premature infants to infection with type 3 attenuated poliovirus, J. PEmAT. 65: 165, 1964.
e. f. g. h. i. j. k.
Cost of immunization Simplicity of the immunization procedure Cost of rehabilitation Economic burden of disabled Availability of the antigen Popular demand Cost of alternative methods of prophylaxis
Editorial comment The observations presented here by Katz and Plotkin appear to be a practical contribution to the prevention of poliomyelitis in developing countries. This article points up the question of the most suitable immunization schedule for such countries, where geographical and cultural difficulties in making contact with young children assume considerable importance, where staff and finance are in short supply, and where one must take every opportunity that offers of immunizing children, provided only that the vaccine will be effective at that age. The schedule recently suggested by Stanfield1 is a feasible one which we can now modify to include the first dose of oral poliomyelitis vaccine at birth. By adopting the plan of Katz and Plotldn, the infant may leave the maternity ward immunized with poliomyelitis vaccine as well as BCG and possibly also smallpox vaccine. Even if no further opportunity for immunization arises, at least something will have been achieved. However, one should seek to add an additional dose of poliomyelitis vaccine at a Iater date in infancy, and before 6 months of age, triple vaccine, and as soon as possible after 9 months of age, measles vaccine. Cook 2, s has described a plan by which the majority of young children in areas with only a limited health service can be cheaply immunized. In determining schedules of immunization for developing regions it is of value to bear in mind the following considerations4: 1. D I S E A S E
a. b. c. d.
PRIORITIES
Incidence of disease Mortality rate from disease "Conditioning" infection or not Cost of treatment
2. A G E - G R O U P
PRIORITIES
a. Above considerations by age group b. Accessibility of the group (e.g., newborn, schools) c. Ability of group to respond to antigen (e.g., BCG compared with measles in newborn) 3. T E C H N I Q U E S a. Number of doses required, minimum and optimum b. Dosage c. Methods of administration (oral, hypodermic, intradermal) d. Choice of preparations available e. Refrigeration requirements at base and in field 4. R E S U L T S a. Intermediate results of successful immunization b. Long-term results of successful immunization c. Number of adverse individual reactions to be expected R.
COOK~
IVf.D.
D. B. JELLIFFE~ M.D.
REFERENCES
1. Stanfield, J. P.: Organization of MCH services in devoloping regions. VI. Special programmes in immunization, J. Trop. Pediat. 13: 102, 1967. 2. Cook, R.: Comprehensive immunization in rural areas, J. Trop. Pediat. 2: 13, 1966. 3. Cook, R.: The Ankole pre-school protection programme 1964-67, Mimeographed document, Department of Paediatrics, Makerere Medical School. 4. JelIiffe, D. B.: Recent experience in maternal and child health in East Africa, J. Trop. Pediat. 2: 15, 1966.