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Factors influencing immunity against diphtheria in adults
PII: S0264-410X(97)00148-5
Vaccine, Vol. 16, No. 1, pp. 70–75, 1998 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264–410X/98 $19+0.00
Factors influencing immunity against diphtheria in adults Hans-Martin Hasselhorn*§, Matthias N¨ ubling†, Friedrich W. Tiller‡ and Friedrich Hofmann* In about 50% of all adult Germans, serological immunity to diphtheria is lacking even though a vaccination history exists in most cases. In this study, the factors contributing to diphtheria immunity, up to 43 years after the last diphtheria vaccination, were analysed. Serum samples were taken from 287 adults whose complete vaccination history was available, and who had received a standard primary vaccination. The sera were tested by tissue culture neutralization assay for diphtheria antitoxin levels.The time lapse since the last diphtheria vaccination proved to be the most important factor influencing the protection rate. Secondly, the factor last vaccination within the first 3 years of life and, finally, female gender were found to correlate significantly with a low protection rate. No direct effect could be established for age, number of previous vaccinations, occupational contact with patients or travel within any endemic region during the last 10 years. Since diphtheritic infection can occur among previously vaccinated persons, the immunity gap observed among adults should be closed by regular diphtheria boosters. Special attention should be paid to the three groups mentioned. Further investigation is required to determine whether or not a single booster vaccination is sufficient to provide long-term protection for those in the risk groups indicated above. © 1997 Elsevier Science Ltd. Keywords: diphtheria; booster; vaccination; adults
During the last 40 years diphtheria has begun to appear increasingly in the older age groups1. Indeed, in the Russian Federation and the Ukraine, adults accounted for approximately 73 to 77% of all reported cases in 19922. In Germany, adult vaccination against diphtheria has practically ceased since the early 1970s, in spite of the official recommendation of one booster vaccination every 10 years. Today, in this country as in many other industrialized countries, full serological protection against diphtheria is only detectable in less than 50% of the adult population3–5, even though most adults have some history of vaccination. In 1993, the European Advisory Group (EAG) of the WHO Expanded Programme on Immunization (EPI) demanded that by 1995 the diphtheria immune status of the adult population should be assessed in all countries2. The present study assesses factors influ*Department of Occupational Health-FB 14, Arbeitsmedizin, University of Wuppertal, D-42097 Wuppertal, Germany. †Freiburger Forschungsstelle Arbeits- und Sozialmedizin, Freiburg, Germany. ‡Medizinisch-Immunologische Laboratorien, M¨ unchen, Germany. §To whom correspondence should be addressed. Department of Occupational Health-FB 14, Arbeitsmedizin University of Wuppertal, Gauss-Strasse 20, D-42097 Wuppertal. Tel: ++49/202/439-2112; Fax: ++49/202/439-2068. (Received 11 November 1996; revised version received 26 May 1997; accepted 28 May 1997)
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encing protection against diphtheria by determining diphtheria antitoxin levels in sera from 287 adults and also by taking into account their complete vaccination history.
MATERIALS AND METHODS A total of 287 healthy adults (154 women and 133 men: mean age 26.4 years; range 17–54, ¹6.1) were recruited in the course of an occupational health survey at the University Hospital, Freiburg. Apart from obtaining informed consent, participation was limited to those for whom a complete record of diphtheria vaccination was available and who had received a correct basic immunization. Serology Blood samples were taken and the serum stored at µ20°C. A modified tissue culture toxin neutralization assay (NT) according to Miyamura et al.6,7 was used to determine the diphtheria antitoxin levels. The tests were carried out on microplates with 96 wells. As culture medium, Eagle’s minimal essential medium (MEM) enriched with 10% calf serum was used. Titration was carried out with 50 ml micropipettes in 2-fold steps from 1:2 up to 1:4096. A total of 25 ml of challenge toxin (100 tissue culture toxic doses, SIGMA) were added to each well. After 1 h at 20°C
Diphtheria immunity: H.-M. Hasselhorn et al. each well received 100 ml culture medium containing 105 viable Vero cells per milliliter. The dilution step of the serum neutralizing toxin in 90% of the cultures was given as titre. The potency of the toxin in each test was determined after 3 days’ incubation at 37°C (5% CO2) by titration against diphtheria antitoxin (Behringwerke AG). Our NT showed a very high correlation when 27 samples were counter-tested by a tissue culture neutralization assay carried out by Behringwerke AG, Marburg (r = 0.97 for correlation of the dilution steps, Ps0.001). The tissue culture neutralization assay is regarded as the most accurate in vitro procedure for measuring diphtheria antitoxin1,8,9,11,12, whereas the ELISA and passive haemagglutination methods are known to be inaccurate in the low antitoxin range8. The following internationally accepted definitions of diphtheria protection8–10 were used in this study: persons with antitoxin values below 0.01 IU mlµ1 were rated as ‘probably susceptible’, from 0.01 to s0.1 IU mlµ1 as ‘basically protected’ and with values E0.1 as ‘fully protected’. Negative values were rated as 0.001 and values above 9 IU mlµ1 as 9.5. Data analysis and statistics Statistical analysis included analysis of variance, correlation analysis, multiple linear regression, path analysis and logistic regression13–15. In all instances, full protection against diphtheria was used as the dependent variable. Independent factors thought to be potentially influential were measured and tested: gender; age; time interval since last vaccination; number of previous vaccinations; occupational contact with patients and travel within any endemic region during the last 10 years. A complete record of previous vaccination dates was used to generate the variable: last vaccination within/after the first 3 years of life.
RESULTS The participants (n = 287) had received a mean of 4.4 diphtheria vaccinations (range: 3–8, ¹1.1), the last vaccination being 19.2 years (mean) before (range: 3 months–43.0 years, ¹7.8 years). No significant gender differences were found in either mark. The lowest values measured were 0.002 IU mlµ1, the highest > 9.0 IU mlµ1. Ninety (31.4%) out of the 287 participants were ‘probably susceptible’ to diphtheria, 76 (26.5%) had ‘basic protection’ and 121 (42.2%) had ‘fully protective’ antitoxin levels. The geometric mean antitoxin level (GMT) was 0.04 IU mlµ1. Women were fully protected in 35.7%, and men in 49.6% of cases. No significant influence of the factors occupational contact with patients and travel within any endemic region during the last ten years on the protection rates could be observed. On a bivariate level significant correlations with diphtheria antitoxin levels and the protection rate was found for the factors time interval since the last vaccination (Figure 1); age (Figure 2); number of previous vaccinations (Figure 3); last vaccination within/after the first 3 years of life and gender (Figure 4). Since these last factors—except gender—are highly correlated, multivariate analysis was necessary to exclude confounders and redundant predictors.
Figure 1 Diphtheria antitoxin levels by time interval since last vaccination. Stages: s0.01 IU mlµ1 = probably susceptible; 0.01–0.1 IU mlµ1 = basic protection; E0.1 IU mlµ1 = full protection. GMT = 0.04 IU mlµ1; negative values treated as 0.001. ntotal = 287
Multiple linear regression analysis produced a model (multiple R = 0.53) which included the factors shown in Figure 5. The factor number of past vaccinations was dropped, because it did not have a genuine supplementary effect in the multivariate model. As can be seen from the arrows und their thickness in the path model, three genuine predictor-variables appeared. The most important of these is the time interval since the last vaccination (bpartial = µ0.388, Ps0.001) followed by booster after the third year of life (bpartial = 0.227, Ps0.001) and female gender (bdirect = µ0.140, Ps0.05). The factor age does not have a direct influence on the diphtheria protection rate in this multivariate model, but since age is very closely related to the first two predictors, its indirect influence is shown in our path model. The estimated probabilities for a protective antibody level depending upon the three factors (obtained by logistic regression modelling with LR-test; model statistics: Gmodel = 93.2, df = 3, pmodel s0.001; µ2 Log likelihood = 298, goodness of fit = 285) are given in Figure
Figure 2 Diphtheria protection rates (E0.1 IU mlµ1) by age groups. etabivariat = 0.30, Ps0.001, no significant direct effect in the multivariate model. ntotal = 287, n17–20 = 39, n21–23 = 68, n24–26 = 63, n27–29 = 49, n30–32 = 31, n33–40 = 27, n > 40 = 10
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Figure 3 Diphtheria protection rates (E0.1 IU mlµ1) by number of previous vaccinations. etabivariat = 0.37, Ps0.001, no significant effect in the multivariate model. ntotal = 287, n3 vaccin. = 63, n4 vaccin. = 100, n5 vaccin. = 81, n6–8 vaccin. = 43
4. One example may clarify this result. For a man, the probability of still being protected 20 years after the last vaccination is around 50%. After the same time interval, a woman is less likely to be still protected (35%). If the person had received his or her last diphtheria vaccination within the first 3 years of life, the probability of still being protected is less than 20%, without any significant gender difference.
DISCUSSION Diphtheria vaccine and vaccination policies in Germany Those responsible for a German vaccination policy recommend basic immunization against diphtheria during infancy, followed by regular booster vaccina-
Figure 4 Estimated probability for diphtheria protection by the predictors: time interval since last vaccination, gender and last vaccination in early childhood. ntotal = 287, nmale = 133 (n = 32 last vaccination in early childhood), nfemale = 154 (n = 37 last vaccination in early childhood)
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Figure 5 Path model of factors influencing protection against diphtheria (E0.1 IU mlµ1). Thickness of arrows equals partial path coefficient. No direct influence of age. ntotal = 287
tions at 6 years of age, 10–12 years of age and thereafter (as in other countries, e.g. the USA16) with one booster vaccination every 10 years17. The common diphtheria vaccine used from 1925 to 1960 contained 75 IU toxoid per dose, since then this has been reduced to 50 IU. In 1984, a reduced dose for adults and adolescents (from 7 years of age) containing about 5 IU toxoid per dose was introduced. Protection rates In our study, 42.2% of the participants had full serological protection against diphtheria (E0.1 IU lµ1) and 31.4% were ‘probably susceptible’ (s0.01 IU mlµ1). These results lie within the same range as other German3,4 and international studies assessing diphtheria protection among adults9,18–27 (range ‘full protection’ 20.5–49.3%, ‘probably susceptible’ 15.3–41%). Much higher full protection rates of 74.7 and 87% were found in Poland28 and Israel29 respectively. Variations between the studies may be explained by differing vaccination schedules. However, varying selection criteria for participants and the different serological test systems used must also be taken into account. Therefore a reliable comparison of these data is not easily obtainable. Factors influencing diphtheria protection In the present study, a detailed analysis of the factors assumed to contribute to the decline of diphtheria antitoxin levels was facilitated by the fact that, in contrast to other investigations, only participants who could present unbroken and complete written documentation of previous diphtheria vaccinations from birth on were included in the study. Vaccination records do not always reflect the real vaccination history. In Germany, diphtheria vaccine was not until recently given during military service or as part of the treatment of traumatic injury. The records of diphtheria vaccination can therefore be regarded as reliable in cases where the complete vaccination documents are available.
Diphtheria immunity: H.-M. Hasselhorn et al. Time interval since last vaccination. Our results support previous findings that diphtheria antitoxin levels wane with the time elapsed since the last vaccination1,5,8,10,22,30–35. The clinical consequences of this became evident in a recent retrospective study in Moskow, where the risk of acquiring diphtheria was 2.5 times higher for children who had received the last vaccination 4–7 years earlier than for those whose most recent immunization was within the last year36. Previous vaccinations. During the course of the present diphtheria epidemic in Eastern Europe it was found that people with four or five previous vaccinations were more resistant to clinical diphtheria than those with 1–3 vaccinations2. Our multiple linear regression analysis indicates that these findings rather may be explained by the fact that those with the greater number of vaccinations are likely to have received the last booster more recently, and it is this time interval since last vaccination which has proved to be the most influential protective factor. Even a differential comparison between people with basic immunization alone and those with additional booster doses revealed no significant difference in the protection rates when controlled for time interval since last vaccination. As our results demonstrate, the factor last vaccination within the first 3 years of life has a significant influence on diphtheria protection, since those who were last vaccinated before the age of three were significantly less well protected. A high level of maternally acquired diphtheria antibodies hinders optimal vaccination in infants8. According to Barkin et al.37 and Edwards et al.38, revaccination following basic vaccination produced higher diphtheria antitoxin levels than primary vaccination alone. Gender. In our study, women had a significantly lower protection rate than men even when several other potential confounder variables were checked. A survey of the literature shows that lower antitoxin levels and protection rates among women were more or less pronounced in other studies3,4,9,10,19,22,26,35,39–41. The opposite was reported in one German24 and one Italian study42 and no clear difference was found in Poland18. These differing results are difficult to explain, but it is noteworthy that in these three studies the passive haemagglutination method was used to determine the antitoxin levels. This test was used in only one of the studies reporting lower protection among women22. In some countries other than Germany, the lower diphtheria antitoxin levels found among women might be explained by the routine administration of combined tetanus and diphtheria vaccines following injury (known to occur more frequently in boys and men10) or for military personnel10,19,22,26,39–41. A lower protection rate among women which could not be explained by either of these ways was reported by Maple et al.9, Simonsen10 and finally by Rieger and Kuhlmann3, who observed a similar gender difference even among children and adolescents. Kjeldsen et al. also conjectured that diphtheria immunity following vaccination might be less long-lasting among women23. The results of this study have established that, in the long term, diphtheria vaccination offers less effective serological protection to women than to men.
It is possible that vaccination is less efficient in women, or that decline of the antitoxin levels in women after vaccination is more pronounced than in men. Age. Galazka reported that whereas in the prevaccination era diphtheria immunity in the population constantly increased with age1, the postvaccination era was associated with a high prevalence of immunity among children, an immunity gap in young adults and thereafter increased immunity in older people5. This immunity gap was reported in several studies3–5,12,24,26, but was not in others9,19,40,41. In our study the effect of age on diphtheria protection disappeared when checked against time interval since last vaccination. Other studies have demonstrated the dependence of the protection rate upon the age factor; but since age merely indicates that a longer time interval is more likely to have elapsed without a booster vaccination, this relationship cannot not be interpreted causally. The increase in immunity among older people is commonly explained by their repeated natural contact with C. diphtheriae toxin. We believe that both early vaccinations and/or natural contacts with Corynebacterium diphtheriae among people born before 1950, in combination with additional vaccinations during the 1950s and 1960s, have contributed to the higher protection rate found in elderly people today. A high protection rate among adults is necessary Previously vaccinated children and adults can acquire clinical diphtheria1,2,5,43–45. For example, 70% of all cases in St Petersburg at the beginning of the outbreak in Russia had previously been vaccinated2, but previous vaccination appeared to reduce the severity of the disease and the likelihood of a fatal outcome2. No level of antitoxin can provide absolute protection against diphtheria46, because such factors as virulence, the dose of the acquired bacillus19 and the general immune status of the infected person2,41,47–49 also determine whether or not symptomatic disease will occur. In Germany, a relatively low but increasing immunization coverage among children (the immunization coverage of three doses of DPT vaccine had reached 75%50 in 1993) and an immunity gap among young and middle-aged adults were found. Even though extensive diphtheria epidemics are unlikely to occur under the present epidemiological circumstances, an increased risk of outbreaks does exist5. According to the WHO, over 90% of all children and more than 75% of adults ought to be protected to prevent an epidemic outbreak of the disease2. Booster vaccinations for adults are necessary to provide collective immunity and prevent local outbreaks of diphtheria among adults. To prevent further immunity gaps among adults, it is necessary to ascertain whether or not one booster suffices to provide 10 years protection in all cases.
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WHO. Diphtheria epidemic in Europe: Emergency and response. Report on a WHO Meeting in St Petersburg, Russia, 5–7 July 1993. EUR/ICP/EPI 038 Rieger, J. and Kuhlmann, D. : Diphtherieimmunit¨ at der Bev¨ olkerung in Deutschland. Ges. Wes. 1994, 56, 667–671. Thilo, W. Immunit¨ at gegen Diphtherie in Deutschland-Populationsimmunit¨ at weist L¨ ucken auf. T.W. P¨ adiatrie 1993, 6, (5), 350–354. Galazka, A.M. and Robertson, S.E. Immunization against diphtheria with special emphasis on immunization of adults. Vaccine 1996, 14, 845–857. Miyamura, K., Nishio, S., Ito, A., Murata, R. and Kono, R. Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells. I. Studies on factors affecting the toxin and antitoxin titration. J. Biol. Standard. 1974, 2, 189–201. Miyamura, K., Tajiri, E., Ito, A., Murata, R. and Kono, R. Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells. II. Comparison with the rabbit skin method and practical application for seroepidemiological studies. J. Biol. Standard. 1974, 2, 203–209. Galazka, A.M. The Immunological Basis for Immunization; Module 2: Diphtheria. WHO 1993, WHO/EPI/GEN/93.12 Maple, P.A., Efstratiou, A., George, R.C., Andrew, N.J. and Sesardic, D. Diphtheria immunity in UK blood donors. Lancet 1995, 345, 963–965. Simonsen, O. Vaccination against tetanus and diphtheria. Dan. Med. Bull. 1989, 36, 24–47. Bj¨ orkholm, B. Clinical and immunological aspects of diphtheria and diphtheria vaccination. Dissertation, Department of Infectious Diseases, University of G¨ oteborg, Sweden, 1990 Naumann, P., Hagedorn, H.J. and Paatz, R. DiphtherieImmunit¨ at und ihre epidemiologische Bedeutung. Dtsch. Med. Wschr. 1983, 108, 1090–1096. Hosmer, Jr., D.W. and Lemeshow, S. Applied Logistic Regression. John Wiley, New York, 1989 Hennekens, C.H. and Buring, J.E. Epidemiology in Medicine. Boston/Toronto, 1987 Fisher, R.A. and Yates, F. Statistical Tables for Biological, Agricultural and Medical Research. 6th edn, Oliver & Boyd, Edinburgh, 1963 ACIP. Diphtheria, tetanus, and pertussis: recommendations for vaccine use and other preventive measures—recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR 1991, 40, (RR-10) St¨ andige Impfkommission. Impfempfehlungen der St¨ andigen Impfkommission am Robert Koch-Institut/STIKO, RKI, Robert Koch Institut. Deutsches Gr¨ unes Kreuz. 1. Aufl., Stand Oktober 1995, Marburg. Kilian Verlag, 1996 Galazka, A.M. and Kardymowicz, B. Immunity against diphtheria in Poland. Epidem. Inf. 1989, 103, 587–593. Christenson, B. and B¨ ottiger, M. Serological immunity to diphtheria in Sweden in 1978 and 1984. Scand. J. Infect. Dis. 1986, 18, 227–233. Bj¨ orkholm, B., Wahl, M., Granstr¨ om, M. and Hagberg, L. Immune status and booster effects of low doses of diphtheria toxoid in Swedish medical personnel. Scand. J. Infect. Dis. 1989, 21, 429–434. Gil, A., Lasberas, I., Gonz´ ales, A. and Dal-R´ e, R. Study to evaluate immunogenicity and reactogenicity of tetanusdiphtheria (Td) vaccine administered as booster dose in adults. In 6th International Congress for Infectious Diseases, Prague, Tschechia, 26–30 April 1994 Kjeldsen, K., Simonsen, O. and Heron, I. Immunity against diphtheria 25–30 years after primary vaccination in childhood. Lancet 1985, i, 900–902. Kjeldsen, K., Simonsen, O. and Heron, I. Immunity against diphtheria and tetanus in the age group 30–70 years. Scand. J. Infect. Dis. 1988, 20, 177–185. Pilars de Pilar, C.E. and Spiess, H. Auffrischimpfungen gegen Diphtherie und Tetanus. In: Neue Schutzimpfungen-Impfempfehlungen, Aufkl¨ arung, Widerst¨ ande (Eds Spiess, H. and Maass, G.). Verlag Deutsches Gr¨ unes Kreuz/DVV, 1992, pp. 179–188 Rappuoli, R., Podda, A., Giovannoni, F., Nencioni, L., Peragallo, M. and Francolini, P. Absence of protective immunity against diphtheria in a large proportion of young adults. Vaccine 1993, 11, 576–577.
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Factors influencing immunity against diphtheria in adults Hans-Martin Hasselhorn*§, Matthias N¨ ubling†, Friedrich W. Tiller‡ and Friedrich Hofmann* In about 50% of all adult Germans, serological immunity to diphtheria is lacking even though a vaccination history exists in most cases. In this study, the factors contributing to diphtheria immunity, up to 43 years after the last diphtheria vaccination, were analysed. Serum samples were taken from 287 adults whose complete vaccination history was available, and who had received a standard primary vaccination. The sera were tested by tissue culture neutralization assay for diphtheria antitoxin levels.The time lapse since the last diphtheria vaccination proved to be the most important factor influencing the protection rate. Secondly, the factor last vaccination within the first 3 years of life and, finally, female gender were found to correlate significantly with a low protection rate. No direct effect could be established for age, number of previous vaccinations, occupational contact with patients or travel within any endemic region during the last 10 years. Since diphtheritic infection can occur among previously vaccinated persons, the immunity gap observed among adults should be closed by regular diphtheria boosters. Special attention should be paid to the three groups mentioned. Further investigation is required to determine whether or not a single booster vaccination is sufficient to provide long-term protection for those in the risk groups indicated above. © 1997 Elsevier Science Ltd. Keywords: diphtheria; booster; vaccination; adults
During the last 40 years diphtheria has begun to appear increasingly in the older age groups1. Indeed, in the Russian Federation and the Ukraine, adults accounted for approximately 73 to 77% of all reported cases in 19922. In Germany, adult vaccination against diphtheria has practically ceased since the early 1970s, in spite of the official recommendation of one booster vaccination every 10 years. Today, in this country as in many other industrialized countries, full serological protection against diphtheria is only detectable in less than 50% of the adult population3–5, even though most adults have some history of vaccination. In 1993, the European Advisory Group (EAG) of the WHO Expanded Programme on Immunization (EPI) demanded that by 1995 the diphtheria immune status of the adult population should be assessed in all countries2. The present study assesses factors influ*Department of Occupational Health-FB 14, Arbeitsmedizin, University of Wuppertal, D-42097 Wuppertal, Germany. †Freiburger Forschungsstelle Arbeits- und Sozialmedizin, Freiburg, Germany. ‡Medizinisch-Immunologische Laboratorien, M¨ unchen, Germany. §To whom correspondence should be addressed. Department of Occupational Health-FB 14, Arbeitsmedizin University of Wuppertal, Gauss-Strasse 20, D-42097 Wuppertal. Tel: ++49/202/439-2112; Fax: ++49/202/439-2068. (Received 11 November 1996; revised version received 26 May 1997; accepted 28 May 1997)
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encing protection against diphtheria by determining diphtheria antitoxin levels in sera from 287 adults and also by taking into account their complete vaccination history.
MATERIALS AND METHODS A total of 287 healthy adults (154 women and 133 men: mean age 26.4 years; range 17–54, ¹6.1) were recruited in the course of an occupational health survey at the University Hospital, Freiburg. Apart from obtaining informed consent, participation was limited to those for whom a complete record of diphtheria vaccination was available and who had received a correct basic immunization. Serology Blood samples were taken and the serum stored at µ20°C. A modified tissue culture toxin neutralization assay (NT) according to Miyamura et al.6,7 was used to determine the diphtheria antitoxin levels. The tests were carried out on microplates with 96 wells. As culture medium, Eagle’s minimal essential medium (MEM) enriched with 10% calf serum was used. Titration was carried out with 50 ml micropipettes in 2-fold steps from 1:2 up to 1:4096. A total of 25 ml of challenge toxin (100 tissue culture toxic doses, SIGMA) were added to each well. After 1 h at 20°C
Diphtheria immunity: H.-M. Hasselhorn et al. each well received 100 ml culture medium containing 105 viable Vero cells per milliliter. The dilution step of the serum neutralizing toxin in 90% of the cultures was given as titre. The potency of the toxin in each test was determined after 3 days’ incubation at 37°C (5% CO2) by titration against diphtheria antitoxin (Behringwerke AG). Our NT showed a very high correlation when 27 samples were counter-tested by a tissue culture neutralization assay carried out by Behringwerke AG, Marburg (r = 0.97 for correlation of the dilution steps, Ps0.001). The tissue culture neutralization assay is regarded as the most accurate in vitro procedure for measuring diphtheria antitoxin1,8,9,11,12, whereas the ELISA and passive haemagglutination methods are known to be inaccurate in the low antitoxin range8. The following internationally accepted definitions of diphtheria protection8–10 were used in this study: persons with antitoxin values below 0.01 IU mlµ1 were rated as ‘probably susceptible’, from 0.01 to s0.1 IU mlµ1 as ‘basically protected’ and with values E0.1 as ‘fully protected’. Negative values were rated as 0.001 and values above 9 IU mlµ1 as 9.5. Data analysis and statistics Statistical analysis included analysis of variance, correlation analysis, multiple linear regression, path analysis and logistic regression13–15. In all instances, full protection against diphtheria was used as the dependent variable. Independent factors thought to be potentially influential were measured and tested: gender; age; time interval since last vaccination; number of previous vaccinations; occupational contact with patients and travel within any endemic region during the last 10 years. A complete record of previous vaccination dates was used to generate the variable: last vaccination within/after the first 3 years of life.
RESULTS The participants (n = 287) had received a mean of 4.4 diphtheria vaccinations (range: 3–8, ¹1.1), the last vaccination being 19.2 years (mean) before (range: 3 months–43.0 years, ¹7.8 years). No significant gender differences were found in either mark. The lowest values measured were 0.002 IU mlµ1, the highest > 9.0 IU mlµ1. Ninety (31.4%) out of the 287 participants were ‘probably susceptible’ to diphtheria, 76 (26.5%) had ‘basic protection’ and 121 (42.2%) had ‘fully protective’ antitoxin levels. The geometric mean antitoxin level (GMT) was 0.04 IU mlµ1. Women were fully protected in 35.7%, and men in 49.6% of cases. No significant influence of the factors occupational contact with patients and travel within any endemic region during the last ten years on the protection rates could be observed. On a bivariate level significant correlations with diphtheria antitoxin levels and the protection rate was found for the factors time interval since the last vaccination (Figure 1); age (Figure 2); number of previous vaccinations (Figure 3); last vaccination within/after the first 3 years of life and gender (Figure 4). Since these last factors—except gender—are highly correlated, multivariate analysis was necessary to exclude confounders and redundant predictors.
Figure 1 Diphtheria antitoxin levels by time interval since last vaccination. Stages: s0.01 IU mlµ1 = probably susceptible; 0.01–0.1 IU mlµ1 = basic protection; E0.1 IU mlµ1 = full protection. GMT = 0.04 IU mlµ1; negative values treated as 0.001. ntotal = 287
Multiple linear regression analysis produced a model (multiple R = 0.53) which included the factors shown in Figure 5. The factor number of past vaccinations was dropped, because it did not have a genuine supplementary effect in the multivariate model. As can be seen from the arrows und their thickness in the path model, three genuine predictor-variables appeared. The most important of these is the time interval since the last vaccination (bpartial = µ0.388, Ps0.001) followed by booster after the third year of life (bpartial = 0.227, Ps0.001) and female gender (bdirect = µ0.140, Ps0.05). The factor age does not have a direct influence on the diphtheria protection rate in this multivariate model, but since age is very closely related to the first two predictors, its indirect influence is shown in our path model. The estimated probabilities for a protective antibody level depending upon the three factors (obtained by logistic regression modelling with LR-test; model statistics: Gmodel = 93.2, df = 3, pmodel s0.001; µ2 Log likelihood = 298, goodness of fit = 285) are given in Figure
Figure 2 Diphtheria protection rates (E0.1 IU mlµ1) by age groups. etabivariat = 0.30, Ps0.001, no significant direct effect in the multivariate model. ntotal = 287, n17–20 = 39, n21–23 = 68, n24–26 = 63, n27–29 = 49, n30–32 = 31, n33–40 = 27, n > 40 = 10
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Figure 3 Diphtheria protection rates (E0.1 IU mlµ1) by number of previous vaccinations. etabivariat = 0.37, Ps0.001, no significant effect in the multivariate model. ntotal = 287, n3 vaccin. = 63, n4 vaccin. = 100, n5 vaccin. = 81, n6–8 vaccin. = 43
4. One example may clarify this result. For a man, the probability of still being protected 20 years after the last vaccination is around 50%. After the same time interval, a woman is less likely to be still protected (35%). If the person had received his or her last diphtheria vaccination within the first 3 years of life, the probability of still being protected is less than 20%, without any significant gender difference.
DISCUSSION Diphtheria vaccine and vaccination policies in Germany Those responsible for a German vaccination policy recommend basic immunization against diphtheria during infancy, followed by regular booster vaccina-
Figure 4 Estimated probability for diphtheria protection by the predictors: time interval since last vaccination, gender and last vaccination in early childhood. ntotal = 287, nmale = 133 (n = 32 last vaccination in early childhood), nfemale = 154 (n = 37 last vaccination in early childhood)
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Figure 5 Path model of factors influencing protection against diphtheria (E0.1 IU mlµ1). Thickness of arrows equals partial path coefficient. No direct influence of age. ntotal = 287
tions at 6 years of age, 10–12 years of age and thereafter (as in other countries, e.g. the USA16) with one booster vaccination every 10 years17. The common diphtheria vaccine used from 1925 to 1960 contained 75 IU toxoid per dose, since then this has been reduced to 50 IU. In 1984, a reduced dose for adults and adolescents (from 7 years of age) containing about 5 IU toxoid per dose was introduced. Protection rates In our study, 42.2% of the participants had full serological protection against diphtheria (E0.1 IU lµ1) and 31.4% were ‘probably susceptible’ (s0.01 IU mlµ1). These results lie within the same range as other German3,4 and international studies assessing diphtheria protection among adults9,18–27 (range ‘full protection’ 20.5–49.3%, ‘probably susceptible’ 15.3–41%). Much higher full protection rates of 74.7 and 87% were found in Poland28 and Israel29 respectively. Variations between the studies may be explained by differing vaccination schedules. However, varying selection criteria for participants and the different serological test systems used must also be taken into account. Therefore a reliable comparison of these data is not easily obtainable. Factors influencing diphtheria protection In the present study, a detailed analysis of the factors assumed to contribute to the decline of diphtheria antitoxin levels was facilitated by the fact that, in contrast to other investigations, only participants who could present unbroken and complete written documentation of previous diphtheria vaccinations from birth on were included in the study. Vaccination records do not always reflect the real vaccination history. In Germany, diphtheria vaccine was not until recently given during military service or as part of the treatment of traumatic injury. The records of diphtheria vaccination can therefore be regarded as reliable in cases where the complete vaccination documents are available.
Diphtheria immunity: H.-M. Hasselhorn et al. Time interval since last vaccination. Our results support previous findings that diphtheria antitoxin levels wane with the time elapsed since the last vaccination1,5,8,10,22,30–35. The clinical consequences of this became evident in a recent retrospective study in Moskow, where the risk of acquiring diphtheria was 2.5 times higher for children who had received the last vaccination 4–7 years earlier than for those whose most recent immunization was within the last year36. Previous vaccinations. During the course of the present diphtheria epidemic in Eastern Europe it was found that people with four or five previous vaccinations were more resistant to clinical diphtheria than those with 1–3 vaccinations2. Our multiple linear regression analysis indicates that these findings rather may be explained by the fact that those with the greater number of vaccinations are likely to have received the last booster more recently, and it is this time interval since last vaccination which has proved to be the most influential protective factor. Even a differential comparison between people with basic immunization alone and those with additional booster doses revealed no significant difference in the protection rates when controlled for time interval since last vaccination. As our results demonstrate, the factor last vaccination within the first 3 years of life has a significant influence on diphtheria protection, since those who were last vaccinated before the age of three were significantly less well protected. A high level of maternally acquired diphtheria antibodies hinders optimal vaccination in infants8. According to Barkin et al.37 and Edwards et al.38, revaccination following basic vaccination produced higher diphtheria antitoxin levels than primary vaccination alone. Gender. In our study, women had a significantly lower protection rate than men even when several other potential confounder variables were checked. A survey of the literature shows that lower antitoxin levels and protection rates among women were more or less pronounced in other studies3,4,9,10,19,22,26,35,39–41. The opposite was reported in one German24 and one Italian study42 and no clear difference was found in Poland18. These differing results are difficult to explain, but it is noteworthy that in these three studies the passive haemagglutination method was used to determine the antitoxin levels. This test was used in only one of the studies reporting lower protection among women22. In some countries other than Germany, the lower diphtheria antitoxin levels found among women might be explained by the routine administration of combined tetanus and diphtheria vaccines following injury (known to occur more frequently in boys and men10) or for military personnel10,19,22,26,39–41. A lower protection rate among women which could not be explained by either of these ways was reported by Maple et al.9, Simonsen10 and finally by Rieger and Kuhlmann3, who observed a similar gender difference even among children and adolescents. Kjeldsen et al. also conjectured that diphtheria immunity following vaccination might be less long-lasting among women23. The results of this study have established that, in the long term, diphtheria vaccination offers less effective serological protection to women than to men.
It is possible that vaccination is less efficient in women, or that decline of the antitoxin levels in women after vaccination is more pronounced than in men. Age. Galazka reported that whereas in the prevaccination era diphtheria immunity in the population constantly increased with age1, the postvaccination era was associated with a high prevalence of immunity among children, an immunity gap in young adults and thereafter increased immunity in older people5. This immunity gap was reported in several studies3–5,12,24,26, but was not in others9,19,40,41. In our study the effect of age on diphtheria protection disappeared when checked against time interval since last vaccination. Other studies have demonstrated the dependence of the protection rate upon the age factor; but since age merely indicates that a longer time interval is more likely to have elapsed without a booster vaccination, this relationship cannot not be interpreted causally. The increase in immunity among older people is commonly explained by their repeated natural contact with C. diphtheriae toxin. We believe that both early vaccinations and/or natural contacts with Corynebacterium diphtheriae among people born before 1950, in combination with additional vaccinations during the 1950s and 1960s, have contributed to the higher protection rate found in elderly people today. A high protection rate among adults is necessary Previously vaccinated children and adults can acquire clinical diphtheria1,2,5,43–45. For example, 70% of all cases in St Petersburg at the beginning of the outbreak in Russia had previously been vaccinated2, but previous vaccination appeared to reduce the severity of the disease and the likelihood of a fatal outcome2. No level of antitoxin can provide absolute protection against diphtheria46, because such factors as virulence, the dose of the acquired bacillus19 and the general immune status of the infected person2,41,47–49 also determine whether or not symptomatic disease will occur. In Germany, a relatively low but increasing immunization coverage among children (the immunization coverage of three doses of DPT vaccine had reached 75%50 in 1993) and an immunity gap among young and middle-aged adults were found. Even though extensive diphtheria epidemics are unlikely to occur under the present epidemiological circumstances, an increased risk of outbreaks does exist5. According to the WHO, over 90% of all children and more than 75% of adults ought to be protected to prevent an epidemic outbreak of the disease2. Booster vaccinations for adults are necessary to provide collective immunity and prevent local outbreaks of diphtheria among adults. To prevent further immunity gaps among adults, it is necessary to ascertain whether or not one booster suffices to provide 10 years protection in all cases.
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