key: cord-0731548-6bli4z4m authors: Breuer, Adin; Raphael, Allon; Stern, Hagay; Odeh, Ma'aran; Fiszlinski, Judith; Algur, Nurit; Magen, Sophie; Megged, Orli; Schlesinger, Yechiel; Barak‐Corren, Yuval; Heiman, Eyal title: SARS‐CoV‐2 antibodies started to decline just four months after COVID‐19 infection in a paediatric population date: 2021-07-23 journal: Acta Paediatr DOI: 10.1111/apa.16031 sha: 5eaf5de9d8b4c67ff357e498b50132a86e675866 doc_id: 731548 cord_uid: 6bli4z4m AIM: We evaluated the prevalence of paediatric severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections using antibody testing and characterised antibody titres by time from exposure. METHODS: This was a single‐centre, prospective, cross‐sectional cohort study. Patients under 18 years old were eligible to participate if they attended the paediatric emergency department at the tertiary Shaare Zedek Medical Center, Jerusalem, Israel, from 18 October 2020 to 12 January 2021 and required blood tests or intravenous access. SARS‐CoV‐2 seropositivity and antibody levels were tested by a dual‐assay model. RESULTS: The study comprised 1138 patients (56% male) with a mean age of 4.4 years (interquartile range 1.3–11.3). Anti‐SARS‐CoV‐2 antibodies were found in 10% of the patients. Seropositivity increased with age and 41% of seropositive patients had no known exposure. Children under 6 years of age had higher initial antibody levels than older children, followed by a steeper decline. The seropositivity rate did not vary during the study, despite schools re‐opening. The findings suggest that children's immunity may start falling 4 months after the initial infection. CONCLUSION: Immunity started falling after just 4 months, and re‐opening schools did not affect infection rates. These findings could aid decisions about vaccinating paediatric populations and school closures. A prospective, cross-sectional epidemiological survey was conducted between 18 October 2020 and 12 January 2021 in the paediatric emergency department (PED) of the Shaare Zedek Medical Center, a public, tertiary medical centre in Jerusalem, Israel. The area covered by the hospital had a high prevalence at the time of the study, with more than 4500 cumulative cases per one hundred thousand population. All patients under 18 years of age who presented to the PED during the study period were eligible to participate if they required blood tests or intravenous access for any clinical reason. Parents and, or, legal guardians gave oral consent for subjects to participate in the study. The study was approved by the hospital's Institutional Research Ethics Board (reference number 0387-20-SZMC). Children under the age of three months were later excluded from the analysis, as antibodies detected in their blood may have been maternal antibodies that transferred though the placenta in-utero. Patients with blood samples that were unsuitable for laboratory testing, for technical reasons, were also excluded from the final analysis ( Figure 1 ). The accompanying parent or guardian filled out a comprehensive questionnaire in Hebrew or Arabic for each patient. This covered the patient's demographic background, their past medical history and specific COVID-19 information, such as their exposure to any individuals with the virus, previous PCR testing for SARS-CoV-2 and symptoms ( Figure S1 ). All patients with positive serology results were contacted by telephone and given their results. Details from the questionnaire were confirmed during the same phone call, in particular the timing and source of the confirmed, or suspected, SARS-CoV-2 infection. All the blood samples were collected in a clot-activator test tube. Initial testing was conducted using the Abbott Architect SARS-CoV-2 IgG assay (Abbott Laboratories, Illinois, USA) according to the manufacturer's instructions. The presence or absence of IgG antibodies against the SARS-CoV-2 nucleocapsid protein was calculated by the system as an index of the chemiluminescent relative light unit in the reaction to the calibrator relative light unit. An index of 1.4 or greater was considered a positive result. All samples with positive or borderline (index 0.5-1.4) results were retested using the DiaSorin Liaison SARS-CoV-2 S1/S2 IgG assay (DiaSorin Spa, Piedmont, Italy), which tests antibodies against the spike protein of the virus. This assay calculates antibody concentrations, expressed as arbitrary • This prospective, cross-sectional study found antisevere acute respiratory syndrome coronavirus 2 antibodies in 10% of the children sampled, and 41% of the seropositive patients had no known exposure to the virus. • Antibody levels began to fall four months after the initial infection and the seropositivity rate did not rise when schools re-opened. units per millilitre (AU/mL): an AU/mL of 15 or greater is considered a positive result and 12-15 AU/mL is borderline. We used a dual-assay model in this study, which means that a patient was only considered positive for SARS-CoV-2 IgG antibodies if both assay tests produced a positive result. A positive Abbott assay, with a subsequent negative or borderline DiaSorin assay, or vice-versa, were considered inconclusive. All other results were considered negative. The manufacturers established that the tests' sensitivity and specificity approached 100% after 14 days from infection. Accordingly, seropositivity rates were not adjusted for imperfect test performance. The statistical analyses were conducted using SPSS Statistics for There were 6264 patients treated in the PED during the 90-day study period. Blood tests and, or, intravenous access were obtained for 2235 patients (35%) and the parent or guardian of 1249 patients F I G U R E 1 Study flow diagram. History of a positive PCR indicates a prior positive SARS-CoV-2 PCR on a nasopharyngeal swab test at the time of enrolment. Serology was only considered positive if antibodies were detected by both the Abbott Architect (anti-n protein IgG antibodies) and DiaSorin Liaison (antispike IgG antibodies) assays (56%) provided consent to participate in the study. We excluded 111 patients (9%) and these comprised 94 infants under the age of 3 months and 17 with blood samples that were rejected by the laboratory. This means that there were 1138 patients (56% male) in the final study group (Figure 1) , with a median age of 4.4 years (IQR 1.3-11.3). The cohort was similar to the general PED population during the study period (median age 4.6 [IQR 1.3-11.6] and 55.9% male, p = 0.49 and p = 0.78 respectively). Additional socio-demographic information is presented in Table 1 . Overall, 114 (10%) patients had anti-SARS-CoV-2 antibodies in both assays and another 16 (1.6%) had inconclusive test results. A positive serology test result was associated with older age (median 7.6 vs. 3.8 years, p < 0.001), self-identifying as a Muslim (21% vs. 14%, p = 0.05) or an ultra-orthodox Jew (69% vs. 50%, p < 0.001) and a larger family size (mean 7.08 ± 3.2 vs. 6.09 ± 2.5, p = 0.004) ( Table 1 ). The rate of seropositivity increased by age, from 13/219 (5.9%) samples from infants under one year of age to 68/490 (13.8%) from adolescents aged 12 to 18 years ( Figure S2 ). When these variables were adjusted using a logistic regression model, only four variables were significantly associated with the virus: older age, larger family size and Muslim and ultra-orthodox Jewish religions. Other patient characteristics, such as gender, housing density and whether the child's school was open or closed during lockdowns were insignificant (Table S1 ). A history of PCR-confirmed SARS-CoV-2 infection was reported in 71 (6%) patients and only 50 (70%) of those had anti-SARS-CoV-2 antibodies. The 26 (37%) of those who were asymptomatic at the time of their PCR-confirmed infection were more likely to be seronegative, but this was not statistically significant (p = 0.34) ( Table 2) . Of note, the five patients with multisystem inflammatory syndrome in children had higher antibody levels than all the other seropositive patients (Abbot index 5.3 vs. 3.9, p = 0.053 and DiaSorin 108.9 vs. 84.8 AU/mL, p = 0.096), but this was not statistically significant. The time interval between the primary infection, established by the initial positive SARS-CoV-2 PCR test, and the serology testing significantly affected the sensitivity of the anti-SARS-CoV-2 antibody assay. There was a peak sensitivity rate of 47/54 (87%) when it was taken 22-119 days after the infection, (p < 0.001, Table 2 ). The same temporal effect was also noted in the levels of antibodies (Figure 2A , Table S2 ). The antibody levels were also affected by the age of the patient. Children under the age of 6 years had higher antibody levels than older children in the first 60 days after infection, followed by a steeper decline in antibody levels ( Figure 2B and C, Table S2 ). Attending school was not associated with an increased risk of infection. The weekly rate of seropositive cases did not vary over the study period, even though the re-opening of schools coincided with the start of the study ( Figure S3 ). In addition, only 9/71 (12%) of the patients with a history of a SARS-CoV-2 infection reported that they contracted the infection at school. Finally, 152/816 (19%) respondents said they still attended school during lockdown. The rate of SARS-CoV-2 seropositive cases in this cohort was not different to the children who stayed at home during lock-down: 3% versus 7.5% for day care or preschool children aged 0-6 years old (p = 0.19), 14% versus 13% for elementary school children aged 6 to 12 (p = 0.82) and 15% versus 14% for high school children aged 12 to 18 (p = 0.83). Overall, there were no differences in the seroprevalence rate between children who went to school, stayed at home during lockdown or whose parents did not answer this question (13%, 13%, and 12%, respectively, p = 0.809) ( Table 1 ). We found an overall 10% seropositivity rate among children in this large, prospective cohort of paediatric patients presenting to the PED in a region with a high prevalence of COVID-19 cases. Seroprevalence rates varied from 7.7% for children under the age of 6 years to 16.4% in older children. Of the seropositive patients, 41% had no known past exposure to COVID-19, indicating a significant rate of undetected, asymptomatic infection and carriage in children. 14 This was similar to the rate in a large meta-analysis. 15 The paediatric seroprevalence rate found in this study was higher than previously reported in Israel 16 and similar to other areas worldwide that have been heavily affected by This was in contrast to studies from earlier in the pandemic, which demonstrated significantly lower seroprevalence in children. 18 The high seropositivity rate we observed among ultra-orthodox Jews and Muslims contributed to the total seroprevalence rate found in this study. Previous studies have described how religious affiliation and communal ceremonies may have influenced SARS-CoV-2 transmission. 19 We also found that a larger family size was associated with seropositivity and this may provide another explanation for the high seroprevalence rate in this group. Less than a third (29%) of the patients with a PCR-confirmed virus infection did not have conclusive positive serology test results. The nucleocapsid-based antibody test was chosen as the primary screening antibody test for this study, based on preliminary studies that found it showed superior sensitivity to spike-based antibody kits. 20 This was in accordance with the Israeli Ministry of Health guidelines for SARS-CoV-2 serology testing at the time of the study. Overall, our dual-assay model only had a sensitivity of 70% for TA B L E 1 Demographics and characteristics of the patients, with a comparison of seropositive and seronegative patients (7) Other/no answer 116 (12) 11 (15) 104 (12) Muslim 160 (14) 24 (21) had a dual-assay sensitivity of 87%, which was slightly lower than the 90%-97% described in adults. 12 However, it is important to note that a lack of detectable antibodies does not necessarily correlate to a lack of neutralising ability and specific, acquired SARS-CoV-2 immunity may still exist. 21, 22 Of note, no correlation was found between symptomatic and asymptomatic COVID-19 cases and the level of anti-SARS-CoV-2 antibodies. 23 We found that the temporal trend of anti-SARS-CoV-2 antibodies over time in children was different to the trends in adults. Children experienced a delayed initial rise in antibody levels, followed by a rapid decline and even the disappearance of detectable antibodies 4 months after the initial infection. This was particularly pronounced in children under the age of 6 years ( Figure 2 ). These findings are in contrast to the current evidence in adults, 24 Our study began immediately after a month-long nationwide The strengths of this study included its large, prospective, population-based cohort in a region with high rates of SARS-CoV-2 infections and a diverse paediatric population. This enabled us to identify asymptomatic carriers and to characterise the serological response over time. We also noted that previous studies found lower rates of unknown, asymptomatic infections in children. 32 A novel coronavirus from patients with pneumonia in China Epidemiology of COVID-19 among children in China Systematic review of COVID-19 in children shows milder cases and a better prognosis than adults Symptomatic and asymptomatic viral shedding in pediatric patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): under the surface Analysis of Covid-19 in children COVID-19 WORKING GROUP. COVID-19 disease severity risk factors for pediatric patients in Italy Pediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): clinical presentation, infectivity, and immune responses Overcoming COVID-19 Investigators; CDC COVID-19 Response Team. Multisystem inflammatory syndrome in U.S. children and adolescents Interim guidelines for collecting, handling, testing clinical specimens from persons for coronavirus disease 2019 (COVID-19) Challenges in laboratory diagnosis of the novel coronavirus SARS-CoV-2 Kinetics of viral clearance and antibody production across age groups in children with severe acute respiratory syndrome coronavirus 2 infection Evaluation of SARS-CoV-2 IgG antibody response in PCR positive patients: comparison of nine tests in relation to clinical data Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody responses in children with multisystem inflammatory syndrome in children (MIS-C) and mild and severe coronavirus disease Seroprevalence of anti-SARS-CoV-2 IgG antibodies in children with household exposure to adults with COVID-19: preliminary findings The proportion of SARS-CoV-2 infections that are asymptomatic: a systematic review The national sero-epidemiological survey of Covid-19 Assessment of SARS-CoV-2 infection by reverse transcription-PCR and serology in the Paris area: a cross-sectional study Prevalence of SARS-CoV-2 infection in children and their parents in Southwest Germany COVID-19 and religion: risks and opportunities Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019 Weak correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity The comprehensive clinic, laboratory, and instrumental evaluation of children with COVID-19: a 6-months prospective study Robust neutralizing antibodies to SARS-CoV-2 infection persist for months Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum Preliminary evidence on long COVID in children Kriemler S Long-term symptoms after SARS-CoV-2 infection in school children: population-based cohort with 6-months follow-up Bidirectional legal socialization and the boundaries of law: the case of enclave communities' compliance with COVID-19 regulations Household SARS-CoV-2 transmission and children: a network prospective study SARS-CoV-2 transmission among children and staff in daycare centres during a nationwide lockdown in France: a cross-sectional, multicentre, seroprevalence study Schools closures during the COVID-19 pandemic: a catastrophic global situation Alberta childhood COVID-19 cohort (AB3C) aim 3: longitudinal sero-epidemiology study first interim report SARS-CoV-2 antibodies started to decline just four months after COVID-19 infection in a paediatric population