**4. Discussion**

This serological assessment using specimens previously collected in 2018 from over 30,000 children aged less than 15 years provided the first estimates of tetanus and diphtheria seroprotection among children in Nigeria. Overall, both tetanus and diphtheria seroprotection were below the recommended vaccination coverage levels of at least 80% for tetanus [25] and 90% for diphtheria [26] to prevent infections in the population, especially among younger age groups. Full and long-term seroprotection against tetanus and diphtheria were low across all age groups and geographies. The survey also identified demographic and socio-economic factors that were associated with decreased seroprotection to tetanus and diphtheria. Tetanus seroprotection varied significantly by age, sex, geopolitical zone, state, urban/rural, and wealth quintile. Diphtheria seroprotection varied mainly by age, sex, geopolitical zone, and state.

Comparisons of tetanus seroprotection by age showed that children aged 2–5 years had the highest proportions of children who lacked tetanus seroprotection and the lowest geometric mean antibody levels; this finding correlates with studies reporting decreases in tetanus seroprotection starting at the age of 2 years [25]. Similarly, diphtheria seroprotection and geometric mean antibody levels were lowest in children who were 2–6 years old. When comparing tetanus and diphtheria seroprotection by age group and state, we observed that proportions of children with diphtheria seroprotection among older age groups were highest in the areas that had the lowest tetanus seroprotection (Figure 1), which highlighted the important contribution of natural infection to diphtheria seroprotection in children ages five years and older. For example, Kano State had one of the lowest minimal seroprotection levels against tetanus (49%) and diphtheria (52%) among children aged <4 years, reflecting low routine immunization coverage, yet diphtheria immunity reached 87% in children aged 10–14 years. Similar observations were noted in Borno and other northern states, likely reflecting the frequent occurrence of diphtheria outbreaks mainly in Northern Nigeria [17,18]. The most recent outbreak in Kano during 2022–2023 caused over 25 deaths, mostly among children [19]. Hence, in addition to improving DTP3 vaccination coverage, Nigeria needs to consider introducing the WHO-recommended booster doses for tetanus and diphtheria at the ages of 12–23 months, 4–7 years, and 9–15 years to ensure long-term protection across the life-course and prevent recurring diphtheria outbreaks, as well as sustain maternal and neonatal tetanus elimination [20,21].

Given that tetanus seroprotection is an indirect measure of DTP3 vaccination coverage especially in younger age groups, tetanus seroprotection levels highlighted areas and populations requiring urgent attention to improve vaccination coverage. Girls had significantly lower levels of full and long-term seroprotection to tetanus and diphtheria compared to boys, indicating the need to ensure that girls are reached with immunization services and are adequately protected against VPDs. Gender-based inequity in access to vaccination services and protection from VPDs has been noted in multiple countries with associations in some cases with maternal education [11,30]. In Nigeria, higher maternal education was associated with higher childhood vaccination coverage based on analysis of the 2018 DHS [31] and with higher utilization of essential maternal and child health services based on analysis of five national household surveys [32].

Geographic variations in tetanus and diphtheria seroprotection highlighted zones and states that require remediation to decrease the number of children susceptible to VPDs. The north west and north east geopolitical zones had the lowest proportions of tetanus and diphtheria seroprotection indicating the need for catch-up vaccination as well as strengthening infant immunization coverage. However, heterogeneity in seroprotection among children aged 0–4 years was also observed between states within the same geopolitical zone reflecting variation in routine immunization coverage. For example, Adamawa state in the north east had >80% minimal seroprotection against tetanus and diphtheria among children aged 0–4 years, indicating better routine immunization performance relative to the north east zone, which had <70% tetanus seroprotection overall. Similarly, inequities in seroprotection against tetanus were also observed in states located in the south south and south east geopolitical zones which were better performing overall than other geopolitical zones. Therefore, serosurvey results highlighted subnational geographies that would benefit from intensive support to improve vaccination coverage and remediations efforts to

prevent diphtheria outbreaks. Our findings were similar to a mapping of areas at risk for measles in Nigeria, which indicated that susceptibility to multiple VPDs tended to cluster in similar states and geopolitical zones [33]. Inequities in seroprotection against tetanus were noted in rural versus urban areas and lower versus higher wealth quintiles. These factors have been shown to be associated with tetanus seroprotection, vaccination coverage or access to maternal and child health services in Nigeria and other countries [11,30–32,34,35].

Triangulating different data sources enabled further interpretation of serosurvey findings. When comparing seroprevalence and vaccination coverage data, DTP3 coverage underestimated seroprotection to tetanus across children of all ages. Immunity can result from a partial series of a multidose vaccine (i.e., DTP2) [14]. Vaccination coverage surveys are also at risk of information and recall bias, and both card documentation and recall have been found to underestimate actual vaccination coverage [9]. The limitations of measuring vaccination coverage based on card or recall have been noted in other tetanus serosurveys [5,7,10,11]. We observed increased tetanus seroprotection among children aged 10–14 years compared with those aged 5–9 and 0–4 years, unlike the gradual decrease in tetanus seroprotection with age documented in other countries related to waning immunity and not providing childhood booster doses against tetanus and diphtheria [8,11,12]. The increased tetanus seroprotection in children aged 10–14 years and the gradual increase in geometric mean antibodies starting at the age of 6 years in Nigeria is most likely a result of the multiple meningitis vaccination campaigns targeting ages 1–29 years in states at highrisk for meningitis in Northern Nigeria during 2011–2014 [36,37]. These campaigns used the meningococcal A conjugate vaccine (MACV) which is conjugated to tetanus toxoid [38] and has been shown to boost tetanus immunity in other countries in the African meningitis belt [39,40]. A tetanus serosurvey conducted in Mali before and after the MACV campaigns showed increase in geometric mean concentrations and tetanus immunity among people aged 1–29 years from 57% to 88% [39]. In addition, clinical studies on MACV showed robust tetanus serologic response in people aged 1–29 years after MACV vaccination [40]. Therefore, the four rounds of MACV campaigns in 17 states in northern Nigeria might have helped boost tetanus immunity and contribute to the higher proportion of minimal, full, and long-term tetanus seroprotection in older children compared to younger age groups.

This serologic assessment used existing stored specimens from NAIIS, which was designed and implemented to make the survey as representative as possible. However, any deviations from protocol implementation may limit generalizability of the findings to the Nigerian population. For example, 72 (1.8%) enumeration areas out of a total of 4035 selected for NAIIS were unable to be visited because of security challenges and one area was not visited due to flooding, potentially limiting representativeness of survey estimates in these areas. The response rate for blood collection in children aged 0–9 years (68.5%) was lower than in children aged 10–14 years (92.3%), and while the distribution of children whose parents refused use of specimens for other tests was similar across gender, age, zone and cluster compared to those who were tested for tetanus and diphtheria, more of these children were in Lagos (15.6%) and Kano (11.0%) [22]. However, the survey included a large sample size which allowed for precise estimates of seroprotection down to the state level, and the overall population distribution of the NAIIS survey was similar to the population of Nigeria [41]. Finally, while the serosurvey results represent a specific time point (2018), results are still relevant in 2023 as vaccination coverage in Nigeria has not changed significantly compared to 2018. DTP3 coverage estimates were 56% during 2019–2021 compared to 55% in 2018 based on WUENIC estimates [27] and 57% in the MICS 2021 compared to 50% in the 2018 DHS [28,42]. These vaccination coverage estimates were still below the seroprotective levels needed to prevent infections. Hence, results in this serosurvey are still relevant to inform public health interventions and policy. In the future, it would be beneficial if testing for vaccine-preventable diseases could be integrated in parallel with other disease-specific testing in large population-based surveys, such as NAIIS, instead of waiting until completion of testing for other diseases to be able to access the specimens. This would enable timely availability of data showing granular differences

in seroprotection in specific demographic and socio-economic groups and would help inform immediate targeted public health actions to address inequities in immunization.
