Emerging Infectious Diseases [Volume 5 No.5 / September - October 1999] Dispatch Diphtheria Antitoxin Levels in the Netherlands: a Population-Based Study H.E. de Melker, G.A.M. Berbers, N.J.D. Nagelkerke, and M.A.E. Conyn-van Spaendonck National Institute of Public Health and the Environment, Bilthoven, the Netherlands ----------------------------------------------------------- In a population-based study in the Netherlands, diphtheria antitoxin antibodies were measured with a toxin-binding inhibition assay in 9,134 sera from the general population and religious communities refusing vaccination. The Dutch immunization program appears to induce long-term protection against diphtheria. However, a substantial number of adults born before the program was introduced had no protective diphtheria antibody levels. Although herd immunity seems adequate, long-term population protection cannot be assured. As more than 60% of orthodox reformed persons have antibody levels lower than 0.01 IU/ml, introduction of diphtheria into religious communities refusing vaccination may constitute a danger of spread of the bacterium. The recent diphtheria epidemics in eastern Europe are a warning that diphtheria can make a comeback in susceptible populations (1). The World Health Organization (WHO) recommends the assessment of diphtheria immunity in nonepidemic countries, to prevent any indigenous cases in the European region by the year 2000 (2). In the Netherlands, the last diphtheria epidemic occurred during World War II (220,000 cases in 1940 to 1946). Diphtheria vaccination was introduced in 1952 for persons born after 1945. Under the current schedule, children are vaccinated at ages 3, 4, 5, and 11 months with diphtheria, tetanus, pertussis, and inactivated polio vaccine (DTP-IPV) and at ages 4 and 9 years with DT-IPV. For the past 25 years, the vaccine coverage for at least three vaccinations at the age of 12 months has been 97%. Rare exposure to Corynebacterium diphtheriae may have led to lack of boosting opportunities (1). As in other industrialized countries, lack of immunity in older persons is a reason for concern (3,4). Furthermore, in the Netherlands, the immune status of sociogeographically clustered members of religious communities who refuse vaccination may be even more unfavorable. Inadequate herd immunity to diphtheria in these groups could lead to outbreaks similar to the poliomyelitis outbreaks in the Netherlands (5). A large population-based serum bank allowed us to assess the diphtheria immunity in the general Dutch population and in persons refusing vaccination (6). The Study From October 1995 through December 1996, a population-based serum bank with specimens from 9,948 persons was established (6). Our objective was to select 40 municipalities with samples proportional to population size. In each of five regions, eight municipalities were included. For each of these 40 municipalities, an age-stratified sample of 380 persons was drawn from the population register (7). Participants were requested to have a blood sample drawn, complete a questionnaire, and provide immunization and military service records. Participants were also selected from eight additional municipalities with low vaccine coverage to assess the immunity of members of religious communities that refuse vaccination. The nationwide sample had 8,357 (55%) participants, and the low vaccine coverage sample had 1,589 (52.5%). Sufficient serum was available for testing 7,715 of the nationwide participants and 1,419 of the participants in the sample with low vaccine coverage. Methods Sera were stored at -86°C. The level of diphtheria antitoxin antibodies was measured with a toxin-binding inhibition assay (8). In brief, twofold serum dilution series were incubated with a fixed amount of toxin, and the nonneutralized toxin was measured in an enzyme-linked immunosorbent assay (ELISA) with equine antitoxin purified from hyperimmune serum as coat and peroxidase-labeled horse antidiphtheria IgG as conjugate. International units were calculated according to the WHO reference standard serum (10 IU/ml) by the four-parameter fit method in Kineticalc (KC4, Biolyse) with a Bio-Tek plate reader (EL312d). The minimum level of detection was 0.01 IU/ml, and samples below this level were set to 0.005 IU/ml for calculating geometric mean titers. The correlation of this method with the Vero neutralization assay has been confirmed recently (r >/= 0.95) (9). Antitoxin antibody levels were classified according to international standards as < 0.01 IU/ml (no protection), 0.01 IU/ml to 0.1 IU/ml (basic protection) and >0.1 IU/ml (full protection) (10). Analysis Frequencies and geometric mean titers in each municipality were weighted by the proportion of the age group in the population. To produce national estimates, the weighted frequencies and geometric mean titers were averaged over the 40 municipalities (7). For the low vaccine coverage sample, they were averaged by weighting the population of the municipality. Data on age, sex, marital status, country of nationality, degree of urbanization, region, and contact information for all participants and nonparticipants were available. The effect of differential probabilities of response for these variables on both sample estimates was less than one standard error and was therefore disregarded. Linear regression analysis was used to study the persistence of diphtheria antitoxin antibodies after full immunization in the national immunization program. The association between diphtheria antibody titer (2log) and age in 2log years was studied for persons who received the sixth documented vaccination at 8 to 9 years of age, without self-reported or documented revaccination or history of military service. Age-Specific Immunity Levels to Diphtheria Antitoxin In the nationwide sample, 58.1%, 30.0%, and 11.9% of persons /= for greater than or equal to.