key: cord-0787782-4rp3p3tn authors: Cooper, D. M.; Messaoudi, i. R.; Aizik, S.; Camplain, R. L.; Lopez, N. V.; Ardo, J.; Boden-Albala, B.; Chau, C.; Condon, C.; Golden, C.; Coimbra Ibraim, I.; Jankeel, A.; Kasper, K.; Meyer, A.; Stehli, A.; Stephens, D.; Weiss, M.; Zulu, M. Z.; Ulloa, E. R. title: SARS-CoV-2 Acquisition and Immune Pathogenesis Among School-Aged Learners in Four K-12 Schools date: 2021-03-26 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.03.20.21254035 sha: a03a4dbae5cf0b4a9e586d7347892a95780203f4 doc_id: 787782 cord_uid: 4rp3p3tn Objectives. To directly measure SARS-CoV-2 infection in diverse schools with either remote or onsite learning. Methods. 4 schools participated. Schools A and B served low-income Hispanic learners, school C special needs, and all three provided predominantly remote instruction. School D served middle and upper-middle income, White learners, with predominantly onsite instruction. 320 learners [10.5+/-2.1(SD); 7-17 y.o.]; 86% had phlebotomy. Testing occurred early in the fall (2020), at lower levels of COVID-19, and 6-8 weeks later during the fall-winter surge (tenfold increase in COVID-19 cases). Results: Nasal RT-qPCR for SARS-CoV-2 and 21 respiratory pathogens was performed. Phlebotomy was obtained for circulating immunity. Face covering and physical distancing fidelity was measured by direct observation. 17 learners were SARS-CoV-2 positive during the surge. School A (97% remote) had the highest infection rate (9/70, 12.9%, p<0.01) and IgG positivity rate (13/70, 18.6%). School D had the lowest infection and IgG positive rate (1/86, 1.2%). Mitigation compliance [physical distancing (mean 87.4%) and face covering (91.3%)] was high at all schools. Learners with documented SARS-CoV-2 infection had neutralizing antibodies (94.7%), broad and robust interferon-gamma T cell responses, reduced frequencies of monocytes, and lower levels of circulating inflammatory mediators. Conclusions: Infection in the schools reflected regional rates rather than remote or onsite learning modalities. Schools can implement successful mitigation strategies across a wide range of income, school-type, and student diversity. Reduced monocyte and immune mediator concentrations coupled with robust humoral and cellular immunity may explain the generally milder symptoms in school-aged children. An urgent need for data on SARS-CoV-2 incidence, immune mechanisms, and mitigation fidelity in the unique setting of K-12 schools was recognized at the earliest stages of the COVID-19 pandemic, 1 when K-12 schools closed in the U.S. and across the world. In this report, we summarize the results of the Healthy School Restart Study, a prospective study of four diverse, K-8 schools in Orange County, California, at two distinct phases of the COVID-19 pandemic: 1) early in the fall (2020) school semester, at a relatively low level of community COVID-19 disease incidence of approximately 3-4 cases per 100,000 across the county (in September, surveillance rates for the county were estimated at 12% but 17% in communities of color 2 ), and 2) approximately 6-8 weeks later in the midst of the fall-winter surge in which COVID-19 had increased to about 40 cases per 100,000. We tested the assumption used to support school closures, namely, that learners would be less susceptible to viral infection if they avoided onsite learning. [3] [4] [5] Key objectives were 1) to begin to understand SARS-CoV-2 infection by identifying schools that reflected the diversity of our region; 2) to gain insight into the serological and cellular mechanisms in the pediatric population in response to SARS-CoV-2 infection; and 3) to measure the fidelity of SARS-CoV-2 mitigation procedures. A total of 320 learners [mean age 10.5±2.1(SD)] and 99 school staff enrolled in our study across four schools for two testing cycles. Participants were allowed to enroll in the study at the second cycle even if they did not participate in the first visit. During the first cycle, 181 students aged 7-15 y.o. were enrolled. During the second cycle, 161 learners returned and 139 new All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; learners aged 7-17 y.o. were enrolled to accommodate additional requests for testing by the schools. At each of the testing cycles, each participant underwent: 1. Brief medical history and a COVID-19 symptom screening 2. Anterior nasal swab for SARS-CoV-2 and co-circulating respiratory pathogens 3. Optional non-fasting phlebotomy for serological and other immunologic markers of SARS-CoV-2 infection, and at cycle 2, non-fasting lipid screening. We partnered with four schools that reflected the diverse population of Orange County ensuring adequate representation of low-income, minority, and special-needs learner-participants (Table) . Inclusion criteria for the student participants were age (7-17 y.o.), current enrollment at one of the schools participating in the study, and fluency and literacy in English or Spanish. The criteria for adult school staff participants were age (equal or greater than 18 years), current employment at one of the participating schools, and fluency and literacy in English or Spanish. The study was approved by the institutional review boards at the Children's Hospital of Orange County (CHOC) and the University of California Irvine (UCI). Informed assent from the children and informed consent from parents or legally authorized guardians, or from the adult participants, were obtained remotely or in person. We organized virtual meetings with school staff and teachers to explain the study and answer questions at each school, and town-hall-type meetings with potential students and their parents or authorized legal representatives. Meetings were held in both English and Spanish. We positioned our testing setup out of the way of normal school operations. Anterior nasal swabs were obtained from each participant. The BIOFIRE Respiratory 2.1 Panel was used to identify SARS-CoV-2 in addition to 21 other respiratory pathogens by RT-qPCR done in the CHOC Clinical Laboratory. Per state mandates, positive SARS-CoV-2 findings were reported by the laboratory to the Orange County Health Care Agency (OCHCA) for subsequent confidential action and tracing by county health authorities. Whole blood samples were obtained in 317 learners into CPT tubes. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation. Presence of nucleocapsid protein (N)-specific IgG was determined with the Abbott Architect immunoassay. IgG and IgM antibody titers against N and receptor binding domain from spike protein (RBD) were measured using standard ELISA. 6 Specific SARS-CoV-2 neutralizing antibodies were measured using focus reduction neutralizing assays. 6 IFN-γ producing Tcells following stimulation with overlapping peptide pools were determined using mAB ELISpot plates and PBMCs from 17 learners positive for SARS-CoV-2 and 17 age-and sex-matched samples. 7 Circulating immune and inflammatory mediators (e.g., TNF-a, IL-6) were measured using the Human 45-Plex kit from R&D. 7 Immunophenotyping to identify innate and adaptive cells was done using flow cytometry. 7 Non-fasting lipid screening (highly recommended for children and adolescents but underutilized) 8 was offered to our participants as an added benefit to the risk of phlebotomy, and measured using enzymatic reflectance spectrophotometery. OCHCA collaborators routinely collect COVID-19 case rates across the county. To better understand the specific regional impact of COVID-19 at each of the four schools, we collated the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. countywide COVID-19 case data according to the zip codes of the learners at each of the schools during the two testing cycles ( Figure 1 ). We modified the original System for Observing Play and Leisure Activity in Youth (SOPLAY) and the System for Observing Play and Recreation in Communities (SOPARC) 9,10 to quantify the fidelity of face covering and physical distancing (≥ 6 ft.) mitigation in schools. 11 SOCOM used momentary time sampling techniques in which systematic and periodic scans of individuals and contextual factors within pre-determined target areas in physical activity (e.g., recess, physical education classes) and school environments (e.g., classroom, communal dining) were made. Trained observers visited each of the 4 schools 3-5 times and quantified mitigation in classrooms, recess, communal dining, and physical education (PE) classes. All serology measurements and symptom ratings were treated as binary (yes/no) across the two visits. CBC and lipid measures were each classified as normal or abnormal based on ageappropriate criteria. In the case of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), the abnormal cases were further distinguished as low or high. Site comparisons of proportions, as well as cross-tabulations of two factors, were performed with Chi-Square analysis utilizing the Mantel-Haenszel correction. A a-level of 0.05 was used as the criterion for statistical significance. For learners found to be infected, age-and sex-matched uninfected peers were selected as controls for analysis. Immunological datasets were first tested for normality. To compare differences in various immune cell subsets between infected and age-and sex-matched uninfected participants, we used one-way ANOVA and Holm Sidak's multiple comparisons tests. Group comparisons were tested using an unpaired t-test (Mann-Whitney U-test). For focus All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254035 doi: medRxiv preprint reduction neutralization assay, the half maximum inhibitory concentration (IC50) was calculated by non-linear regression analysis using normalized counted foci; 100% of infectivity was obtained normalizing the number of foci counted in the wells derived from the cells infected with SARS-CoV-2 virus in the absence of plasma. Pearson correlation analyses were done by log transforming antibody end-point titers or neutralization titers. No positive nasal RT-qPCR tests were identified in the first cycle of testing. During the second cycle, a total of 17 SARS-CoV-2 RT-qPCR positive results were observed among learners (5.31%, mean age 9.9±2.1 y.o.). As shown in Figure 1 , school A had the highest number of SARS-CoV-2 infected learners (p<0.01). In the aggregate, there was no statistically significant difference in SARS-CoV-2 positive rates among remote or onsite learners (p=.1468). In school B (the same geographic location as school A), we found 2 of 45 onsite learners (4.4%) and 4 of 89 remote learners (4.5%) had positive SARS-CoV-2 PCR. School D had 1(1.25%) of onsite learners vs. none of remote learners had positive SARS-CoV-2 RT-qPCR. The low number of onsite participants in School A (n=2) prevented us from directly comparing SARS-CoV-2 positivity between remote and onsite instruction at each school site. Among the 99 staff and teachers tested, 6 (6.1%) were SARS-CoV-2 positive, and all positive tests were identified on the second cycle visit. When normalized to data obtained from OCHCA zip code-based case rates, school A showed the highest ratio of learner-to-local SARS-CoV-2 positivity. We found that a significant number of learners in schools A and B had either low HDL or high LDL. In addition, 26% of learners with low HDL (p<0.0001) were also found to be SARS-CoV-2 positive. There was no evidence of either influenza or RSV infection. Rhinovirus/enterovirus All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 4B ). School A had the highest number of learners who reported symptoms associated with COVID-19 at 30% (p=0.0452). Rates for Schools B, C, and D were 14.9%, 7.7%, and 18.6%, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254035 doi: medRxiv preprint preschools and K-12 schools in Australia. All studies concluded that within-school secondary transmission of SARS-CoV-2 was limited. We also found that infection rates reflected those of the community and neither remote learning nor highly mitigated onsite school attendance could eliminate SARS-CoV-2 infection. Comparisons and conclusions among our study and others done to date must be made with caution. For example, Zimmerman and Falk relied on public health agency contact tracing data. SARS-CoV-2 testing in learners or teachers would most likely result from a family reporting either symptoms or a known exposure to a primary care provider or testing center. Positive results would subsequently be linked to a particular school. Some infected individuals who were asymptomatic or symptom-deniers may not have been identified. Although we directly tested school personnel and learners at the school sites, a weakness of our study was that we relied on the participants' willingness to consent, which could have introduced selection bias. We showed that under certain conditions, schools could host onsite learning with relatively low SARS-CoV-2 infection rates. The private school in our study (school D) remained open with a majority of onsite learners from July through December 2020, with few SARS-CoV-2 cases and low IgG positivity despite a 14-fold increase in regional case rates. School D Laws et al. 16 recently confirmed the widespread observation that SARS-CoV-2 infected children tend to report fewer symptoms than adults. Despite this, we found that school A had All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254035 doi: medRxiv preprint both the highest percentage of learners with symptoms and highest percentage of SARS-CoV-2 positivity. In addition, there was a significant relationship between SARS-CoV-2 positivity and presence of symptoms. These data support the use of limited symptom screening as a mechanism to enhance healthy school reopening. We found that 26% of infected learners had significantly low circulating levels of HDL. Factors relating to obesity and physical activity are known to affect COVID-19 disease's severity in adults and children. [17] [18] [19] Overweight and obesity are associated with these lipid abnormalities, all of which tend to occur with greater incidence in low-income school aged children. 20, 21 Levels of HDL seem to be particularly sensitive to physical activity. 22, 23 The mechanisms responsible for the significant association we found between low HDL levels and SARS-CoV-2 are at present unknown, but may be related to the association of overweight/obesity and chronic inflammation in children. 24 Increases in physical inactivity and weight in children have accompanied school closures over the past year. 25, 26 Widespread implementation of pediatric COVID-19 vaccination 27 is many months away, and it is likely that adherence to COVID-19 mitigation procedures, including physical distancing and face covering, will need to continue for the near future. Previous studies cited above all highlighted the need to achieve high fidelity of COVID-19 mitigation procedures if viral transmission were to be limited. The in-classroom SOCOM data that we collected also revealed high fidelity at all four schools, including School C, which served predominantly children with special needs, presenting additional challenges to COVID-19 mitigation. The successful implementation of mitigation procedures both in onsite settings and in the instruction to remote learners might have played a role in the complete absence of influenza virus that we observed. 28, 29 In contrast, rhinovirus (which has the highest detection rate on school room desks All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254035 doi: medRxiv preprint among any respiratory viruses 30 ) was observed in all schools. Implementation of quantifiable non-intrusive instruments like SOCOM along with testing of several respiratory viruses could help schools implement actionable strategies to limit SARS-CoV-2 transmission. Our immunological analyses revealed patterns that can explain the mild symptomatology that accompanies SARS-CoV-2 infection in most children. Frequencies of circulating total and classical monocytes, expression levels of monocyte activation markers, and concentration of key inflammatory markers were surprisingly lower in the infected compared with uninfected children. This is in contrast to the monocytosis and heightened systemic inflammation observed in adult patients. 31, 32 The reduced systemic inflammatory profile, however, did not indicate a weaker immune response to SARS-CoV-2. Infected children generated robust and broad humoral and cellular immune responses, and had detectable frequency of SARS-CoV-2 specific IFN-γ secreting CD4 + T following exposure to SARS-CoV-2 antigens. The infected children also had increased expression of PD1 on both CD4 and CD8 T cells and higher frequency of proliferating CD4 T cells indicative of a recent history of activation. The frequency of circulating cytolytic NK cells, those that mediate antibody-dependent cell cytotoxicity, was lower in the infected children. This observation corroborates previous studies in both children and adults, and supports the speculation that NK cells may be recruited into the lung. 31, [33] [34] [35] Similarly, frequency of circulating CD4 T cells was reduced, suggestive of potential recruitment into the site of infection. These results support a maturation-dependent immune response to SARS-CoV-2 infection in children, one that specifically leads to milder disease and, possibly, to reduced transmission. The immune dysregulation that occurs in the rare but serious pediatric multisystem inflammatory syndrome in children (MIS-C) remains poorly understood. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 26, 2021. ; This study indicated that neither remote nor onsite learning strategies could eliminate SARS-CoV-2 infection in school-aged children. In the four schools, varied levels of successful mitigation were observed, and the degree of success was related to socioeconomic factors and regional levels of COVID-19 infection. The key challenge, of course, is balancing the damaging effects of school closures, which in the U.S. and throughout the world have adversely impacted low-income school-aged children and those with disabilities, 36, 37 with the consequences of SARS-CoV-2 transmission to other learners and school staff. In retrospect, a larger, arguably national, more comprehensive approach to prospectively collecting SARS-CoV-2 infection patterns in school-aged children, their school staff and faculty, and family contacts would likely have provided the necessary information to achieve the shared goal of the healthiest environment for the continued education and physical and mental health of children and adolescents throughout the country. Finally, we would be remiss in not highlighting the remarkable dedication of the faculty and staff at all four schools who worked tirelessly to continue to provide meaningful learning to their students, and willingly and enthusiastically permitted us to intrude into their sites during an anxiety-provoking and uncertain time. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. *Predominantly special needs learners; **We were unable to confirm onsite or remote status in 14 learners, none of whom were found to be SARS-CoV-2 positive.