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Immunity to diphtheria among children in Northern Norway and North-Western Russia
Vaccine 19 (2001) 197±203
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Immunity to diphtheria among children in Northern Norway and North-Western Russia Vegard Skogen a,*, PaÊl A. Jenum b, Elena Danilova c, Vera N. Koroleva c, Dag S. Halvorsen a, Haakon Sjursen d a
Department of Medicine, Institute of Clinical Medicine, University of Tromsù, Tromsù, Norway b Department of Bacteriology, National Institute of Public Health, Oslo, Norway c Institute for Infectious Diseases of Children, Regional Hospital, Arkhangelsk, Russia d Institute of Medicine, University of Bergen, Haukeland Hospital, Norway Received 12 October 1999; received in revised form 27 April 2000; accepted 11 May 2000
Abstract In 1990, diphtheria re-emerged in Russia and during the following four-year period the resulting epidemic reached all the Newly Independent States (NIS) of the former Soviet Union. Several neighbouring countries of NIS, Norway included, have experienced sporadic imported cases. A sero-epidemiological study among children in Northern Norway and North-Western Russia was performed in order to evaluate protection against diphtheria and how dierences in vaccination programmes aect immunity. A total of 664 sera, 400 from Norwegian and 264 from Russian children, were examined for antibodies against diphtheria, using an in vitro toxin neutralisation method. The Russian children studied had satisfactory protection in all age groups examined. The Norwegian children had poor protection against diphtheria from the age of 7 years until they received the booster dose at the age of 11. Therefore, a revision of the Norwegian vaccination programme, including a booster dose at school-entry age, seems warranted. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Diphtheria; Sero-epidemiology; Children
1. Introduction Diphtheria was a major cause of death in the ®rst half of this century. In 1923, formalin treated toxin, the toxoid, was introduced as an immunising agent by G. Ramon [1]. This was a breakthrough in the prevention of diphtheria. The vaccine became generally available from the late 1920s and was increasingly used during the following decades. A signi®cant decrease in the number of clinical cases and carriers was seen in the countries where vaccination against diphtheria was introduced [2]. * Corresponding author. Tel.: +47-776-45479; fax: +47-77644650. E-mail address: [email protected] (V. Skogen).
In the former Soviet Union diphtheria vaccination started as early as the late 1920s in some areas, but was not included in the general immunisation programme until 1958 [3]. The largest diphtheria outbreak in the developed world since the 1960s began in the Russian Federation in 1990 and reached all the Newly Independent States (NIS) of the former Soviet Union during the following fouryear period [4,5]. The European Regional Oce of the World Health Organization (WHO), now consider the diphtheria outbreak to be nearly under control in most of the NIS [2]. This epidemic caused more than 150,000 diphtheria cases and more than 4000 deaths. The highest incidence rates were among school-aged children and adolescents (12.4±18.2 per 100,000) and in adults 40±49 years of age (16.7 per 100,000) [2,6]. Factors such as an
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V. Skogen et al. / Vaccine 19 (2001) 197±203
increased proportion of susceptible population, migration and a deterioration of health service infrastructure including vaccination against diphtheria have been proposed as catalysts for this epidemic [4]. Since World War I a slow but steady decline in the number of diphtheria cases was observed in Norway. However, during World War II the diphtheria incidence increased and peaked in 1943±44 (4.94±4.77 per 1000 inhabitants, respectively) [7]. The diphtheria vaccine was introduced in 1942 and a general vaccination programme for infants with a combined DTP vaccine started in 1952. During the following decade the number of new diphtheria cases decreased rapidly. Since 1962, no case has been diagnosed in Norway, until 1992 when a 15 year old Norwegian boy in the northern part of Norway became ill with clinical diphtheria after contact with a Russian group visiting his community [8]. Several neighbouring countries of NIS, have experienced sporadic imported cases of diphtheria. However, no secondary cases have been reported in these countries [9]. In December 1998, two closely related cases of diphtheria were reported from Copenhagen, Denmark [10]. This incident adds to some few minor diphtheria outbreaks in North-Western Europe in the 1980s [11±13]. The resurgence of diphtheria in NIS and the evident threat it represents have again called attention to Corynebacterium diphtheriae and the protection level against diphtheria. These recent outbreaks have shown the importance of good immunity and infection surveillance, even in countries where vaccination against diphtheria has been widely practised. Knowledge about optimal diphtheria±tetanus±pertussis (DTP) vaccination schemes is of crucial importance, in order to obtain satisfactory short and long term protection. The timing and number of doses of diphtheria toxoid vaccine vary among dierent countries and an optimal schedule may also be dependent on the toxoid dosage used. In Europe, vaccination schedules dier considerably but almost all countries complete the primary immunisation of children by the age of 1 year [14]. During the last years several sero-epidemiological studies have been conducted in order to assess whether the recommendations endorsed by WHO, trying to eliminate diphtheria in Europe, were ful®lled [15]. We have performed a sero-epidemiological study comparing immunity to diphtheria among children in Northern Norway and North-Western Russia in order to evaluate how dierences in vaccination programmes aect immunity and to investigate the present potential for the spread of a diphtheria epidemic to Norway.
2. Materials and methods 2.1. Study population In the period from 1994 to 1997 a total of 664 sera were collected from hospitalised and outpatient children living in Northern Norway and the Arkhangelsk Region in Russia. The Norwegian study population n 400 covered the age group from 1 to 12 years of age, while the Russian sera n 264 covered the age group up to 10 years of age (Table 1). The Norwegian children were recruited from the three most northern counties (Finnmark, Troms and Nordland). The Russian children were from both urban and rural areas of the Region. Detail information on the individual vaccination status among all the infants included was not available. None of the participants had suered from diphtheria or diseases that could have interfered with the antibody response to vaccination. The serum samples were stored at ÿ208C before being analysed at the National Institute of Public Health, Oslo, Norway. 2.2. Serology The sera were analysed by an in vitro toxin-neutralisation assay based on the method described by Miyamura et al. [16] and performed as described earlier [17]. In brief, two-fold dilutions of serum were mixed with a 4 times minimal cytotoxic dose of diphtheria toxin (Statens Seruminstitut, Copenhagen, Denmark) and incubated at 378C for 1 h before Vero cells were added. An antitoxin-positive control serum was run for every 17th serum sample. The pH-mediated colour change from red to yellow, caused by growing Vero cells, was recorded after ®ve days of incubation at 378C. The antitoxin level of each sample was deterTable 1 Number of sera n 664 from Northern Norway and North-Western Russia examined for diphtheria antitoxin by age and gendera Age (years)
Norwegian children
Russian children
1 2 3 4 5 6 7 8 9 10 11 12 Total
48 (0.8) 36 (1.0) 30 (1.3) 27 (1.3) 26 (1.0) 36 (0.5) 32 (0.9) 31 (0.9) 40 (1.5) 30 (0.8) 35 (1.2) 29 (1.1) 400 (0.98)
15 (0.2) 20 (0.3) 22 (1.0) 28 (0.6) 29 (1.2) 30 (0.7) 35 (0.8) 27 (1.1) 25 (0.4) 33 (0.7) ± ± 264 (0.65)
a
Females to males ratio is shown in brackets.
V. Skogen et al. / Vaccine 19 (2001) 197±203
199
Fig. 1. Protection against diphtheria among Russian children n 264 according to age.
mined by comparing the colour change breakpoint to that of a WHO standard (Statens Seruminstitut) analysed simultaneously [17]. The results were expressed in international units per ml (IU/ml). The children were grouped according to the following protection levels against diphtheria in serum: unprotected (<0.01 IU/ ml), relatively or short-term protected (r0.01, <0.1 IU/ml) and protected (r0.1 IU/ml) [18]. 2.3. Statistical methods We examined diphtheria antitoxin levels in relation to nation, age and gender using analysis of covariance based on type I sums of squares (PROC GLM, SAS Institute, 1991). Interaction terms were included after the main eects. Geometric mean titre (GMT) of antidiphtheria toxin with the corresponding 95% con®dence interval (CI) was calculated for each age group by year and country. ( p-values <0.05 were considered signi®cant). This work was approved by the local Ethical Health Committee in Northern Norway, by local Health Authorities in Arkhangelsk, Russia and by the Norwegian Data Inspectorate.
3. Results
sian children were fully protected against diphtheria, 121 (30.3%) Norwegians and 31 (11.8%) Russians were relatively protected, while 21 (5.3%) and 3 (1.1%) were unprotected. The proportions of dierent degrees of protection according to age are shown in Figs. 1 and 2. In each age group more than 75% of the Russian children were found to be protected, except for infants one year of age (Fig. 1). Children not protected were only sporadically detected. Among Norwegian children above 2 years of age, the number of protected children was clearly lower. This dierence in protection among Norwegian and Russian children was most evident for children from 6 to 10 years of age (Fig. 2). Table 2 Analysis of covariance (Proc GLM) by Type I Sums of Squares indicating dierent main eects and their interactions related to the level of diphtheria antibody (log) Variable
dfa
Fb
p
Nation Age Agec Nation Gender GenderNation GenderAge GenderAgeNation
1,560 9,560 9,560 1,560 1,560 9,560 9,560
228.66 2.63 8.94 4.47 0.67 0.69 0.58
0.0001 0.0055 0.0001 0.0349 0.4128 0.7183 0.8160
a
Of the total 664 children examined, 258 (64.4%) of the Norwegian children and 230 (87.1%) of the Rus-
Degrees of freedom. F distribution. c Interaction.
b
200
V. Skogen et al. / Vaccine 19 (2001) 197±203
Fig. 2. Protection against diphtheria among Norwegian children n 400 according to age.
Table 2 illustrates the dierent independent variables like nation, age, gender and its dierent interactions related to the diphtheria antitoxin. Russian children were signi®cantly better protected than the Norwegian children p 0:0001). A signi®cant gender dierence was seen, boys had a signi®cantly higher protection to diphtheria than girls p 0:035). However, no signi®cant gender dierence was seen in interaction with nation p 0:41 or age p 0:72). We found dierent eects of age in the two countries p 0:0001). The GMT serum levels of diphtheria antitoxin among Norwegian children were r0.1 IU/ml until the age of 6 years (Table 3). From 7 to 10 years the GMT values were low (<0.1 IU/ml), however, from the age of 11 years, when a booster dose is given according to the standard vaccination programme, the GMT increased signi®cantly. The GMT levels among the Russian children were signi®cantly higher than among the Norwegian children for all age groups except for children 1 year of age (Table 3). From 2 to 6 years of age the GMT values of Russian children were 3±10 times higher. After 6 years of age, when Russian children normally receive a booster vaccination, the GMT values were 13±36 times higher for Russian than for Norwegian children. These dierences were highly signi®cant (Table 3).
4. Discussion
humoral and cell-mediated immunity to multiple antigens of C. diphtheriae [19]. In practice, however, only the level of diphtheria antitoxin (IgG) is measured. For epidemiological purposes the minimum protection level is considered to be 0.01 IU/ml of diphtheria antitoxin. The higher level of 0.1 IU/ml is desirable for individual protection [20]. No level of antitoxin provides absolute protection against clinical diphtheria, however, the severity of the illness is often proportional with the lack of antitoxin [18]. In order to achieve suf®cient herd immunity, a minimum immunity rate of Table 3 Geometric mean titre against diphtheria among Norwegian n 400 and Russian n 264 children according to age Age (years)
1 2 3 4 5 6 7 8 9 10 11 12 a
Immunity to diphtheria presumably involves both
b
Norwegian children
Russian children
GMTa
(95% CI)b
GMT
(95% CI)
0.523 0.274 0.180 0.127 0.239 0.183 0.082 0.070 0.083 0.055 0.603 1.109
0.347, 0.158, 0.010, 0.065, 0.162, 0.114, 0.046, 0.040, 0.051, 0.029, 0.220, 0.519,
0.184 0.874 1.165 0.975 0.688 1.896 3.000 0.894 1.114 1.791 ± ±
0.051, 0.295, 0.517, 0.502, 0.352, 1.034, 2.100, 0.401, 0.516, 1.030, ± ±
geometric mean titre. 95% Con®dence interval.
0.787 0.475 0.324 0.248 0.352 0.295 0.146 0,123 0.134 0.105 1.657 2.372
0.658 2.590 2.623 1.893 1.343 3.478 4.284 1.994 2.408 3.115
p-value
0.5171 0.0329 0.0003 < 0.0001 0.0075 < 0.0001 0.0001 < 0.0001 < 0.0001 < 0.0001 ± ±
V. Skogen et al. / Vaccine 19 (2001) 197±203
90% in children and 75% in adults is required [20]. It is also a fact that other factors than immunity to diphtheria are important in the spread of diphtheria, as poor socio-economical conditions, and the amount of circulating diphtheria strains in the community. During the diphtheria outbreaks in the US in 1971± 1981 and the Swedish outbreak in the 1980s, a substantial proportion of the patients were middle-aged homeless men abusing alcohol [11,21]. All studies on immunity to diphtheria have to be evaluated in relation to the vaccination programme that has been accomplished in the preceding years. In 1984 the Norwegian vaccination scheme was changed, reducing the number of doses from ®ve to four, with basis immunisation including 3 doses followed by a booster dose given at the age of 11 years. The old scheme included four doses of toxoid at 3, 4, 5 and 17 months of age followed by a booster dose at 11 years. In a previous study, we found no signi®cant dierence in the overall protection against diphtheria, when comparing immunity among adults living in Northern Norway and in North-Western Russia [17]. However, in the present study comparing children only, the Norwegian children were found to have a signi®cantly lower degree of protection in almost all age groups compared to the Russian children. The immunity among the younger Norwegian population is vaccine induced, since no circulating C. diphtheriae strains has been identi®ed for more than 30 years, except for the one imported case in 1992. Generally, the Norwegian children had some protection until the age of 6, but insuf®cient protection from 7 to 10 years of age. It therefore seems that the primary immunisation, including three vaccine doses given at 3, 5 and 10 months of age, is unable to maintain an adequate protection level until the booster-dose (dT) is given at the age of 11 years. The general trend among the Russian children was a high antibody titre indicating a markedly better immunity against diphtheria than among the Norwegian children of corresponding age. This was most pronounced for the older age groups. This seems to be due to a compulsory vaccination programme, which in general includes more booster doses than in Norway. It is also a fact that the sera used in this sero-epidemiological study were collected in a period when the epidemic peaked. At this time a great eort was made in the NIS to achieve high vaccination coverage, in order to control the diphtheria epidemic. None of the Russian children included in this study had been part of a catch up activity that took place in some areas as a response to the outbreak. The Russian children have lived in an environment with circulating C. diphtheriae strains for many years, even before the outbreak. Thus, the immunity is probably not induced by vaccination alone, despite the fact that we excluded children
201
with a known diphtheria diagnosis. In 1994, the school-entry booster dose was reintroduced and the Russian vaccination scheme now includes a booster dose at 2, 6 and 16 years of age. In some areas like the Arkhangelsk Region, the vaccination scheme also includes one booster dose at 9 years of age. A study investigating the risk of diphtheria among school children in Russia in relation to the time elapsed since last vaccination, showed evidence for the ecacy of a booster dose given at the age of school entry in preventing diphtheria among school aged children [22]. Based on these results, an additional booster dose in childhood should be considered included in the Norwegian vaccination programme if a general protection against diphtheria should be obtained. A Swedish study showed that diphtheria antitoxin levels among Swedish children were unsatisfactory [23]. Among 6 year old children who had received primary vaccination with a three-dose schedule DT before one year of age, as many as 15% were unprotected i.e. they had a serum antitoxin level of less than 0.01 IU/ ml. Among 10 year old children as many as 48% were unprotected. In Sweden as in Norway the compliance to the vaccination programme is above 90% [23,24]. The low protection rate among Swedish children was explained by a too long interval between the primary doses given during the ®rst year of life and the booster dose given at 10 years of age [23]. Another explanation could be a less potent vaccine, since the pertussis component, that is regarded to act as an adjuvant, was not part of the Swedish vaccine [23]. Most countries have now adopted the recommendations from WHOs Expanded Programme on Immunization (EPI), which includes a short interval between the second and the third dose of the primary immunisation scheme [25]. However, in some countries, like Norway, Sweden and Italy, the ®rst two doses of the primary immunisation are given at 3 and 5 months of age and the third at 10±12 months of age. Sero-epidemiological studies conducted in Italy have shown that children receiving a booster at the age of 6 years have good protection until early adolescence [26±28]. A serological study performed among school children in Denmark in 1987 demonstrated that the proportions of 8 and 10 year old children not protected against diphtheria were 14% and 21%, respectively [29]. This Danish population did not receive any booster doses after their primary immunisation (at the 5th, 6th and 15th month of age). The authors concluded that a booster should be included during school age. In 1996, the booster was introduced at the age of 5 years [10]. Our results, clearly demonstrate that the situation in Norway is similar to that of Denmark. The gender-related dierence showing that boys had a higher level of antibodies to diphtheria toxin than girls was unexpected. A similar ®nding was
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V. Skogen et al. / Vaccine 19 (2001) 197±203
recently published by Mark et al. [30] and by Rieger et al. [31]. We reported a gender dierence among adults in Norway [17]. However, this was explained by the booster immunisation of men entering military service. We have no explanation for the gender dierence observed among children. Further attention and studies are needed, in order to evaluate the gender-related dierence to vaccineinduced immunity. Until recently, the pertussis component in the DTP vaccine for primary immunisation contained whole cell antigen (Pw). New acellular pertussis vaccines (Pa) containing from one to ®ve dierent single antigens are now more commonly used, mainly due to the lower frequency of adverse vaccine reactions [25]. However, this change from the whole cell to the acellular pertussis component in the DTP may in¯uence the immune response to the other two components of the vaccine, since the Pw component might have a stronger adjuvant eect than some of the Pa components in the vaccines [32]. In January 1998, the Norwegian health authorities introduced the DTPa vaccine giving three primary doses at 3, 5 and 12 months of age and a booster dose at the age of 11 as stated previously. Consequently, protection against diphtheria among the Norwegian population that will be vaccinated according to the new programme may become even weaker in the coming years than shown in this study. An additional dose of diphtheria toxoid given during childhood may reduce this tendency. In conclusion, the Russian children studied had a satisfying degree of protection in all age groups examined. This indicate that the eort to reach high vaccination coverage, in order to control the diphtheria epidemic has been successful. The Norwegian children had a poor protection level from the age of 7 years until they received the booster dose at the age of 11. A revision of the Norwegian vaccination programme, including a booster dose of diphtheria toxoid at school-entry age seems warranted.
Acknowledgements We wish to thank Anne-Gerd Gundersen at the National Institute of Public Health, Oslo for excellent technical assistance. We also thank Dr. J. Nielsen at Hammerfest Hospital and Professor T. Flñgstad at the University Hospital of Tromsù for providing the Norwegian sera. This work was supported by grants from the Research Council of Norway (Medical Science and Health), the Barents secretary and the Norwegian Ministry of Foreign Aairs.
References [1] Ramon G. Sur la toxine et sur lÂanatoxine diphteÂriques, porvoir ¯oculant et proprieÂteÂs immunisantes. Ann Inst Pasteur 1924;38:1±22 (in French). [2] Vitek CR, Wharton M. Diphtheria in the former Soviet Union: reemergence of a pandemic disease. Emerging Infectious Diseases 1998;4:539±50. [3] Khazanov MI. On the problem of the elimination of diphtheria. Vestn Ross Akad Medi Nauk 1964;8:13±20. [4] Hardy IRB, Dittman S, Slutter RW. Current situation and control strategies for resurgence of diphtheria in newly independent states of the former Soviet Union. Lancet 1996;347:1739±44. [5] Centers for Diseases Control and Prevention. Diphtheria epidemic-new independent states of the former Soviet Union, 1990±1994. Morbid Mortal Weekly Rep 1995;44:177±81. [6] Markina SS, Maksimova NM, Bogatyreva EY, et al. Incidence of diphtheria in Russia in 1993. In: Monisov AA, Podunova LG, Tyasto AS, Emeljanov OV, Churchill RE, editors. The health of the population and the environment. Moscow: Russian Federation State Committee on Sanitary Epidemiologic Surveillance, 1994. p. 10±5 (in Russian). [7] Vogelsang TM. Diphtheria antitoxin estimation in the blood of the adult population of Bergen, Norway, just after a diphtheria epidemic. J Infect Dis 1949;85:17±24. [8] Lystad A. Diphtheria in Finnmark. Communicable Disease Report, vol. 20. Oslo, Norway: National Institute of Public Health, 1992. p.53. (in Norwegian). [9] Popovic T, Wharton M, Wenger JD, McIntyre L, Wachsmuth IK. Are we ready for diphtheria? In: A Report from the Diphtheria Diagnostic Workshop, Atlanta, 11 and 12 July 1994. J Infect Dis, 71. 1995. p. 765±7. [10] Rùnne T. Lundstedt C. Pedersen K. Hùiby N. Bruun B. Diphtheria in Copenhagen. Statens Seruminstitut: EPI-nyt 1999; Uge 1: 1±2 (in Danish). [11] Bjùrkholm B, Bùttiger M, Christenson B, Hagberg L. Antitoxin antibody levels and outcome of illness during an outbreak diphtheria among alcoholics. Scand J Infect Dis 1986;18:235±9. [12] Rappuoli R, Perugini M, Falsen E. Molecular epidemiology of the 1984±1986 outbreak of diphtheria in Sweden. N Engl J Med 1988;318:12±4. [13] Naumann P. Diphtheria in West Germany. Com Dis Rep 1983;18:3. [14] Prospero E, Rao M, Bagnoli M, Appignanesi R, D'Errico MM. Diphtheria: epidemiological update and review of prevention and control strategies. Eur J Epidemiol 1997;13:527±34. [15] Report on a WHO meeting. The control of diphtheria in Europe. Report EUR/ICP/EPI 024 5882r; Geneva, Switzerland, 1990 April. [16] Miyamura K, Nishio S, Ito A, Murata R, Kono R. Micro cell culture method for determination of diphtheria toxin and antitoxin titers using Vero cells. I. Studies on factors aecting the toxin and antitoxin titration. J Biol Stand 1974;2:189±201. [17] Jenum PA, Skogen V, Danilova E, Eskild A, Sjursen H. Immunity to diphtheria in Northern Norway and NorthWestern Russia. Eur J Clin Microbiol Infect Dis 1995;14:794±8. [18] Ipsen J. Circulating antitoxin at the onset of diphtheria in 425 patients. J Immunol 1946;54:325±47. [19] Tasman A, Lansberg H. Problems concerning the prophylaxis, pathogenesis and therapy of diphtheria. Bull World Health Organ 1975;16:939±73. [20] Begg N. Manual of management and control of diphtheria in the European region. The expanded programme on immunization in the European region of WHO. ICP/EPI/038 (B);1994. [21] Chen RT, Broome CV, Weinstein RA, Weaver R, Tsai TF.
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Immunity to diphtheria among children in Northern Norway and North-Western Russia Vegard Skogen a,*, PaÊl A. Jenum b, Elena Danilova c, Vera N. Koroleva c, Dag S. Halvorsen a, Haakon Sjursen d a
Department of Medicine, Institute of Clinical Medicine, University of Tromsù, Tromsù, Norway b Department of Bacteriology, National Institute of Public Health, Oslo, Norway c Institute for Infectious Diseases of Children, Regional Hospital, Arkhangelsk, Russia d Institute of Medicine, University of Bergen, Haukeland Hospital, Norway Received 12 October 1999; received in revised form 27 April 2000; accepted 11 May 2000
Abstract In 1990, diphtheria re-emerged in Russia and during the following four-year period the resulting epidemic reached all the Newly Independent States (NIS) of the former Soviet Union. Several neighbouring countries of NIS, Norway included, have experienced sporadic imported cases. A sero-epidemiological study among children in Northern Norway and North-Western Russia was performed in order to evaluate protection against diphtheria and how dierences in vaccination programmes aect immunity. A total of 664 sera, 400 from Norwegian and 264 from Russian children, were examined for antibodies against diphtheria, using an in vitro toxin neutralisation method. The Russian children studied had satisfactory protection in all age groups examined. The Norwegian children had poor protection against diphtheria from the age of 7 years until they received the booster dose at the age of 11. Therefore, a revision of the Norwegian vaccination programme, including a booster dose at school-entry age, seems warranted. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Diphtheria; Sero-epidemiology; Children
1. Introduction Diphtheria was a major cause of death in the ®rst half of this century. In 1923, formalin treated toxin, the toxoid, was introduced as an immunising agent by G. Ramon [1]. This was a breakthrough in the prevention of diphtheria. The vaccine became generally available from the late 1920s and was increasingly used during the following decades. A signi®cant decrease in the number of clinical cases and carriers was seen in the countries where vaccination against diphtheria was introduced [2]. * Corresponding author. Tel.: +47-776-45479; fax: +47-77644650. E-mail address: [email protected] (V. Skogen).
In the former Soviet Union diphtheria vaccination started as early as the late 1920s in some areas, but was not included in the general immunisation programme until 1958 [3]. The largest diphtheria outbreak in the developed world since the 1960s began in the Russian Federation in 1990 and reached all the Newly Independent States (NIS) of the former Soviet Union during the following fouryear period [4,5]. The European Regional Oce of the World Health Organization (WHO), now consider the diphtheria outbreak to be nearly under control in most of the NIS [2]. This epidemic caused more than 150,000 diphtheria cases and more than 4000 deaths. The highest incidence rates were among school-aged children and adolescents (12.4±18.2 per 100,000) and in adults 40±49 years of age (16.7 per 100,000) [2,6]. Factors such as an
0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 1 7 6 - 6
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V. Skogen et al. / Vaccine 19 (2001) 197±203
increased proportion of susceptible population, migration and a deterioration of health service infrastructure including vaccination against diphtheria have been proposed as catalysts for this epidemic [4]. Since World War I a slow but steady decline in the number of diphtheria cases was observed in Norway. However, during World War II the diphtheria incidence increased and peaked in 1943±44 (4.94±4.77 per 1000 inhabitants, respectively) [7]. The diphtheria vaccine was introduced in 1942 and a general vaccination programme for infants with a combined DTP vaccine started in 1952. During the following decade the number of new diphtheria cases decreased rapidly. Since 1962, no case has been diagnosed in Norway, until 1992 when a 15 year old Norwegian boy in the northern part of Norway became ill with clinical diphtheria after contact with a Russian group visiting his community [8]. Several neighbouring countries of NIS, have experienced sporadic imported cases of diphtheria. However, no secondary cases have been reported in these countries [9]. In December 1998, two closely related cases of diphtheria were reported from Copenhagen, Denmark [10]. This incident adds to some few minor diphtheria outbreaks in North-Western Europe in the 1980s [11±13]. The resurgence of diphtheria in NIS and the evident threat it represents have again called attention to Corynebacterium diphtheriae and the protection level against diphtheria. These recent outbreaks have shown the importance of good immunity and infection surveillance, even in countries where vaccination against diphtheria has been widely practised. Knowledge about optimal diphtheria±tetanus±pertussis (DTP) vaccination schemes is of crucial importance, in order to obtain satisfactory short and long term protection. The timing and number of doses of diphtheria toxoid vaccine vary among dierent countries and an optimal schedule may also be dependent on the toxoid dosage used. In Europe, vaccination schedules dier considerably but almost all countries complete the primary immunisation of children by the age of 1 year [14]. During the last years several sero-epidemiological studies have been conducted in order to assess whether the recommendations endorsed by WHO, trying to eliminate diphtheria in Europe, were ful®lled [15]. We have performed a sero-epidemiological study comparing immunity to diphtheria among children in Northern Norway and North-Western Russia in order to evaluate how dierences in vaccination programmes aect immunity and to investigate the present potential for the spread of a diphtheria epidemic to Norway.
2. Materials and methods 2.1. Study population In the period from 1994 to 1997 a total of 664 sera were collected from hospitalised and outpatient children living in Northern Norway and the Arkhangelsk Region in Russia. The Norwegian study population n 400 covered the age group from 1 to 12 years of age, while the Russian sera n 264 covered the age group up to 10 years of age (Table 1). The Norwegian children were recruited from the three most northern counties (Finnmark, Troms and Nordland). The Russian children were from both urban and rural areas of the Region. Detail information on the individual vaccination status among all the infants included was not available. None of the participants had suered from diphtheria or diseases that could have interfered with the antibody response to vaccination. The serum samples were stored at ÿ208C before being analysed at the National Institute of Public Health, Oslo, Norway. 2.2. Serology The sera were analysed by an in vitro toxin-neutralisation assay based on the method described by Miyamura et al. [16] and performed as described earlier [17]. In brief, two-fold dilutions of serum were mixed with a 4 times minimal cytotoxic dose of diphtheria toxin (Statens Seruminstitut, Copenhagen, Denmark) and incubated at 378C for 1 h before Vero cells were added. An antitoxin-positive control serum was run for every 17th serum sample. The pH-mediated colour change from red to yellow, caused by growing Vero cells, was recorded after ®ve days of incubation at 378C. The antitoxin level of each sample was deterTable 1 Number of sera n 664 from Northern Norway and North-Western Russia examined for diphtheria antitoxin by age and gendera Age (years)
Norwegian children
Russian children
1 2 3 4 5 6 7 8 9 10 11 12 Total
48 (0.8) 36 (1.0) 30 (1.3) 27 (1.3) 26 (1.0) 36 (0.5) 32 (0.9) 31 (0.9) 40 (1.5) 30 (0.8) 35 (1.2) 29 (1.1) 400 (0.98)
15 (0.2) 20 (0.3) 22 (1.0) 28 (0.6) 29 (1.2) 30 (0.7) 35 (0.8) 27 (1.1) 25 (0.4) 33 (0.7) ± ± 264 (0.65)
a
Females to males ratio is shown in brackets.
V. Skogen et al. / Vaccine 19 (2001) 197±203
199
Fig. 1. Protection against diphtheria among Russian children n 264 according to age.
mined by comparing the colour change breakpoint to that of a WHO standard (Statens Seruminstitut) analysed simultaneously [17]. The results were expressed in international units per ml (IU/ml). The children were grouped according to the following protection levels against diphtheria in serum: unprotected (<0.01 IU/ ml), relatively or short-term protected (r0.01, <0.1 IU/ml) and protected (r0.1 IU/ml) [18]. 2.3. Statistical methods We examined diphtheria antitoxin levels in relation to nation, age and gender using analysis of covariance based on type I sums of squares (PROC GLM, SAS Institute, 1991). Interaction terms were included after the main eects. Geometric mean titre (GMT) of antidiphtheria toxin with the corresponding 95% con®dence interval (CI) was calculated for each age group by year and country. ( p-values <0.05 were considered signi®cant). This work was approved by the local Ethical Health Committee in Northern Norway, by local Health Authorities in Arkhangelsk, Russia and by the Norwegian Data Inspectorate.
3. Results
sian children were fully protected against diphtheria, 121 (30.3%) Norwegians and 31 (11.8%) Russians were relatively protected, while 21 (5.3%) and 3 (1.1%) were unprotected. The proportions of dierent degrees of protection according to age are shown in Figs. 1 and 2. In each age group more than 75% of the Russian children were found to be protected, except for infants one year of age (Fig. 1). Children not protected were only sporadically detected. Among Norwegian children above 2 years of age, the number of protected children was clearly lower. This dierence in protection among Norwegian and Russian children was most evident for children from 6 to 10 years of age (Fig. 2). Table 2 Analysis of covariance (Proc GLM) by Type I Sums of Squares indicating dierent main eects and their interactions related to the level of diphtheria antibody (log) Variable
dfa
Fb
p
Nation Age Agec Nation Gender GenderNation GenderAge GenderAgeNation
1,560 9,560 9,560 1,560 1,560 9,560 9,560
228.66 2.63 8.94 4.47 0.67 0.69 0.58
0.0001 0.0055 0.0001 0.0349 0.4128 0.7183 0.8160
a
Of the total 664 children examined, 258 (64.4%) of the Norwegian children and 230 (87.1%) of the Rus-
Degrees of freedom. F distribution. c Interaction.
b
200
V. Skogen et al. / Vaccine 19 (2001) 197±203
Fig. 2. Protection against diphtheria among Norwegian children n 400 according to age.
Table 2 illustrates the dierent independent variables like nation, age, gender and its dierent interactions related to the diphtheria antitoxin. Russian children were signi®cantly better protected than the Norwegian children p 0:0001). A signi®cant gender dierence was seen, boys had a signi®cantly higher protection to diphtheria than girls p 0:035). However, no signi®cant gender dierence was seen in interaction with nation p 0:41 or age p 0:72). We found dierent eects of age in the two countries p 0:0001). The GMT serum levels of diphtheria antitoxin among Norwegian children were r0.1 IU/ml until the age of 6 years (Table 3). From 7 to 10 years the GMT values were low (<0.1 IU/ml), however, from the age of 11 years, when a booster dose is given according to the standard vaccination programme, the GMT increased signi®cantly. The GMT levels among the Russian children were signi®cantly higher than among the Norwegian children for all age groups except for children 1 year of age (Table 3). From 2 to 6 years of age the GMT values of Russian children were 3±10 times higher. After 6 years of age, when Russian children normally receive a booster vaccination, the GMT values were 13±36 times higher for Russian than for Norwegian children. These dierences were highly signi®cant (Table 3).
4. Discussion
humoral and cell-mediated immunity to multiple antigens of C. diphtheriae [19]. In practice, however, only the level of diphtheria antitoxin (IgG) is measured. For epidemiological purposes the minimum protection level is considered to be 0.01 IU/ml of diphtheria antitoxin. The higher level of 0.1 IU/ml is desirable for individual protection [20]. No level of antitoxin provides absolute protection against clinical diphtheria, however, the severity of the illness is often proportional with the lack of antitoxin [18]. In order to achieve suf®cient herd immunity, a minimum immunity rate of Table 3 Geometric mean titre against diphtheria among Norwegian n 400 and Russian n 264 children according to age Age (years)
1 2 3 4 5 6 7 8 9 10 11 12 a
Immunity to diphtheria presumably involves both
b
Norwegian children
Russian children
GMTa
(95% CI)b
GMT
(95% CI)
0.523 0.274 0.180 0.127 0.239 0.183 0.082 0.070 0.083 0.055 0.603 1.109
0.347, 0.158, 0.010, 0.065, 0.162, 0.114, 0.046, 0.040, 0.051, 0.029, 0.220, 0.519,
0.184 0.874 1.165 0.975 0.688 1.896 3.000 0.894 1.114 1.791 ± ±
0.051, 0.295, 0.517, 0.502, 0.352, 1.034, 2.100, 0.401, 0.516, 1.030, ± ±
geometric mean titre. 95% Con®dence interval.
0.787 0.475 0.324 0.248 0.352 0.295 0.146 0,123 0.134 0.105 1.657 2.372
0.658 2.590 2.623 1.893 1.343 3.478 4.284 1.994 2.408 3.115
p-value
0.5171 0.0329 0.0003 < 0.0001 0.0075 < 0.0001 0.0001 < 0.0001 < 0.0001 < 0.0001 ± ±
V. Skogen et al. / Vaccine 19 (2001) 197±203
90% in children and 75% in adults is required [20]. It is also a fact that other factors than immunity to diphtheria are important in the spread of diphtheria, as poor socio-economical conditions, and the amount of circulating diphtheria strains in the community. During the diphtheria outbreaks in the US in 1971± 1981 and the Swedish outbreak in the 1980s, a substantial proportion of the patients were middle-aged homeless men abusing alcohol [11,21]. All studies on immunity to diphtheria have to be evaluated in relation to the vaccination programme that has been accomplished in the preceding years. In 1984 the Norwegian vaccination scheme was changed, reducing the number of doses from ®ve to four, with basis immunisation including 3 doses followed by a booster dose given at the age of 11 years. The old scheme included four doses of toxoid at 3, 4, 5 and 17 months of age followed by a booster dose at 11 years. In a previous study, we found no signi®cant dierence in the overall protection against diphtheria, when comparing immunity among adults living in Northern Norway and in North-Western Russia [17]. However, in the present study comparing children only, the Norwegian children were found to have a signi®cantly lower degree of protection in almost all age groups compared to the Russian children. The immunity among the younger Norwegian population is vaccine induced, since no circulating C. diphtheriae strains has been identi®ed for more than 30 years, except for the one imported case in 1992. Generally, the Norwegian children had some protection until the age of 6, but insuf®cient protection from 7 to 10 years of age. It therefore seems that the primary immunisation, including three vaccine doses given at 3, 5 and 10 months of age, is unable to maintain an adequate protection level until the booster-dose (dT) is given at the age of 11 years. The general trend among the Russian children was a high antibody titre indicating a markedly better immunity against diphtheria than among the Norwegian children of corresponding age. This was most pronounced for the older age groups. This seems to be due to a compulsory vaccination programme, which in general includes more booster doses than in Norway. It is also a fact that the sera used in this sero-epidemiological study were collected in a period when the epidemic peaked. At this time a great eort was made in the NIS to achieve high vaccination coverage, in order to control the diphtheria epidemic. None of the Russian children included in this study had been part of a catch up activity that took place in some areas as a response to the outbreak. The Russian children have lived in an environment with circulating C. diphtheriae strains for many years, even before the outbreak. Thus, the immunity is probably not induced by vaccination alone, despite the fact that we excluded children
201
with a known diphtheria diagnosis. In 1994, the school-entry booster dose was reintroduced and the Russian vaccination scheme now includes a booster dose at 2, 6 and 16 years of age. In some areas like the Arkhangelsk Region, the vaccination scheme also includes one booster dose at 9 years of age. A study investigating the risk of diphtheria among school children in Russia in relation to the time elapsed since last vaccination, showed evidence for the ecacy of a booster dose given at the age of school entry in preventing diphtheria among school aged children [22]. Based on these results, an additional booster dose in childhood should be considered included in the Norwegian vaccination programme if a general protection against diphtheria should be obtained. A Swedish study showed that diphtheria antitoxin levels among Swedish children were unsatisfactory [23]. Among 6 year old children who had received primary vaccination with a three-dose schedule DT before one year of age, as many as 15% were unprotected i.e. they had a serum antitoxin level of less than 0.01 IU/ ml. Among 10 year old children as many as 48% were unprotected. In Sweden as in Norway the compliance to the vaccination programme is above 90% [23,24]. The low protection rate among Swedish children was explained by a too long interval between the primary doses given during the ®rst year of life and the booster dose given at 10 years of age [23]. Another explanation could be a less potent vaccine, since the pertussis component, that is regarded to act as an adjuvant, was not part of the Swedish vaccine [23]. Most countries have now adopted the recommendations from WHOs Expanded Programme on Immunization (EPI), which includes a short interval between the second and the third dose of the primary immunisation scheme [25]. However, in some countries, like Norway, Sweden and Italy, the ®rst two doses of the primary immunisation are given at 3 and 5 months of age and the third at 10±12 months of age. Sero-epidemiological studies conducted in Italy have shown that children receiving a booster at the age of 6 years have good protection until early adolescence [26±28]. A serological study performed among school children in Denmark in 1987 demonstrated that the proportions of 8 and 10 year old children not protected against diphtheria were 14% and 21%, respectively [29]. This Danish population did not receive any booster doses after their primary immunisation (at the 5th, 6th and 15th month of age). The authors concluded that a booster should be included during school age. In 1996, the booster was introduced at the age of 5 years [10]. Our results, clearly demonstrate that the situation in Norway is similar to that of Denmark. The gender-related dierence showing that boys had a higher level of antibodies to diphtheria toxin than girls was unexpected. A similar ®nding was
202
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recently published by Mark et al. [30] and by Rieger et al. [31]. We reported a gender dierence among adults in Norway [17]. However, this was explained by the booster immunisation of men entering military service. We have no explanation for the gender dierence observed among children. Further attention and studies are needed, in order to evaluate the gender-related dierence to vaccineinduced immunity. Until recently, the pertussis component in the DTP vaccine for primary immunisation contained whole cell antigen (Pw). New acellular pertussis vaccines (Pa) containing from one to ®ve dierent single antigens are now more commonly used, mainly due to the lower frequency of adverse vaccine reactions [25]. However, this change from the whole cell to the acellular pertussis component in the DTP may in¯uence the immune response to the other two components of the vaccine, since the Pw component might have a stronger adjuvant eect than some of the Pa components in the vaccines [32]. In January 1998, the Norwegian health authorities introduced the DTPa vaccine giving three primary doses at 3, 5 and 12 months of age and a booster dose at the age of 11 as stated previously. Consequently, protection against diphtheria among the Norwegian population that will be vaccinated according to the new programme may become even weaker in the coming years than shown in this study. An additional dose of diphtheria toxoid given during childhood may reduce this tendency. In conclusion, the Russian children studied had a satisfying degree of protection in all age groups examined. This indicate that the eort to reach high vaccination coverage, in order to control the diphtheria epidemic has been successful. The Norwegian children had a poor protection level from the age of 7 years until they received the booster dose at the age of 11. A revision of the Norwegian vaccination programme, including a booster dose of diphtheria toxoid at school-entry age seems warranted.
Acknowledgements We wish to thank Anne-Gerd Gundersen at the National Institute of Public Health, Oslo for excellent technical assistance. We also thank Dr. J. Nielsen at Hammerfest Hospital and Professor T. Flñgstad at the University Hospital of Tromsù for providing the Norwegian sera. This work was supported by grants from the Research Council of Norway (Medical Science and Health), the Barents secretary and the Norwegian Ministry of Foreign Aairs.
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