Serum antibodies to poliomyelitis in Italy

Bulletin ofthe World Health Organization, 62 (4): 591 - 595 (1984) i World Health Organization 1984 Serum antibodies to poliomyelitis in Italy R. SANTORO,' F. LOMBARDI,' F. NOVELLO,' C. AMATO,' F. M. RUGGERI,2 M. E. GRANDOLFO, I. ANNESI,3 C. M. CORTELLESSA,3 F. TIMPERI,3 & R. INGLESE4 A seroepidemiological survey was conducted to assess the effectiveness of the Italian nationwide immunization programme againstpoliomyelitis. Over3800serum samples were collectedfrom persons, aged between 6 months and 79years and residing in urban and rural areas with different socioeconomic conditions and geographic locations. A very high level of protection was found in the target population, confirming the almost 100% history of complete immunization. Almost all subjects over 30 years of age were found to have poliovirus antibodies, although they had never been vaccinated. In Naples, a higher proportion ofsubjects susceptible to all 3 types of virus wasfound in the age group 6-23 months, suggesting a delay in the administration of vaccine. This was con- firmed by the analysis of the immunization history. No difference was observed between urban and rural areas, in either serological results or history of immunization. The results confirmed the higher immunogenicity ofpoliovirus type 2 with respect to the other two types, the effectiveness of the vaccine, and, finally, the efficiency of the operational procedures adoptedfor its administration. Immunization with Sabin oral poliovirus vaccine (OPV) has been practised in Italy since 1964, when a mass immunization programme was started. Before that year, an average of over 3000 cases of polio- myelitis were reported annually in Italy; in com- parison, there were only 60 reported cases in the period 1972-80, and only 6 cases, all in unvaccinated children, in 1981-83. The nationwide poliomyelitis control programme aims to immunize children aged 3-12 months with 3 doses of trivalent OPV, with revaccination at the age of 3 years. The service is provided all year round, under the control of local health authorities, which regularly receive a list of live births from local mayors. Each infant is individually sought out for immunization. Since 1966, immunization against poliomyelitis has been compulsory for all Italian children, and a certificate ofimmunization is required for admission to school. The apparent absence of poliomyelitis in the country is a good indicator of the effectiveness of the vaccination control programme; nevertheless, sero- logical surveys can provide useful information, com- plementing data on immunization coverage and l Virology Laboratory, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy. 2 Ultrastructure Laboratory, Istituto Superiore di Sanita, Rome, Italy. 3 Epidemiology and Biostatistics Laboratory, Istituto Superiore di Sanita, Rome, Italy. 4 Virology Service, Bambino Gesu Hospital, Rome, Italy. disease incidence obtained through routine reporting systems (1). In recent years, only partial immunological surveys have been carried out to confirm the favourable trend of poliomyelitis in Italy (2, 3). The present collabor- ative studya was planned to assess the immune status against poliomyelitis of the Italian population, by the analysis of sera collected in five geographical areas, representative of different socioeconomic con- ditions. MATERIALS AND METHODS City and sample selection Five cities, representing different socioeconomic environments and geographical areas (4), were chosen for sampling: Turin, in northern Italy; Rome and Perugia, in central Italy; and Naples and Bari, in southern Italy. For each city, an age-stratified sample was defined, the size of each age group being deter- mined so as to achieve a precision of 5% in the esti- mation of the prevalence of people susceptible to all three types of poliovirus, at a 95% confidence level. Collection ofsamples Blood specimens were obtained from people aged between 6 months and 79 years, attending outpatient a The participants in the study are listed in Annex 1. 4435 -591- 592 R. SANTORO ET AL. departments of city hospitals with both urban and rural catchment areas. From 1978 to 1982, sera were collected using a quota sampling method. Each centre was given an annual quota for each age group, and adopted a systematic selection of subjects based on the total number of outpatients. Each participant also completed a survey questionnaire, giving date of birth, vaccination history (with reference, if possible, to the immunization card), area of residence, educa- tional level, and occupational status (or that of the parents). Serology Neutralizing antibody titrations were carried out in tissue culture plates,b according to the method of Oberhofer et al. (5), with minor modifications. Serum and virus dilutions were made in Eagle's mini- mum essential medium (MEM) with 20 ml/litre fetal calf serum (FCS). Twofold dilutions of sera (from 1:2 to 1:256) were placed in the wells; 0.05 ml of virus (80-200 TCIDo5C) was added to each well and the mixture was incubated at 37 °C for 1 hour. Aliquots of 0.1 ml of a human heteroploid (HEp - 2) cell sus- pension, in MEM with FCS, containing 1.2 x 104 cells, were added to each well. The plates were covered with pressure-sensitive film and incubated at 37 °C for 7 days. Readings were made every other day using an inverted microscope. Antibody titre was taken as the highest positive serum dilution that com- pletely inhibited the cytopathic effect. Samples were considered negative if the antibody titre was less than 1:2; reciprocal geometric mean titres (GMT) were computed for positive sera. Analyses were performed b Microtest II, Falcon Division, Becton Dickinson and Co., Oxnard, CA 99030, USA. ' Median tissue culture infective dose. in the laboratories of each collaborating city centre following a standadrdized procedure. Sera collected in Rome were tested in the Istituto Superiore di SanitA. In each run, reference sera were included as controls. Statistical analysis An analysis of variance was performed on the GMT values in the various age groups. The x2 test was used to evaluate the vaccination status by area of resi- dence. RESULTS A total of 3834 sera were collected from people attending outpatient departments, 60%o of whom came from urban areas and the remainder from rural areas. Over 9907 of individuals in the age group 4-20 years had a history of complete immunization, with no significant difference between the groups from urban and rural areas. Table 1 shows the number of sera tested by age group, together with the percentages of subjects sus- ceptible to 1, 2 or 3 types of poliovirus. Overall, a very low percentage of subjects were susceptible to 2 or 3 poliovirus types, with a decrease with age. Higher proportions of sera in all age groups were found to lack antibodies against a single type. For those sus- ceptible to all 3 types, the vaccination status is given in Table 1, showing that in most cases vaccination was incomplete. A higher proportion of subjects sus- ceptible to all 3 types was observed in children aged 6-23 months in Naples (32.1%7o, 8 out of 9 with no history of immunization) than in any other city. Table 2 shows the distribution of subjects sus- ceptible to one poliovirus type, by age group. Since no Table 1. Distribution of subjects susceptible to poliovirus, by age group Age Subjects without antibodies (%) group Vaccination (years) No. tested One type Two types Three types status' 0.5-1 156 8.3 5.1 7.1 8nv, 2nk, lv 1 225 3.5 0.4 2.7 6 nv, 1 nk 2 205 3.9 0.5 0.5 1 nk 3 230 7.0 0.4 0.4 1 v2 4 234 1.3 0.4 0.4 1 nv 5-10 1195 3.4 0.7 0.2 2 v4 11-20 650 3.7 0.5 0.2 1 nv 21-30 437 4.1 0.2 0 > 30 502 6.2 0.2 0.2 1 nk ° Vaccination status of those susceptible to all 3 types: nv = not vaccinated, nk = not known, v = vaccinated, v2 = received 2 doses of vaccine, v4 = received 4 doses of vaccine. POLIOMYELITIS ANTIBODIES IN ITALY Table 2. Distribution of subjects susceptible to one type of poliovirus, by age group Age Subjects without one type of antibody 1%) group (years) No. tested Type 1 Type 2 Type3 0.5-1 156 3.2 0.6 4.5 1 225 1.3 0 2.7 2 205 1.5 0.5 2.0 3 230 2.6 0.4 3.9 4 234 0.9 0 0.4 5-10 1195 0.9 0.3 2.3 11-20 650 1.5 0.8 1.4 21-30 437 1.4 0.2 2.5 > 30 502 2.2 1.4 2.6 significant difference was observed among the cities, the data are grouped together. There was a higher prevalence of antibodies against type 2 poliovirus than against the others; this is consistent with the known higher immunogenicity of this type of virus. Fig. 1 shows the cumulative proportions of sero- positive subjects by titre, for age groups 6 months to 4 years, 5-10 years, 11-20 years, and over 20 years. The median neutralizing titre against type 2 was ap- proximately twice those against types 1 and 3 in all groups. The median titre in the oldest age group was half that in the youngest group, for all virus types. These results are confirmed in Table 3, which shows the percentage of seropositive subjects and GMT by age group for the three types of poliovirus. An analysis of variance of the GMT values in the various age groups gave a highly significant F value for each virus type. 100 TYPE ~80 60 _ X '440 E = 20 0 100 TP 80 Or 60 m_ 40 E 3 20 0 100 TP 80. Z 60 40 E _z 20 0 I:2 1 :4 1 8 1:16 1:32 1 :64 1 :128 1:256 Titre 0.5 4years LhI- years E1 120years = :_->20years Fig. 1. Cumulative distribution of antibody titres by age group for poliovirus types 1, 2 and 3. Table 3. Distribution of subjects seropositive to types 1, 2, and 3 polio"irsi". hy age group. Type 1 Type 2 Type 3 Age group No. tested % GMTa GMTa % GMT0 (years) positive positive positive 0.5-1 156 85.3 46.3 (37.9-56.6) 91.7 60.7 (51.8-71.0) 83.3 47.7 (38.9-58.6) 1 225 95.1 40.5 (37.5-43.9) 96.9 52.4 (46.5-59.0) 93.8 40.0 (34.4-46.6) 2 205 97.6 32.3 (27.9-37.5) 99.0 47.4 (41.1-53.8) 97.1 33.7 (29.1-39.1) 3 230 96.5 28.3 (24.3-33.0) 99.1 43.4 (37.7-49.8) 95.2 28.6 (24.4-33.5) 4 234 98.3 24.5 (21.1-28.4) 99.6 50.2 (43.7-57.6) 98.7 24.4 (20.7-28.8) 5-10 1195 98.4 25.0 (23.5-26.7) 99.3 43.3 (40.6-46.1) 97.0 22.7 (21.3-24.3) 11-20 650 98.0 20.5 (18.6-22.5) 98.9 35.1 (31.9-38.5) 98.0 20.5 (18.7-22.4) 21-30 437 98.4 27.4 (24.6-30.4) 99.5 35.3 (32.2-38.6) 97.5 22.0 (19.8-24.5) >30 502 97.2 23.9 (21.5-26.5) 97.8 36.3 (32.7-40.3) 96.6 17.1 (15.4-18.9) a Reciprocal geometric mean titre. Figures in parentheses give 95% confidence limits. 593 R. SANTORO ET AL. A detailed analysis of seropositive subjects over 30 years of age showed that the GMT for antibody to type 1 poliovirus decreased from 27.7 in the group aged 31-40 years to 13.2 in the group aged 71-79 years; the corresponding values for type 2 and type 3 polioviruses were 38.7-19.4 and 16.1-12.7, respec- tively. DISCUSSION A very small proportion of the subjects over 1 year of age in five cities in Italy were susceptible to 2 or 3 types of poliovirus. This suggests that, at this age, vir- tually all children have been vaccinated; this was con- firmed by the analysis of questionnaires concerning the immunization history. The only exception was in Naples, where a higher percentage of children aged 6-23 months were susceptible to all 3 types. These serological results were supported by the immuniza- tion history, as most of this group had not received any vaccine; only 70% of children aged 12-23 months had received three doses of OPV, compared with almost 1000/ in the other areas. In the older age groups, 100% vaccination coverage was reported in all cities, including Naples, indicating a delay in immunization in this city. Our findings are in good agreement with the reports of the national health authority indicating immunization coverage close to 100% throughout the country. In view of the finding of a very low prevalence of subjects susceptible to all 3 types among persons over 30 years old, who had never received vaccine, it is conceivable that immunity in this group is related to subclinical infection with wild strains of polioviruses in the prevaccination era. Immunity might also be partly due to spread of vac- cine virus after the introduction of Sabin vaccine. The finding of comparable GMT values in the 11-20-year age group and in the 31-79-year age group suggests that a similar immunity is conferred by OPV and wild viruses. The finding of people susceptible to one type of poliovirus in all age groups seemed to suggest a failure ofthe OPV and was therefore investigated further. By analysing data on these subjects for each single type of virus, it was found that fewer than 5% of subjects lacked antibody to any one type, confirming the cor- rectness of the OPV composition. Further, our data show that seroconversion is elicited immediately after the first cycle of vaccination. The slightly lower pro- portion of subjects susceptible to type 2 poliovirus in all age groups reflects the higher immunogenicity of this poliovirus with respect to the other two. This was further confirmed by the twofold higherGMT of anti- bodies to type 2 compared with types 1 and 3. The results of the present seroepidemiological study show that in Italy a very high prevalence of immunity to poliovirus has been attained among the whole population during an 18-year period of mass immunization with Sabin live attenuated oral polio- virus vaccine. The data are consistent with the very low number of reported paralytic cases of polio- myelitis in unvaccinated children in 1981-83: there was one imported case from the Libyan Arab Jamahiriya, two cases from whom two type 2 Sabin- like strains were isolated, two cases from whom two type 1 strains were isolated and characterized as fully virulent, and one case with a seroconversion against type 1. All these cases were studied in our laboratory. Altogether, this suggests that an adequate herd immunity has been achieved in Italy, which has greatly reduced the spread of wild virus. No difference was observed between rural and urban populations, either in serological results or in history of immunization, strongly suggesting that an efficient national network has been established for the administration of the vaccine. It must be borne in mind that wild polioviruses have not yet been eliminated and that an appropriate level of immunity should be maintained. The immune status of the population should be tested periodically. Where necessary, attempts should be made to ensure that the time schedule of vaccination is more closely adhered to. Finally, particular care should be taken with respect to imported cases of poliomyelitis. ACKNOWLEDGEMENTS This work was supported in part by CNR-ISS agreement no. 82.02422.52, and by the Italian Ministry of Health. RESUME ETAT IMMUNITAIRE DE LA POPULATION VIS-A-VIS DE LA POLIOMYtLITE EN ITALIE Plus de 3800 echantillons de serum ont et preleves chez rurales et urbaines de differentes regions d'Italie, dans des des sujets sains ages de 6 mois a 79 ans, vivant dans des zones conditions socio-economiques variees. On a recherche les 594 POLIOMYELITIS ANTIBODIES IN ITALY 595 anticorps neutralisants diriges contre les trois types de polio- virus. Pour chaque sujet, on a rempli un questionnaire indi- quant sa date de naissance, son lieu de r6sidence et ses ante- c6dents de vaccination. En gen6ral la prevalence des sujets sensibles aux 3 types de poliovirus 6tait tres faible dans tous les groupes d'age. Naples fait toutefois exception, 32,1% des enfants ag6s de 6 a 23 mois y etant depourvus d'anti- corps; la plupart d'entre eux n'avaient pas e vaccines. En ce qui concerne les enfants plus ages on n'a pas observe de variation de la prevalence des anticorps avec le lieu d'echantillonnage, ce qui suggere que la vaccination est pratiqu6e plus tardivement a Naples qu'ailleurs. La proportion de sujets non immuns s'est revele legere- ment plus elevee pour les poliovirus des types 1 et 3 que pour le type 2, observation qui confirme la plus grande immuno- genicite du virus de type 2. Neanmoins, on a constate que la prevalence des sujets sensibles a l'un ou l'autre des 3 types de virus 6tait tres faible a la fin du cycle de vaccination, ce qui d6montre a la fois l'efficacit6 du vaccin antipoliomy6litique buccal et celle du calendrier vaccinal. Le titre moyen geom6- trique des sujets seropositifs est compris entre 46,3 (groupe d'Age 6 a 11 mois) et 20,5 (groupe d'age 11 a 20 ans) pour le virus de type 1, entre 60,7 et 35,1 pour le virus de type 2 et entre 47,7 et 20,5 pour le virus de type 3, chiffres qui temoignent de la plus grande immunogenicite du virus de type 2. En conclusion, cette enquete sero-epid6miologique a confirme la grande efficacite du programme italien de vaccination contre la poliomyelite, comme le laissait supposer l'absence manifeste de cas de paralysie observ6e ces dernieres annees. REFERENCES 1. EVANS, A. S. The need for serologic evaluation of immu- nization programs. American journal of epidemiology, 112: 725-731 (1980). 2. SQUERI, L. ET AL. Osservazioni sulla situazione immuni- taria a distanza di 7 anni dall'introduzione in Italia della vaccinazione antipoliomielitica a V.V.A. Annali Sclavo, 15: 1-18 (1973). 3. VOLPI, A. ET AL. Seroimmunity to polioviruses in an urban population of Italy. Bulletin of the World Health Organization, 54: 275-278 (1976). 4. SANTORO, R. ET AL. Measles epidemiology in Italy. International journal of epidemiology, 13: (1984) (in press). 5. OBERHOFER, T. R. ET AL. Seroimmunity to poliomyelitis in an American community. American journal of epidemiology, 101: 333-339 (1975). Annex I COLLABORATING CENTRES The participants in the study were: Professor 0. Schiraldi and Dr E. Jandolo, Istituto di Clinica delle Malattie Infettive, University of Bari, Bari Professor M. Piazza and Professor L. Picciotto, Istituto di Clinica delle Malattie Infettive, Univer- sity of Naples, Naples Professor S. Paoluzzi and Professor G. Stagni, Istituto di Clinica delle Malattie Infettive, Univer- sity of Perugia, Perugia Dr D. Menichella, Virology Service, Bambino Gestu Hospital, Rome Professor G. Giuliani, Virology Laboratory, Amedeo di Savoia Hospital, Turin