{'fraction_non_alphanumeric': 0.037248787248787246, 'fraction_numerical': 0.030405405405405407, 'mean_word_length': 5.200322234156821, 'pattern_counts': {'":': 0, '<': 7, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Seroepidemiology of SARS‐CoV‐2 in pediatric population during a 16‐month period prior to vaccination

Abstract Limited prospective serosurveillance data in children regarding severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection have been reported. We prospectively investigated SARS‐CoV‐2 seropositivity in children during a 16‐month period of the coronavirus disease 2019 (COVID‐19) pandemic, including the four waves of the pandemic, before SARS‐CoV‐2 adolescents' vaccination. Serum samples from children admitted to the major tertiary Greek pediatric hospital for any cause, except for COVID‐19 infection, were randomly collected from 05/2020 to 08/2021. The study period was divided into four 4‐month periods representing relevant epidemic waves. Total SARS‐CoV‐2 antibodies for nucleocapsid protein were determined using the Elecsys® Anti‐SARS‐CoV‐2 reagent. A total of 3099 children (0–16 years) were included in the study. A total of 344 (11.1%) seropositive children were detected (males: 205 [59.5%]; median age [interquartile range [IQR]]: 3 years [0.6–10]). Seropositivity rates (%) increased during the four 4‐month periods: 1.4%, 8.6%, 17.2%, and 17.6%, respectively. A correlation of seropositivity rates in children with new diagnosed SARS‐CoV‐2 cases in the community was detected. No significant differences were detected between males and females. Seropositivity was significantly higher in hospitalized than in nonhospitalized children and in non‐Greek compared to Greek children (p < 0.001). The lowest seropositivity rate before school opening (9/2021) was detected in the age groups 6–12 years (14.4%) and 12–16 years (16.1%). However, compared with the other age groups, the lowest median antibody titers were observed in children 0–1 year (median [IQR]: 13.9 cut‐off index: [4.5–53.9] [p < 0.001]). Although the seropositivity of children was related to the community epidemic waves, the exposure was limited. Low seropositivity rates in school‐age children support the need for SARS‐CoV‐2 immunization.


| INTRODUCTION
Since the first reports of coronavirus disease 2019  and the identification of the novel coronavirus that causes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infection has spread at an unprecedented pace and caused more than 200 million confirmed cases in adults and children worldwide. 1,2 Although most pediatric COVID-19 cases present as asymptomatic, mild, or moderate, there are rare cases of severe clinical presentation of the postinfection multisystem inflammatory syndrome, when children require hospitalization to prevent life-threatening complications of the infection. [3][4][5] It is difficult to estimate the exact incidence rate of SARS-CoV-2 infection in children, mainly due to the increased number of asymptomatic cases. 6 Children are commonly infected after close contact with an infected family member within the same household, even though viral transmission rates have also been reported in several other activities, including schools. 7 Serological studies have been critical in tracking the evolution of the COVID-19 pandemic by deciphering the true extent of transmission in different populations. 8 Estimating the true rate of SARS-CoV-2 infection allows public health specialists to predict the likely future course of the epidemic in specific locations or populations and to better design interventions to control the epidemic. 8,9 The aim of this study was to prospectively evaluate the SARS-CoV-2 seropositivity rates of children 0-16 years old during a 16month period of the pandemic in Athens, Greece before adolescents' immunization. In the study, the role of epidemiological parameters, including sex, age, origin, and hospitalization status, was also evaluated.

| Study design and participants
This was a prospective cohort study involving children who presented in the emergency department or were admitted for any reason to the ''Aghia Sophia'' Children's Hospital, a 750-bed tertiary pediatric hospital, which is the largest hospital for children in Greece.
To evaluate the seropositivity of children in the Athens metropolitan area, serum samples were prospectively collected from 05/ 2020 to 08/2021. Each month, approximately 200 serum samples were randomly collected from the Department of Clinical Biochemistry of "Aghia Sophia" Children's Hospital. Serum samples were residual sera that were ordered from paediatricians for any medical reason from hospitalized children or children from the emergency department. Children with proven COVID-19 infection either with SARS-CoV-2 PCR or with the rapid test were excluded from the study. Epidemiological parameters, which included age, sex, origin, and hospitalization status, were recorded. If a child was admitted to the hospital more than once within the study period and the results of his antibody test results were positive, only those of the first positive result were included in the analysis. Further laboratory testing of the serum samples was performed anonymously using an identification code.
Data on the new cases of COVID-19 in Greece were found on the site of the Greek National Public Health Organization (GNPHO). 10

| Ethical approval
The study protocol was in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards and was approved by the scientific and bioethics committee of "Aghia Sophia" Children's Hospital (No. 25609).

| Statistical analysis
The assumption of normality was checked through kurtosis and skewness, Kolmogorov-Smirnov, and Shapiro-Wilk tests.
The seropositivity of SARS-CoV-2 (%) during each month of the study is presented in Figure 1A  The SARS-CoV-2 seropositivity (%) detected in the four different subperiods per age group are presented in Table 1 and Figure 2. In all age groups, there was a statistically significant increase in seropositivity during the four study subperiods (p < 0.001) except for the age group of 4-6 years (p: 0.119). In all four study subperiods, the age group 4-6 years had the highest seropositivity rate, followed by the 0-1 year and the 1-4 years group. The lowest seropositivity rate at T A B L E 1 Distribution of the demographic characteristics of the 3099 children who included in the study in the four different study periods: 1st: 05/2020-08/2020, 2nd: 09/2020-12/2020, 3rd: 01/2021-04/2021, 4th: 05/2021-08/2021  No significant differences were detected between seropositive males and females during the study period (p: 0.09) ( Table 1). Seropositivity was significantly higher in hospitalized children than in non-hospitalized children (p: 0.0004) ( Table 1). Children of non-Greek origin had statistically significant higher seropositivity rates compared to children of Greek origin in all subperiods (p < 0.001) ( Table 1).
Antibody titers per age group are presented in Figure 3. The median (IQR) antibody titer per age group was 13.9 COI (4.5-53.9) in In contrast, the subsequent increase coincides with the release of restriction measures, as well as the circulation of more transmissible variants of SARS-CoV-2. 12,13 In our area, children <17 years old represent almost 17% of the total population. Even taking into account the highest seropositivity rate of children during the study period (21.8%), it means that only an additional 3.7% of the total population has SARS-CoV-2 immunity. At the same period (end of August 2021), the GNPHO reported that people <17 years represent 12.1% of total cases. 10 For this discrepancy between pediatric confirmed cases and seropositivity, we could hypothesize that antibodies are waning over time or there is a delay between cases and antibody detection in the population. Another hypothesis could be that children who have asymptomatic or mild symptomatic infection do not display detectable antibody responses.
No differences in seropositivity rates between sexes were detected, which is consistent with findings regarding confirmed SARS-CoV-2 cases in Greece. 10 Data from the United States on a Nationwide Commercial Laboratory Seroprevalence Survey also support the comparable seroprevalence rates between females and males (20.4% and 20.8%, respectively), as well as the equal incidence of the disease in both women and males <17 years by August 2021. 10,14,15 In all study subperiods, pre-school children (4-6 years old) had the highest seropositivity rate. This finding may possibly be overestimated due to the limited number of children of this age group represented in the study. Nevertheless, it may partially explain the transmission of preadolescent children in kindergartens and playgrounds, in which epidemic control strategies are not always effective due to absence of physical distancing and use of face masks. 16 An increased proportion of seropositive hospitalized compared to non-hospitalized children during the study period (12.3% and 8%, respectively) was detected. This could be a reflection of persistent physical symptoms in children after a known or unknown SARS-CoV-2 infection (long-COVID-19), including dyspnea, cough, headache, joint, or chest pain that may require hospitalization for further evaluation. 17 Another hypothesis could be that COVID-19 infection could affect post-infectious general health status. This has been shown for other infections, such as measles, which has long-term effects in childhood infectious disease morbidity and mortality. 18 However, this is a hypothesis that must be tested in large epidemiological studies.
In the present study, significant differences in seropositivity rates were found between children of Greek and non-Greek origin in all different subperiods. It appears that SARS-CoV-2 alongside other infectious diseases incommensurately affects underrepresented racial and ethnic groups within society. 19,20 This trend may possibly be attributed to poverty, densely populated households, and to limited access to health facilities. 19 Limitations of the study include that it represents only the pediatric population of Athens and not country-wide data. In addition, the distribution of age groups in the study does not necessarily represent the total pediatric population. Furthermore, seroepidemiology studies performed in hospitals may overestimate SARS-CoV-2 seropositivity as some of the children could be at the beginning of the infection or could be exposed because of their hospitalization. These potential limitations are common to most seroprevalence studies and do not degrade the utility of the data during the pandemic, as so far this is the only longitudinal study in our area. 9

| CONCLUSION
As most children have mild symptoms after SARS-CoV-2 infection or remain asymptomatic, it is difficult to determine the exact incidence of COVID-19 in the pediatric population. Through a prospective investigation of seropositivity in children, we showed that seropositivity of children is associated with epidemic waves in the community. Certain groups were affected disproportionately, indicating that public health decisions need to better protect these groups to reduce inequity in the impact of COVID-19. Before the initiation of SARS-CoV-2 immunization for adolescents and younger children, the number of seropositive children is limited, which supports the need for immunization. Continuous surveillance of seroepidemiological data in children can guide and optimize public health policies on the implementation of preventive measures for transmission or immunization in schools and communities.