Data sharing: The data obtained as part of this study are available from the corresponding author upon reasonable request.
As schools plan for re-opening, understanding the potential role children play in the coronavirus infectious disease 2019 (COVID-19) pandemic and the factors that drive severe illness in children is critical.
Study design: Children ages 0-22 years with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presenting to urgent care clinics or being hospitalized for confirmed/suspected SARS-CoV-2 infection or multisystem inflammatory syndrome in children (MIS-C) at Massachusetts General Hospital (MGH) were offered enrollment in the MGH Pediatric COVID-19 Biorepository. Enrolled children provided nasopharyngeal, oropharyngeal, and/or blood specimens. SARS-CoV-2 viral load, ACE2 RNA levels, and serology for SARS-CoV-2 were quantified.
A total of 192 children (mean age 10.2 +/- 7 years) were enrolled. Forty-nine children (26%) were diagnosed with acute SARS-CoV-2 infection; an additional 18 children (9%) met criteria for MIS-C. Only 25 (51%) of children with acute SARS-CoV-2 infection presented with fever; symptoms of SARS-CoV-2 infection, if present, were non-specific. Nasopharyngeal viral load was highest in children in the first 2 days of symptoms, significantly higher than hospitalized adults with severe disease (P = .002). Age did not impact viral load, but younger children had lower ACE2 expression (P=0.004). IgM and IgG to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein were increased in severe MIS-C (P<0.001), with dysregulated humoral responses observed.
This study reveals that children may be a potential source of contagion in the SARS-CoV-2 pandemic in spite of milder disease or lack of symptoms, and immune dysregulation is implicated in severe post-infectious MIS-C.
Supported by the National Heart, Lung, and Blood Institute (5K08HL143183 to L.Y.), the Cystic Fibrosis Foundation (YONKER18Q0 to L.Y.), the National Institute of Child Health and Human Development (K08 HD094638 [to A.N.] and R01HD100022 [to A.E.]), Mark and Lisa Schwartz (to J.L.), the National Institute of Diabetes and Digestive and Kidney Diseases (DK039773, DK072381 [to J.B.] and DK104344 [to A.F.]), the National Institute of Allergy and Infectious Disease (K24AI141036 to I.B.), the Centers for Disease Control and Prevention (U01CK000490 to E.R.), and the Department of Pediatrics and the Department of Obstetrics/Gynecology at Massachusetts General Hospital (to L.Y. and A.E.). The authors declare no conflicts of interest.
As schools plan for re-opening, debates around the role children play in the COVID-19 pandemic persist. Concerns have been raised as to whether allowing children to congregate in the classroom will fuel the spread of the pandemic. On an individual level, families are worried how SARS-CoV-2 infection could affect their children and family. Particular concern is elevated for families belonging to low socio-economic classes, where the prevalence of SARS-CoV-2 infection is higher, and where multi-generational co-habitation is the norm, increasing the risk of transmitting the infection to vulnerable grandparents and older adults(1).
The manner in which children contribute to the spread of SARS-CoV-2 is unclear. Children are less likely to become seriously ill from SARS-CoV-2(2); however, asymptomatic carriers, including children, can spread infection and carry virus into their household.3 Children infected with SARS-CoV-2 tend to have milder symptoms with significantly lower mortality than is seen in adult infection(4). It has been hypothesized that children have reduced incidence of COVID-19 because ACE2 expression in the nasopharynx increases with age(5); however ACE2 expression has not been studied in the upper airways of children infected with SARS-CoV-2. Understanding infectious burden and potential for transmissibility within the pediatric population is critical for developing both short- and long-term responses, including public health policies, to the current pandemic.
) several weeks after possible SARS-CoV-2 infection or exposure, with severe cardiac complications, including hypotension, shock, and acute heart failure(8). Understanding post-infectious immune responses in pediatric SARS-CoV-2 infection(9), especially MIS-C, is critical for designing treatment and prevention strategies.
Here, we describe the pediatric impact of COVID-19, specifically focusing on viral burden, susceptibility to disease, and immune responses.
Table 1online: Description of adult samples included for comparative purposes in virology and antibody assays.
Once informed consent, and if appropriate, assent, were verbally obtained by the patients or parent/guardian in accordance with IRB guidelines, nasopharyngeal and oropharyngeal swabs were obtained and placed in phosphate buffered saline. The samples were immediately aliquoted and stored at -80oC. Venipuncture was performed; plasma and serum were collected and immediately stored at -80oC.
Study definitions: SARS-CoV-2 (+) individuals had a nasopharyngeal swab sample positive for SARS-CoV-2 by clinical quantitative polymerase chain reaction (qPCR) testing. SARS-CoV-2 (-) individuals had negative nasopharyngeal qPCR testing. MIS-C was defined per the Centers for Disease Control and Prevention (CDC) criteria: fever >38oC for >24 hours, laboratory evidence of inflammation, at least two organs involved, and no alternative plausible diagnoses and a positive SARS-CoV-2 test by RT-PCR, serology or antigen test, or exposure to an individual. with COVID-19 within 4 weeks prior to the onset of symptoms.
Data collection: Medical records were reviewed to assess demographic and clinical factors, including age, medical history, presenting features and clinical testing, household contacts, and other possible risk factors at presentation. Data were stored in a REDcap database.
) SARS-CoV-2 pseudoviral reference standards (SeraCare, Milford, MA, USA) were used as positive controls for each run. SARS-CoV-2 viral loads below 40 RNA copies/mL were categorized as undetectable and set at 1.0 log10 RNA copies/mL.
ACE2 expression in the upper airway
cDNA was transcribed from RNA extracted from nasopharyngeal and oropharyngeal swabs using TRIzol-LS reagent (Thermo Fisher) and then purified by isopropanol extraction. qPCR standards were created using a hACE2 plasmid and MEGAscript T7 transcription kit (Thermo Fisher), purified with the RNeasy MinElute spin column kit (Qiagen, the Netherlands), and quantified by nanodrop. ACE2 and IPO8 Gene expression was assessed by qPCR using iTaq Universal SYBR Green mix (Bio-Rad Laboratories, Hercules, CA, USA) with ACE2 primers (FWD AAACATACTGTGACCCCGCAT, REV CCAAGCCTCAGCATATTGAACA) as previously used(15) and IPO8 primers (Bio-Rad Laboratories, Hercules, CA, USA). ACE2 and IPO8 RNA were used to generate standard curve to quantitate copy numbers per sample and ACE2 expression relative to IPO8 was calculated as previous(16).
IgG and IgM titers measured by ELISA: SARS2-CoV2-RBD (in-house, HEK293 cells provided by Aaron Schmidt, Ragon Institute) and SARS2-CoV2-NC (Aalto Bio Reagents Ltd., Ireland) specific plasma antibodies were quantified by ELISA. The average plus 5x or 3x standard deviation of included negative adult plasma controls were defined as negative cutoff for IgG or IgM, respectively. SARS-CoV-2-RBD specific monoclonal human IgG1 or IgM antibody (clone: CR3022) was added in a two-fold dilution curve starting at 2.5ug/ml to each plate and specific IgG or IgM concentrations were calculated.
IgG1 and IgM titers measured by Luminex: SARS2-CoV2-RBD, SARS2-CoV2-NC, SARS2-CoV2-S (provided by Eric Fischer, Dana Farber), and RBD domains of the coronavirus strains NL-63, HKU1, 229E and OC43 (in-house, provided by Aaron Schmidt) specific antibody isotypes were analyzed by Luminex multiplexing(17). Antigens were carboxy-coupled to Luminex microspheres (Luminex Corp, TX, USA) and incubated with polyclonal plasma samples containing IgM and IgG1. Isotypes were probed with fluorophore-tagged secondary antibody and relative concentrations analyzed by flow cytometry.
Statistical Analyses: Mann-Whitney U-test assessed statistical significance between two outcomes; Kruskal-Wallis test assessed comparisons of continuous variables. For all categorical comparisons, the Fisher exact test was used. The Spearman rank correlation tested relationships between two variables. Prism software was used to analyze and graph data.
Table 2online: Patient characteristics of children not infected with SARS-CoV-2, children with SARS-CoV-2 infection, and children diagnosed with MIS-C. Age, sex, socioeconomic status, race and ethnicity, past medical history, vaccination status, COVID-19 household exposures, and daycare/school levels are presented.
Children ages 0-22 years participated in this study, with children ages 11-16 years most highly represented in the SARS-CoV-2 (+) cohort (16, 34%) and children ages 1-4 years most highly represented in the MIS-C cohort (7, 39%). Only 2 (4%) of the SARS-CoV-2 (+) cohort were <1 year of age, although this was previously reported as a higher risk age-group(18). Sex was equally distributed between children with and without acute SARS-CoV-2 infection, although there was a male predominance in the MIS-C group (14, 78%). Latino/Hispanic children were most highly represented in both the SARS-CoV-2 (-) and SARS-CoV-2 (+) groups. Twenty-five (51%) of children infected acutely with SARS-CoV-2 came from low-income communities, as compared with 1 (2%) from high-income communities (Fisher exact test, P<0.001).
All children enrolled in the Pediatric COVID-19 biorepository had the option of providing nasopharyngeal, oropharyngeal, and blood specimens for research. Eighty-three children provided a nasopharyngeal specimen, 105 provided an oropharyngeal specimen, and 100 provided a blood sample.
Table 3online: Presenting symptoms of enrolled patients. Comparisons between symptoms reported in acute SARS-CoV-2 infection and non-SARS-CoV-2 illnesses, and SARS-CoV-2 (+) and MIS-C are compared by Fisher exact test.
None of the SARS-CoV-2 (+) or MIS-C children had heart disease, hypertension, or diabetes, which are risk factors for infection in the adult population(19); however, 13 (27%) of SARS-CoV-2 (+) children were obese, as compared with 2 (11%) of the MIS-C cohort. Asthma was a common feature in SARS-CoV-2 (-) patients (29, 19%) whereas SARS-CoV-2 (+) and MIS-C patient groups displayed typical population rates of asthma(20) (6, 12% and 2,11%, respectively). Other pulmonary diseases, immune/autoimmune diseases, and neuro/neurodevelopmental diseases were assessed and were not seen in high levels in any cohort.
Nine (18%) SARS-CoV-2 infected children and 10 (56%) children with MIS-C did not have a known infected household contact. Of the children acutely infected with SARS-CoV-2, 26 (53%) attended grade school. None of the 7 preschool/kindergarteners tested positive for SARS-CoV-2 or developed MIS-C.
SARS-CoV-2 viral load
SARS-CoV-2 viral binding sites
SARS-CoV-2 antibody response
We present findings from the largest pediatric COVID-19 biospecimen repository to date, describing viral load, ACE2 expression, and antibody responses as they relate to children with acute SARS-CoV-2 infection and MIS-C. We found that children can carry high levels of virus in their upper airways, particularly early in an acute SARS-CoV-2 infection, yet they display relatively mild or no symptoms. However, there was no age correlation with viral load, indicating that infants through young adults can carry equally high levels of virus. However, SARS-CoV-2 infected children have higher levels of ACE2 expression, which may pre-dispose certain children to infection. Children with MIS-C do not have high levels of viral load on nasopharyngeal or oropharyngeal viral testing, nor do they have detectable viremia, however, they do have hyperactive antibody responses.
From an infection-control perspective, it is critical to identify infected children early for quarantine purposes. One third of school-aged children presenting with illness during the height of the local pandemic were found to have SARS-CoV-2 infection. However, children display relatively mild or no symptoms. Although ACE2 expression was increased in SARS-CoV-2 infected children, ACE2 expression did not impact viral load within the upper airway. Similarly, although younger children had reduced ACE2 expression, age also did not impact viral load. This suggests that regardless of disease susceptibility, children can carry high viral loads, which is a key consideration when opening up schools and daycare centers.
Moreover, when present, the symptoms of SARS-CoV-2 are non-specific and overlap considerably with non-COVID-related illnesses. Identifying SARS-CoV-2 infection in children will become even more challenging during pollen allergy season and influenza season this fall. Further, some children carry very high viral loads even before symptoms develop. On the other hand, children with severe symptoms, e.g. MIS-C, do not have high levels of viral load on nasopharyngeal or oropharyngeal viral testing, nor do they have detectable viremia. Overall, the lack of correlations between viral load and symptoms will complicate infection-control strategies for children.
Children with severe MIS-C have elevated SARS-CoV-2 IgM and IgG levels; IgG levels are not only elevated in SARS-CoV-2 but also in the other coronaviruses, influenza, and RSV. The broad, nonspecific antibody response points to T and B cell over-reactivity, or to auto-antibodies that may be driving an inflammatory process causing MIS-C(22). Elevated ferritin levels in MIS-C, which positively correlate with SARS-CoV-2 serology, also suggest an interplay with macrophage activation. Further, SARS-CoV-2 IgG are positively correlated with NT-proBNP, a marker of heart failure, which could indicate mechanism of disease or provide a correlation with disease severity.
Limiting the spread of SARS-CoV-2 infections in children is of particular concern as schools plan for re-opening. Our findings suggest that it would be ineffective to rely on symptoms or temperature monitoring to identify SARS-CoV-2 infection. Instead, infection control measures should minimize the possibility of viral spread, with focus on strategies including social distancing precautions, mask use, and/or remote learning. Moreover, schools could screen all students for SARS-CoV-2 infection and establish routine screening protocols. Without infection control measures such as these, there is significant risk that the pandemic will persist, and children could carry the virus into the home, exposing adults who are at higher risk of developing severe disease. This risk is particularly high in lower income communities where household size may be larger with multi-generational co-habitation and greater housing density. These recommendations contradict previous reports from the initial phase of the pandemic, which found children to be less likely to be the index case for viral transmission within a household(23). However, in our cohort, nearly 20% of acute SARS-CoV-2 infections and over half of the MIS-C cases did not have a known household exposure to SARS-CoV-2. Although transmissibility was not assessed in this study, children with high viral loads and non-specific symptoms including rhinorrhea and cough can likely transmit SARS-CoV-2 as easily as other viral infections spread by respiratory particles. If schools were to re-open fully without necessary precautions, it is likely that children will play a larger role in this pandemic.
Our initial findings show that although a low expression of ACE2 in younger children (<10 years of age) likely corresponds to reduced infection rates, children of all ages, once infected, can carry high SARS-CoV-2 viral loads. Symptom monitoring is an ineffective strategy for identifying infected children. Children can develop severe illness during the post-infectious stage with a hyperinflammatory antibody response. Potential transmission of SARS-CoV-2 between children and families should be considered when designing strategies to mitigate the COVID-19 pandemic.
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