Early immunization of neonates with trivalent oral poliovirus vaccine.

Early immunization of neonates with trivalent oral poliovirus vaccine* L.Y. Weckx,1 B.J. Schmidt,2 A.A. Herrmann,3 C.H. Miyasaki,4 & N.F. Novo5 Described is the evaluation in Brazil of the immune response of early immunization with trivalent oral poliovirus vaccine (TOPV). A total of 85 normal neonates from Sao Paulo were assigned one of the fol- lowing immunization schedules: group A - one dose of TOPV at birth and subsequent doses at 2, 4, and 9 months of age; or group B - one dose of TOPV at 2, 4 and 6 months of age. Blood samples were collected sequentially from the mother at delivery, from the umbilical cord, and from the child at 2, 4, 6, 9 and 12 months of age for assay of poliovirus neutralizing antibodies. Administration of TOPV at birth, in addition to establishing immunity against poliomyelitis at an earlier stage, produced a superior immune response to poliovirus type 3. At the end of the first year, the proportion of susceptible indivi- duals was 3.7% in group A and 25.9% in group B. When immunization against poliomyelitis is started at birth, excellent seroconversion rates are obtained from the third dose onward. Introduction Immunization of neonates with oral poliovirus vacci- ne (OPV) was studied extensively in the 1950s and 1960s (1-10). Recently, this possibility has been re- evaluated, because in developing countries, where poliomyelitis is not yet under control, early vaccina- tion would permit immunization at a time when there is little interference from intestinal organisms and ensure the administration of at least one dose while the child is still under medical care. In Sao Paulo, Brazil, more than 90% of deliveries occur in hospi- tals. Also, the occurrence in very young childrena of poliomyelitis that is associated with an immunity gap (11) would be prevented by earlier establishment of immunity against the disease. Recently, studies car- ried out in China (12) and in India (13) have reported excellent rates of seroconversion after early adminis- tration of poliovirus vaccine, with higher antibody titres during the first months of life (12,14). On the basis of these data WHO now recom- mends a supplementary dose of polio vaccine at * From Escola Paulista de Medicina, Sao Paulo, Brazil. Associate Professor, Division of Puericulture and Social Paediatrics, and Division of Immunization, Secretary of Health, State of Sao Paulo, Brazil. Requests for reprints should be sent to Dr Weckx at: Escola Paulista de Medicina, Departamento de Pediatria, Rua Botucatu, 740, 04023-Sao Paulo- SP, Brazil. 2 Professor, Division of Puericulture and Social Paediatrics. 3Assistant Professor, Division of Puericulture and Social Paediatrics. 4Associate Professor, Division of Neonatology. 5Associate Professor, Division of Biostatistics. a Neves, W.E. [Some aspects of poliomyelitis in the first 6- months of life. A study of 241 cases]. Doctoral thesis, Faculty of Medicine, University of Sao Paulo, 1972 (in Portuguese). Reprint No. 5251 birth, in addition to the three doses for primary immunization (15,16). We therefore carried out a study to assess the immunological response to early poliomyelitis vaccination of neonates in Sao Paulo. Materials and methods The protocol was approved by the Committee of Ethical Control of Clinical Research, Escola Paulista de Medicina, Sao Paulo. Study population A total of 85 children from Sao Paulo were evaluated from March 1986 to September 1988. The children satisfied the following criteria: they were normal full-term newborn infants, had a birth weight of over 2500g, and a 5-minutes Apgar score of >7. In addi- tion, their mothers had normal pregnancies, without infectious episodes, and amniorrhexis less than 12 hours before delivery. After obtaining the written consent of one of the parents, the neonates were assi- gned at random to one of two groups (A and B), each of which received different poliomyelitis immuniza- tion schedules. All children were followed up in the nursery and afterwards at monthly visits throughout the first year of life. Those who received an extra dose of vaccine as a result of immunization campai- gns were excluded from the study. Of the 85 neo- nates at the beginning of the study, only 54 (27 in each group) followed the protocol until the end. Immunization schedules Trivalent oral poliovirus vaccine (TOPV; SmithKline), which was distributed by the Ministry Bulletin of the World Health Organization, 70 (1): 85-91 (1992) © World Health Organization 1992 85 L.Y. Weckx et al. of Health, was used for both study groups; however, because the composition of the vaccine was modified during the study period, group B children received a higher concentration of vaccine to poliovirus type 3. The schedules and composition of vaccine used with the two study groups are shown below. - Group A: TOPV was given at birth (second day of life) and subsequently at 2, 4, and 9 months of age. Composition of vaccine: poliovirus type 1 (PI) = 1 million TCID50; type 2 (P2) = 100 000 TCID50; and type 3 (P = 300 000 TCID50. - Group B: TOPV was given according to the offi- cial schedule established in Brazil, i.e., at 2, 4 and 6 months of age. Composition of vaccine: P1 = 1 million TCID50; P2 = 100 000 TCID50; and P3 = 500 000 TCID50. Laboratory tests In each case blood samples were collected from the mother at delivery, from the umbilical cord, and from the child, by venepuncture, at 2, 4, 6, 9 and 12 months of age. Sera, which were obtained by centri- fugation of the blood samples, were stored at -20 °C until being analysed. The levels of types 1, 2 and 3 poliovirus antibodies were assayed by the neutraliza- tion technique (17) at dilutions of between 1:5 and 1:10 240. Evaluation parameters Individuals whose poliovirus neutralizing antibody titres were .1:5 were considered to be seropositive (11, 18, 19). Seroconversion was taken to be de- velopment of poliovirus neutralizing antibodies in previously seronegative individuals or at least a four- fold increase in the expected titre caused by a reduc- tion in transplacental antibodies. A maternal anti- body half-life of 30 days was assumed for this purpose (16). Statistical analysis Because of our sample characteristics, the following nonparametric methods were used in the statistical analysis of the results: Wilcoxon's, Friedman's and Cochran's G tests; the level of significance was 0.05 (5%). For calculation of the geometric mean anti- body titres (GMT), titres <1:5 were taken to be equal to 1:2.5 and those >1:10 240 to be 1:20 480 (20). Results With the exception of one child, poliovirus neutrali- zing antibodies were present in all the children at birth; the antibody levels were lower in the umbilical cord (GMT: P, = 109.49; P2 = 71.77; and P3 = 46.88) than in maternal blood (GMT: P1 = 130.03; P2 = 100.45; and P3 = 73.87). The difference was sig- nificant for P2. Sequential determination of poliovirus anti- bodies in a given child throughout the first year of life permits evaluation of the serological response to the vaccine. The results obtained were analysed for antibody level, seropositivity, susceptibility and sero- conversion. Antibody level For group A children (who received TOPV at birth) a slight decrease in the antibody titres to P1 and P3 occurred between birth and the second month of life, but increased thereafter (Fig. 1). In general there was a good serological response to this vaccination schedule, leading at the end of the first year to high antibody titres against the three polioviruses. For group B children (who were immunized first at the age of 2 months) the antibody levels for the three polioviruses decreased significantly until the second month. This was due to the natural loss of maternal antibodies present at birth. The reduction in antibodies to P3 was more pronounced, with the increase in titres occurring only from the fourth month onward. At the end of the first year the mean titres for P2 (557.22) and P1 (153.03) were consider- ably greater than the mean for P3 (40.02). Seropositivity In group A the percentage seropositivity (titre >1:5) for P1 and P2 was rather high and constant during the first year of life. The seropositivity for P3 decreased in the second month, but recovered subsequently, and by the end of the primary immunization sched- ule, the protection rates against the three polioviruses were excellent (Fig. 2) (Pi=100%, P2=100%, and P3=96.3%). In group B, although the seropositivity for P, and P2 remained high, there was a significant decrea- se in that for P3 until the fourth month (minimum seropositivity, 51.9%). At the end of the first year, while high seropositivity rates were obtained for Pi and P2, only 74.1% of the children were protected against poliovirus P3. Susceptibility Individuals who are susceptible to one, two, or three types of poliovirus were classifled as singly, doubly or triply susceptible, respectively. Almost all suscep- tible individuals in group A were singly susceptible, while in group B there were a considerable number 86WHO Bulletin OMS. Vol 70 199286 Early immunization of neonates with oral poliovirus vaccine Fig. 1. Geometric mean titres (reciprocal) for poliovirus neutralizing antibodies in group A and group B children; P1 = poliovirus type 1; P2 = poliovirus type 2; P3 = poliovirus type 3. Group A Group B 5 cn a) 0co 0) C E ._0 E0 a) 0.C ,.. ..~~~~~~~~~~~~~~~~~~~~...... .................. - P1 -- P2 .P3 0.5 O 2 4 6 I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0 2 4 6 Age (months) Antibody titres (reciprocal of GMT) 9 Cord 2 months 4 months 6 months 9 months 12 months 5 0 co 0) 0 aD cc E .0 E 0 a) C, 0.5 12 0 2 4 6 Age (months) Antibody titres (reciprocal of GMT) 00m aQ 129 Cord 2 months 4 months 6 months 9 months 12 months P1 86.45 63.13 145.72 180.08 123.06 205.63 P1 138.69 33.66 102.17 152.44 152.73 153.03 P2 65.46 193.64 840.26 805.20 457.74 532.67 P2 78.69 28.66 495.34 789.74 637.53 557.22 P3 41.06 16.75 84.99 165.96 93.96 97.67 P3 53.54 13.88 9.67 31.02 39.67 40.02 Fig. 2. Percentage of seropositive individuals (antibody titre >1:5) for poliovirus in group A and group B; P1 = polio- virus type 1; P2 = poliovirus type 2; P3 = poliovirus type 3. Group A Group B 100 -N -N011 -1 80 k.....P. ...... 20~ 2 4 6 Age (months) 100 80 1-- t 60 .>4 0. 0 40 a) cn) 20 [ 9 12 0 2 4 6 Age (months) WHO Bulletin OMS. Vol 70 1992 ..... ~~~~~~~~~..... ................................. - P1 -- P2 .P3 0 0- .=>" 60 co0. 2 40a) cn) - P1 -- P2 .P3 0 ...I. ... oit - P1 -- P2 ....P3 p 01 0 9 12 87 L.Y. Weckx et al. of doubly susceptible individuals, in addition to a higher overall proportion of susceptible individuals. In group A, the proportion of susceptibles aged 2-6 months was particularly low. At the end of the two schedules the proportion of susceptible individuals in group A was 3.7%, while in group B it was 25.9% (Fig. 3). Seroconversion Seroconversion, an important parameter in evalua- ting the active response to a vaccine, was determined by assaying the antibody levels in both groups two months after each dose of vaccine had been adminis- tered (Fig. 4). For children in group A, seroconversion increa- sed progressively with each successive dose. After three doses (including dose zero given at birth, the rate of seroconversion for the three polioviruses was high (PI = 92.6%, P2 = 96.3%, P3 = 92.6%), and close to that obtained after four doses (dose 3) (P1 = 100%, P2 = 100%, P3 = 96.3%). High rates of seroconversion for P1 (96.3%) and P2 (100%) were obtained for children in group B; however, only 74.1% of these children had sero- converted to the P3 vaccine virus by the end of the schedule. Discussion One of the problems associated with early immuni- zation with TOPV is the possible interference of maternal antibodies with the response to the adminis- tered vaccine (1-6, 12). In the present study, passive- ly transferred maternal antibodies were present in all but one neonate and did not seem to have influenced the final outcome, since almost all children in group A seroconverted. Dong et al. have shown that the seroconversion is delayed when high levels of mater- nal antibodies are present at birth (12); in contrast, we found this outcome only for P1. Because of the small number of patients in our study, the evaluation for other poliovirus types was difficult. The high gastric acidity during the first 24 hours of life (21,22) and the presence of ingested amniotic fluid (23) could theoretically interfere with vaccines given immediately after birth; the children were therefore vaccinated on their second day of life. Also, the chil- dren in our study were routinely breast-fed. Administration of a supplementary dose of TOPV at birth led to a higher level of polio antibo- dies during the first months of life (Fig. 1), with a greater seropositivity (Fig. 2), and consequently to a decrease in the proportion of susceptible individuals over this period (Fig. 3). Use of a supplementary dose at birth led also to increased protection at the end of the primary immunization schedule: only Fig. 3. Percentage of susceptible individuals (antibody titre <1:5) for poliovirus in group A and group B: single = susceptible to one poliovirus type; double = susceptible to two poliovirus types; total = all susceptibles. Group A Group B 50 0 40 1- 30 C) cj 10 0 2 4 6 Age (months) 9 12 0 2 4 6 Age (months) 0 9 12 WHO Bulletin OMS. Vol 70 1992 - Double -- Single . Total - - ~ 0.,rts....N4 s Il N. 1 4.' 88 Early immunization of neonates with oral poliovirus vaccine Fig. 4. Percentage seroconversion to poliovirus types after each dose of vaccine. Seroconversion was defined as the development of antibodies in formerly seronegative individuals or a .4-fold increase in the level of a type-specific antibody. Assays were performed 2 months after administration of each dose of vaccine. P1 = poliovirus type 1; P2 = poliovirus type 2; P3 = poliovirus type 3. Group A 100 80 Group B Dose 0 Dose 1 Dose 2 Dose 3 Dose 1 Dose 2 Dose 3 (birth) (2 months) (4 months) (9 months) (2 months) (4 months) (6 months) E P1i P2 P3 3.7% of susceptible individuals were in group A, compared with 25.9% in group B, whose schedule began in the second month of life. How can the improved serological response in children who receive TOPV at birth be explained? Why were the same results not observed in 2-month-old children who had already developed immunological mechan- isms, were subject to little interference from maternal antibodies, and who had received a more concen- trated vaccine? In India, John obtained excellent rates of sero- conversion in children who received their first dose of poliovirus vaccine during the first week of life, the rates being slightly higher than those resulting from administration of the vaccine at 5 or 6 weeks of age (13). In Brazil, Neves et al. have reported better rates of seroconversion when administration of TOPV is started in the first rather than in the third month of life (24). It should be noted that these two studies were performed in tropical countries, where the efficacy of the routinely administered oral polio- vaccine is lower than that in countries with tempera- te climates (25, 26). Several factors play a role in accounting for these differences: potency is reduced in tropical countries because of deficiencies in the storage of the vaccine (27); there could be diffe- rences in inhibitory factors in the digestive tract that have an effect on the implantation and replication of the vaccine virus (28); and the interference by other enteroviruses in small children in tropical countries may reduce the immune response (29-32). The inci- dence of enterovirus infections among neonates in Brazil (33) and elsewhere (34) is significantly lower than that among older children, and may ac- count for the better seroconversion rates observed in younger children. Our results indicate that there is WHO Bulletin OMS. Vol 70 1992 89 L.Y. Weckx et al. an improved serological response to TOPV when the schedule is started early, during the neonatal per- iod, particularly as far as poliovirus type 3 is con- cerned. During the outbreak of poliomyelitis in north- east Brazil in 1986, most cases were due to polio- virus type 3. Epidemiological investigations have revealed that the TOPV used to immunize the chil- dren has a low efficacy, particularly in terms of the P3 component. Higher seroconversion rates were obtained by doubling the P3 level (35), and this led the Brazilian Ministry of Health to modify the com- position of the vaccine used, increasing the P3 level from 300 000 TCID50 per dose to 500 000 TCID50 per dose. The children in group B in our study who re- ceived their first dose of TOPV in their second month of life, were immunized from the beginning with a vaccine that had the higher P3 level. Even so, the rate of seroconversion to P3 was lower than that of children who received their first dose at birth using vaccine with a P3 level of 300 000 TCID50 per dose. This indicates that immunization of neonates with TOPV may be a much more relevant factor than use of a higher vaccine concentration. With the official schedule, beginning in the second month of life, primary immunization with three doses of TOPV resulted in about 25% of the children remaining incompletely immunized, and susceptible to poliovirus type 3. In the case of expo- sure to wild poliovirus this proportion of the popula- tion can be considered to be at risk. In contrast, with early immunization, better protection can be achieved and the risk may diminish considerably. When a supplementary dose of TOPV is given at birth (zero dose), excellent rates of seroconversion for the three poliovirus types are produced from the third dose onward, and after the fourth dose, the seroconversion rates are still slightly higher (Fig. 4). No difficulty was experienced in administering the TOPV to neonates. The schedule is feasible operationally and requires no specialized technical knowledge or additional equipment. In view of the results obtained, we propose that immunization against poliomyelitis be initiated early in the neona- tal period; every newborn can receive the first dose of polio vaccine before being discharged from hospi- tal. This measure together with other efforts to improve vaccination will contribute considerably to the control of poliomyelitis. Acknowledgements We are indebted to the Institut Merieux, for carrying out the laboratory tests; the Centre International de l'Enfance, for valuable suggestions; and to CNPq (Brazilian Research Council), for financial support. Resume Vaccination precoce des nouveau-n6s par le vaccin antipoliomyelitique buccal trivalent La vaccination des nouveau-nes contre la polio- myelite offre certains avantages; par exemple, l'enfant recoit la premiere dose de vaccin lorsqu'il est encore a I'hopital, a un age ou il y a peu d'interference avec les germes enteriques et, sur- tout, cela permet d'assurer une protection precoce contre la maladie. Nous avons effectu6 une 6tude visant a quan- tifier la r6ponse immunitaire a une vaccination antipoliomyelitique pr6coce avec un vaccin buccal trivalent (VPOT) a Sao Paulo, au Bresil. Au total, 85 nouveau-nes normaux ont forme la population d'6tude, qui a 6te divisee au hasard en deux groupes soumis a differents calendriers vaccinaux: groupe A - une dose de VPOT a la naissance puis a 2, 4 et 9 mois; groupe B - une dose de VPOT a 2, 4 et 6 mois. Dans chaque cas, on a effectu6 des pr6levements sanguins au moment de l'accou- chement chez la mere et au cordon ombilical, puis chez l'enfant a 2, 4, 6, 9 et 12 mois. On a ainsi obtenu une evaluation s6quentielle de l'immunit6 antipoliomyelitique au cours de la premiere annee. On a titr6 les anticorps des trois souches de polio- virus par neutralisation. L'administration d'une dose suppl6mentaire de VPOT a la naissance, outre qu'elle etablit l'immu- nite antipoliomyelitique a un age plus precoce, produit egalement une meilleure reponse immuni- taire contre le P3. A la fin de la premiere annee, la proportion de sujets sensibles etait de 3,7% dans le groupe A et de 25,9% dans le groupe B. La faible incidence des infections enterovirales chez le nouveau-n6 a peut-etre contribu6 a cette meilleure r6ponse immunitaire. Lorsqu'on com- mence la vaccination antipoliomyelitique des la naissance, on obtient d'excellents taux de sero- conversion a partir de la deuxieme dose c'est-a- dire a 4 mois. Notre calendrier vaccinal est tout a fait appli- cable et chaque enfant a requ sa premiere dose de vaccin avant sa sortie de l'h6pital. Cette facon de proceder devrait se g6neraliser, en particulier dans les pays ou cette maladie s6vit encore. References 1. Koprowski, H. et al. Immunization of infants with living attenuated poliomyelitis virus. Journal of the 90 WHO Bulletin OMS. Vol 701992 Early immunization of neonates with oral poliovirus vaccine American Medical Association, 162: 1281-1288 (1956). 2. Plotkin, S.A. et al. Clinical trials in infants with oral- ly administered attenuated poliomyelitis viruses. Pediatrics, 23: 1041-1062 (1959). 3. Pagano, J.S. et al. Variations in the responses of infants to living attenuated poliovirus vaccines. New England journal of medicine, 264: 155-163 (1961). 4. Krugman, S. et al. Immunization with live attenu- ated poliovirus vaccine. American journal of diseases in children, 101: 23-29 (1961). 5. Lepow, M.L. et al. Effect of Sabin type 1 polio- myelitis vaccine administered by mouth to newborn infants. New England journal of medicine, 264: 1071-1078 (1961). 6. Sabin, A.B. et al. Effect of oral poliovirus vaccine in newborn children. I. Excretion of virus after inges- tion of large doses of type 1 or of mixture of all three types, in relation to level of placentally trans- mitted antibody. Pediatrics, 31: 623-640 (1963). 7. Sabin, A.B. et al. Effect of oral poliovirus vaccine in newborn children. II. Intestinal resistance and anti- body response at 6 months in children fed type 1 vaccine at birth. Pediatrics, 31: 641-650 (1963). 8. Plotkin, S.A. et al. Oral poliovirus vaccination in newborn African infants. American journal of diseases in children, 111: 27-30 (1966). 9. Katz, M. et al. Oral poliovirus vaccination in new- born African infants: relative ineffectiveness of early feeding of vaccine. Tropical and geographical medi- cine, 20: 133-136 (1968). 10. Keller, R. et al. Intestinal IgA neutralizing anti- bodies in newborn infants following poliovirus immu- nization. Pediatrics, 43: 330-338 (1969). 11. Melnick, J.L. et al. Developing gap in immunity to poliomyelitis in an urban area. Journal of the American Medical Association, 209: 1181-1185 (1969). 12. Dong, D.X. et al. Immunization of neonates with tri- valent oral poliomyelitis vaccine (Sabin). Bulletin of the World Health Organization, 64: 853-860 (1986). 13. John, T.J. Immune response of neonates to oral poliomyelitis vaccine. British medical journal, 286: 881 (1984). 14. Expanded Programme on Immunization. Immunization of neonates with trivalent oral live poliovirus vaccine. Weekly epidemiological record, 59(48): 369-376 (1984). 15. Expanded Programme on Immunization: Global Advisory Group. Weekly epidemiological record, 60(3): 13-16 (1985). 16. Halsey, N. & Galazka, A. The efficacy of DPT and oral poliomyelitis immunization schedules initiated from birth to 12 weeks of age. Bulletin of the World Health Organization, 63: 1151-1169 (1985). 17. Rosenbaum, M.J. et al. A simplified method for virus tissue culture procedures in microtitration plates. Proceedings of the Society for Experimental Biology and Medicine, 113: 224-229 (1963). 18. Liberto, M.I.M. et al. Immunological response for poliovirus in cases of poliomyelitis in Rio de Janeiro in 1975. Revista Latinoamericana de microbiologia, 20: 169-176 (1978). 19. Schatzmayr, H.G. et al. Serological evaluation of poliomyelitis oral and inactived vaccines in an urban low-income population at Rio de Janeiro, Brazil. Vaccine, 4: 111-113 (1986). 20. White, C. Statistical methods in serum surveys. In: Paul, J. R. & White, C., ed. Serological epidemiolo- gy, New York, Academic Press, 1973, pp. 19-32. 21. Faber, H. K. & Dong. L. Inactivation of poliomyelitis virus in relation to gastric and intestinal digestion. Proceedings of the Society for Experimental Biology and Medicine, 63: 575-577 (1946). 22. Ebers, D.W. et al. Gastric acidity on the first day of life. Pediatrics, 18: 800-802 (1956). 23. Steigman, A.J. & Lipton, M.H. Neonatal immunity. II. Poliocidal effects of amniotic fluids. Proceedings of the Society for Experimental Biology and Medi- cine, 99: 576-579 (1958). 24. Neves, W.E. et al. [Sabin type poliomyelitis vacci- nation in infants aged 1-3 months of age]. Revista do Instituto de Medicina Tropical de Sao Paulo, 10: 376-382 (1968) (in Portuguese). 25. Drozdov, S.G. & Cockburn, W.H. The state of poliomyelitis in the world. In: The First International Conference on Vaccines against Viral and Rick- ettsial Diseases of Man, Washington. Washington, DC, Pan American Health Organization, 1967, pp. 198-209 (Scientific Publication No. 147). 26. John, T.J. Antibody response of infants in tropics to five doses of oral polio vaccine. British medical jour- nal, 1:812 (1976). 27. Sokhey, J. et al. Stability of oral polio vaccine at different temperatures. Vaccine, 6: 12-13 (1988). 28. Domok, I. et al. Factors affecting the efficacy of live poliovirus vaccine in warm climates. Bulletin of the World Health Organization, 51: 33-47 (1974). 29. Feldman, R.A. et al. Oral poliovirus vaccination in children: a study suggesting enterovirus interfer- ence. Pediatrics, 33: 526-533 (1964). 30. Montefiore, D.G. Problems of poliomyelitis immuni- zation in countries with warm climates. In: Pro- ceedings of The International Conference on the Application of Vaccines against Viral, Rickettsial and Bacterial Diseases of Man, Washington, 14-18 December 1970. Washington, Pan American Health Organization, Washington, 1971, pp. 182-185. (Scientific Publication No. 226). 31. John, T.J. & Jayabal, P. Oral polio vaccination of children in the tropics. I. The poor seroconversion rates and the absence of the viral interference. American journal of epidemiology, 96: 263-269 (1972). 32. Idris, M.Z. et al. Oral polio vaccination and factors affecting its efficacy. Indian journal of medical research, 71: 671-678 (1980). 33. Waldman, E.A. et al. [Occurrence of enterovirus infection among neonates in S&o Paulo (1960)]. Revista do Instituto Adolfo Lutz, 26: 7-12 (1984) (in Portuguese). 34. Moscovici, C. & Maisel, J. Intestinal viruses of newborn and older prematures. American journal of disease in children, 101: 771-777 (1961). 35. Patriarca, P.A. et al. Randomized trial of alternative formulations of oral poliovaccine in Brazil. Lancet, 1: 429-433 (1988). WHO Bulletin OMS. Vol 70 1992 91