Our study, titled Coro-Buddy (Coronavirus and Antibody Buddy), is a longitudinal, prospective, observational study to determine the prevalence of SARS-CoV-2 antibodies as a surrogate for previous infections in children and adolescents, and the change of antibody incidence over 12 months. Saliva samples from each study participant were collected at three time points: time point 1 (T1), after the release of lockdown measures in spring 2020 (ie, summer 2020); time point 2 (T2), before the winter season 2020; and time point 3 (T3), after the winter season 2021. An adult cohort (ie, adult comparator group) was sampled at the same time points to describe the seroepidemiological profile in adults. In addition, to establish and validate the SARS-CoV-2 antibody measurement process in saliva, an additional adult cohort group (ie, adult validation group) will have saliva and peripheral blood samples collected every 2 months at seven time points, over a 1-year follow-up. See Figure 1 for an overview of the study flow.

Children and adolescents were enrolled and sampled via preschools, primary schools, and secondary schools randomly spread over the Tübingen city area. In total, 1850 children and adolescents were enrolled: (1) preschools—infants aged 1 to 3 years (n=350) and young children aged 4 to 6 years (n=350), (1) primary schools—children aged 7 to 10 years (n=350), and (3) secondary schools—children and adolescents aged 11 to 18 years (n=800). Study participants and their families were informed about the study and its aims via their institutions. In addition to all relevant documents, families were also provided with a link to a video that explains the study (Multimedia Appendix 1) and procedures in an easy-to-understand way to ensure broad participation. Saliva was sampled after written informed consent has been given by parents or legal representatives. Assent from children aged 12 years and older was collected on the day of first sampling. Inclusion criteria include the following: aged 1 to 18 years, enrolled in an educational institution within the city of Tübingen, and written informed consent given or written assent given if the child is 12 years or older. Exclusion criteria include not attending a preschool or school in the city of Tübingen.

For the adult comparator group (n=400), any person aged 18 years and older living or working in Tübingen who had given written informed consent could be enrolled. For the adult validation group (n=40), the following individuals can participate in the study: those with a previous SARS-CoV-2 infection, confirmed by either quantitative polymerase chain reaction or by ELISA with a SARS-CoV-2–specific antibody at any time period before enrollment, who have given written informed consent. Blood and saliva samples are being collected every 2 months for 12 months (Figure 1).

Tübingen has a total of 91,656 inhabitants; among them, 4910 are children under the age of 6 years and 8366 are children between the ages of 6 and 18 years (≤6 years: n=4061; 7-18 years: n=6621, as of December 31, 2019). Therefore, in total, 13,276 children were living in Tübingen at the time of the study; of these, we aimed to recruit 1850 children and young adults. In addition, 400 adults will be included as an adult comparator group. At the time of planning, 567 out of 100,000 inhabitants—as determined by the Robert Koch Institute in Berlin, Germany, on June 1, 2020—have had a confirmed SARS-CoV-2 infection in the county of Tübingen, but the number of unreported and asymptomatic infections is believed to be higher. Therefore, we expect to have at least 2.2 positive cases in the adult group; in the event that children are infected at an equal frequency, we expect to find at least 10.4 positive cases in children at T1.

A structured questionnaire with six questions addressing exposure to SARS-CoV-2 infection in the family was administered at each of the three time points to the parents and representatives of the children as well as to the participants in the adult comparator group (Multimedia Appendix 2).

Saliva samples were collected from children 6 years and below using an ORACOL S14 saliva collection device (Malvern Medical Developments) by gently brushing the gumline for 2 minutes. For children 7 years and above, 3 mL of saliva was collected by spitting into a plastic tube; 30-mL multipurpose containers (item No. 201150; Greiner Bio-One) were used for this purpose. Saliva samples were kept on ice for a maximum of 3 hours before further processing. The ORACOL S14 saliva collection device was centrifuged at 2500 rpm for 10 minutes and the 30-mL multipurpose containers were centrifuged for 6 minutes. Supernatant was transferred into a 2-mL microtube and inactivated using a solvent and detergent treatment to a final concentration of 0.3% tri-n-butyl phosphate and 1% Triton X-100. Samples were immediately stored at –20 °C. Blood was collected by vein puncture into 9-mL lithium-heparin Monovettes; the plasma was obtained by centrifugation at 1400 rpm for 10 minutes and stored at –20 °C.

Saliva and blood samples were processed according to standard procedures and stored at –20 °C until analysis by ELISA to identify SARS-CoV-2–reactive antibodies. A previously established and validated in-house ELISA to detect antibodies against SARS-CoV-2 in saliva was performed [20]. Blood samples from the validation group cohort will be analyzed using a certified commercial ELISA by EUROIMMUN to quantify immunoglobulin G reactive to the S1 domain of the of SARS-CoV-2 spike protein.

The case report form is the source document for all personal data. Each participant included in the study received a unique identifier. All data were collected on paper forms and were entered and pseudonymized into an electronic database.

Data will be typed into Excel (version 16.51; Microsoft), and graphics will be generated with Prism (version 9.1; GraphPad) and RStudio (version 1.2.5001) running R (version 4.0.4; The R Foundation). Simple descriptive statistical analyses will be conducted. Depending on the classification of the data, parametric or nonparametric tests will be used. Correlation and regression models will be analyzed using RStudio. The estimated prevalence will be computed and adjusted using the R package epiR (version 0.9-43).

This study was conducted with the approval from, and consent form signed by, the parents and participants, according to the protocol that was reviewed and accepted by the Ethics Committee of the University Hospital Tübingen on June 24, 2020 (reference No. 20-231/BO1). Each study participant was protected against invasion of privacy. Written informed consent was obtained from the participants, with parental consent and participant assent from those 12 years of age and older. The trial was retrospectively registered at ClinicalTrials.gov (NCT04581889) on October 10, 2020.

Data will be available upon the study’s completion.