Lack of seroprotection against diphtheria in the Austrian population, in light of reported diphtheria cases in Europe, 2022

Following an increase in diphtheria cases in Europe since 2022, we retrospectively estimated the prevalence of seroprotection against diphtheria and tetanus in 10,247 Austrian residents (population: 8,978,929) voluntarily tested between 2018 and 2022. Lack of seroprotection against diphtheria was found in 36% compared with 4% against tetanus. The geometric mean antibody concentration against tetanus was 7.9-fold higher compared with that for diphtheria. Raising awareness of regular booster vaccinations against diphtheria in combination with tetanus and pertussis is urgently needed.

Following an increase in diphtheria cases in Europe since 2022, we retrospectively estimated the prevalence of seroprotection against diphtheria and tetanus in 10,247 Austrian residents (population: 8,978,929) voluntarily tested between 2018 and 2022. Lack of seroprotection against diphtheria was found in 36% compared with 4% against tetanus. The geometric mean antibody concentration against tetanus was 7.9fold higher compared with that for diphtheria. Raising awareness of regular booster vaccinations against diphtheria in combination with tetanus and pertussis is urgently needed.
Since August 2022, an increase of diphtheria cases among migrants has been observed in Europe [1]. A recent study found that a high proportion of adults in 18 European Union/European Economic Area (EU/EEA) countries did not have protective antibody concentrations against diphtheria [2]. Although no diphtheria cases among the general population in Austria or the EU/EEA have been detected, low seroprotection in the resident community provides an increased risk for disease introduction and outbreaks. Here, we aimed to estimate the prevalence of seroprotection based on the measurement of antibody concentrations as correlate of seroprotection against diphtheria in Austria among 10,247 voluntarily tested individuals between January 2018 and January 2022 to recommend data-based public health measures.

Seroprotection against diphtheria and tetanus
We included all individuals in Austria who voluntarily provided samples for testing of anti-diphtheria toxoid IgG (DT) and anti-tetanus toxoid IgG (TT) concentrations at the Austrian reference centre for diphtheria, tetanus, and pertussis serology at the Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna between January 2018 and January 2022. In addition, we assessed the extent of DT and TT antibody waning in individuals with antibody results available from a minimum of two time points counted from the latest vaccination. DT and TT antibody concentrations were determined using commercial enzyme-linked immunosorbent assay (ELISA; Binding Site Group Ltd.). Samples with IgG concentrations above the upper limit of quantification (DT: 3 IU/mL, TT: 7 IU/mL) were not further titrated. Antibody concentrations were categorised according to Plotkin et al. [3,4]: non-protective (< 0.01 IU/mL), inadequately protective (≥ 0.01 to < 0.1 IU/mL), adequately protective (≥ 0.1 IU/mL) against diphtheria and tetanus, respectively.
Prevalence of diphtheria and tetanus seroprotection, inadequate seroprotection and non-seroprotection and their multinomial 95% confidence intervals (CI) were estimated by the method of Sison and Glaz [5]. Antibody concentrations were log-transformed and analysed by a generalised linear model, with age categories and sex as independent variables. Geometric mean concentrations (GMC) and 95% CI were calculated based on the least-squares estimates. Waning of antibody titres was analysed by generalised estimating equations model separately for DT and TT antibody concentrations, with age and sex as covariates and time since last vaccination as a within-subject variable.
Between January 2018 and January 2022, 10,247 individuals were included in the analyses of diphtheria seroprotection and 8,034 individuals of tetanus seroprotection (Table 1, see Supplementary Figure S1 for overview of selection of individuals for analyses). For the analysis of antibody waning, 17,468 serum samples from 16,090 individuals, obtained between March 2010 and January 2022 were screened for the availability of vaccination information. Of these, vaccination data and more than one result of anti-DT testing, i.e. from at least two different time points, were available in 89 individuals; in 64 of these individuals, results of anti-TT testing were also available. Overall, test results from 322 samples were analysed for waning of antibody concentrations.

Diphtheria and tetanus toxoid antibody waning
The DT antibody concentrations declined with an average percentage change of 2.9% (95% CI: 0.6-5.0; p < 0.001) in the 89 individuals. The DT antibody concentrations decreased with age, with a percentage change of 16.5% (95% CI: 8.0-24.1; p < 0.001) for each 10-year increase in age (data not shown; of note: individuals were not split up into age groups but entered with their exact age).
The estimated initial GMC of TT antibodies following the latest vaccination was 20.6-fold higher than that of the DT antibodies (GMCs TT: 3.92 IU/mL vs GMCs DT: 0.19 IU/mL). The TT antibody concentrations decreased with an average annual percentage change of 6.9% (95% CI: 4.6-9.1; p = 0.012, data not shown). However, because of the high initial TT antibody concentrations, the average decline of antibodies was estimated not to reach the threshold of inadequate seroprotection of 0.1 IU/mL earlier than 50 years after the last vaccination in the subgroup of the 64 individuals.

Discussion
After the introduction of general childhood vaccination against diphtheria in Austria in 1946, the occurrence of the disease has remarkably decreased by the end of the 1960s. Only sporadic cases of infection with toxigenic Corynebacterium diphtheriae were reported until recently [7], despite an insufficient coverage of primary childhood vaccination (85%, rank 141/192 countries) [8][9][10]. Up to 24 January 2023, 242 imported cases of diphtheria have been reported by eight EU/EEA countries, including 72 in Austria, all among recently arrived migrants [11]. In Austria, one fatal case of respiratory diphtheria was reported in a migrant from outside of the Europe. Thus far, the European Centre for Disease Prevention and Control (ECDC) has no indication of a spread from the currently affected groups to the general population. However, an increased risk of exposure to Corynebacterium species in individuals in contact with the reception centres or their residents lacking sufficient protection against diphtheria needs consideration. Therefore, knowledge on the seroprotection prevalence in the population is crucial for potential implementation of public health measures, such as vaccination campaigns for disease control.
We observed a low prevalence of seroprotection against diphtheria in the tested population, which raises the question whether this is due to missed booster vaccinations, early antibody waning or low vaccine-induced antibody levels. The lower DT vs TT antibody levels might be due to a reduced DT content in the adult booster vaccine formulation compared with the infant formulation [12,13]. In addition, the considerably higher tetanus antibody titres could be a result of a preferred use of monovalent tetanus vaccines (no longer available) in emergency care settings in the past. The DT antibody levels decreased more slowly than the TT antibody levels, but because the levels started from a lower average concentration, participants' diphtheria antibody levels were more likely to fall below the protective level. These findings, albeit based on a small  Antibody titres were interpreted as non-protective (antibody concentration of < 0.01 IU/mL) and protective (antibody concentration of ≥ 0.1IU/mL).
sample, may imply that the national recommendation for regular boosters with combined vaccines, i.e. diphtheria/tetanus/pertussis ± polio vaccines every 10 years until the age of 60 years and thereafter every 5 years, is not followed [14]. The fact that 36% of individuals actively requesting testing of their DT antibody levels lack adequate seroprotection against diphtheria requires immediate attention, especially given the assumption that seroprotection level in the general population may be even lower.
Currently, the most important interventions are (i) to provide information and easy access to primary and booster vaccination to populations at risk, such as migrants from regions with low vaccination coverage due to disruption of medical service in their home countries, (ii) to improve the awareness of high coverage for primary and regular booster vaccinations so herd immunity is achieved in the local population and (iii) to use booster vaccinations with monovalent diphtheria vaccines (as was previously available) to avoid side effects because of unnecessarily increased TT antibodies, or to consider a higher diphtheria toxoid dose for the adult booster vaccine formulation; the latter may need further evidence from larger studies Vaccination and medical history were incomplete for the whole study population because of lack of information on the referral sheets. Although we do not know the intention of the individuals for whom we analysed the anti-TT and anti-DT antibody concentrations, experience indicates that many wish to have their level of seroprotection tested before they decide whether to receive a booster vaccination. For some individuals, the sera were sent in by their treating physicians because of an underlying disease. Thus, the study population is not necessarily representative of the general population and might have a higher health awareness. Thus, we suggest that the observed prevalence of seroprotection is probably higher than in the general population, which may be unaware of the need for regular booster vaccinations.

Conclusions
Our findings, combined with a low coverage of 85% for primary diphtheria vaccination in children in Austria, signal that the detection of diphtheria cases among migrants coincides with an inadequately immunised resident population. In order to protect both migrants and the resident population from diphtheria infections, there is a need to improve seroprotection by vaccination in both groups.

Ethical statement
The Ethics Committee of the Medical University of Vienna approved this study (ethics committee identification number: 2117/2022).

Funding statement
The study was financed via third party funding derived from the serodiagnostic research unit at the Institute of Specific Prophylaxis and Tropical Medicine, Medical University Vienna.

License, supplementary material and copyright
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This article is copyright of the authors or their affiliated institutions, 2023. Median values (yellow line) and interquartile range (yellow dotted line) are represented. Levels of significance in a Kruskal-Wallis test are indicated by asterisks: **** p < 0.0001, ** for p < 0.01 and * for p < 0.05.