ABSTRACT
Background: Most children exposed to SARS-CoV-2 virus present with mild symptoms, but some may experience severe illnesses such as Multisystem inflammatory syndrome (MISC) or respiratory failure. Currently there are no established biomarkers to predict progression to severe disease. Although specific serum cytokines have been found to be higher in adults with severe COVID-19, their role as predictors of severe disease in children remains unclear. Further, the role of salivary cytokines in COVID-19 associated inflammation is unknown. Our objective was to compare cytokine levels in saliva of children with and without severe disease due to SARS-CoV-2 infection. Methods: This prospective observational study, conducted at two tertiary children's hospitals, was supported by a grant from the National Institute of Health RADx Program. Children ≤ 18 years of age with symptoms due to SARS-CoV-2 infection (positive PCR test, serology or immunological link) were enrolled after informed consent. Severe cases were defined as the occurrence of any of the following within 30 days of testing: diagnosis of MISC or Kawasaki disease, requirement for >2L oxygen, inotropes, mechanical ventilation or ECMO, or death. A saliva sample was obtained through passive drool using MicroSAL kits (Oasis Diagnostics) and a viral transport medium (VTM-C19, Biomed). Abundance levels of six cytokines (TNFR1, IL13, IL-15, CCL7, CXCL10 and CXCL9) were measured in triplicate using microfluidic immunoassays (Ella, Protein Simple). Mean concentrations for each sample were determined against a standard curve and corrected for dilution. Levels of the six cytokines were compared between those with severe or nonsevere SARS-CoV-2 symptoms using a non-parametric t-test. The relationship between salivary levels of individual cytokines was assessed among children with severe and non-severe SARS-CoV2 using a Pearson correlation analysis Results: A total of 150 children were enrolled from 03/29/2021 to 05/30/2021 (mean age of 7.1 years ± 5.7 years, 54.6% females). Of the total, 38 (25.3%) children met criteria for severe SARS-CoV-2 infection. CXCL10 displayed significantly (fold change>2, p < 0.05) elevated levels in the saliva of children with severe SARS-CoV-2 (Figure 1). The relationship between levels of CXCL9 (MIG) and CXCL10 showed greater levels association (R2 = 0.93) in children with severe SARS-CoV-2 than in peers with non-severe SARS-CoV-2 (R2 = 0.65;Figure 2). Conclusion: In this preliminary analysis of salivary cytokines among children with SARS-CoV-2 infection, we found CXCL10 displayed differential expression with severe symptoms. These findings may provide critical information about the pathophysiology of severe SARS-CoV-2. Confirmation in further studies is necessary. Saliva concentrations of CXCL10 in children with severe SARSCoV-2 symptoms. The whisker box plots display salivary concentrations of CXCL10 in children with severe (green) and non-severe (red) SARS-CoV-2 infection as measured with next generation enzyme linked immunosorbent assay. Levels of CXCL10 (p < 0.01;fold change = 3.04) were elevated in children with severe SARS-CoV-2 symptoms on Wilcoxon testing. .
ABSTRACT
Background: The majority of children with exposure to SARSCoV-2 virus have mild disease. However severe diseases such as Multisystem inflammatory syndrome (MISC) and pneumonia do occur in children. Currently, there are no established biomarkers that can predict progression to severe disease in children exposed to the virus. MicroRNAs (miRNAs) are non-coding RNAs that can be found in saliva and are thought to play a role in the regulation of inflammation following an infection. Our objective was to compare the miRNA profile in saliva of children with or without severe disease due to SARS-CoV-2 infection. Methods: This prospective observational study was supported by the National Institutes of Health (NIH) RADx Program. Children ≤ 18 years of age presenting to two tertiary care children's hospitals with symptoms of SARS-CoV-2 infection (confirmed by PCR test, serology or epidemiological link) were enrolled between 03/29/2021 and 04/30/2021. Severe infection was defined as any of the following within 30 days of testing: MISC or Kawasaki disease diagnosis, requirement for oxygen > 2L, inotropes, mechanical ventilation or ECMO, or the occurrence of death. Informed consent and a saliva swab were obtained at the time of SARS-CoV-2 diagnosis (DNA Genotek, Ottowa Canada), and RNA was extracted (Qiagen, Germantown, MD). Small RNA species (<50 base pairs) were interrogated via shotgun sequencing (HiSeq 2500, Illumina, San Diego, CA) and miRNAs were quantified through alignment to the human genome (GRCh38). RNA features with sparse counts (<10 in 90% of samples) were filtered, and the data was quantile normalized and mean-center scaled. Salivary miRNA levels were compared between those with severe and non-severe SARS-CoV-2 infection using Wilcoxon tests with Benjamini Hochberg multiple testing corrections. In addition, a logistic regression analysis was used to identify miRNA pairs that could best discriminate severe cases based on a Monte Carlo 100-fold cross-validated area under receiver operating characteristic curve (AUROC). Results: Samples from 33 children were analyzed. Median age was 3 (3, 10) years and 54.5% were males. Of the total, 29 were RT PCR positive, 4 had a positive serology and 6 children had severe infection. Seven miRNAs displayed significant differences (Fold change >2, FDR adjusted p < 0.1) among children with severe SARS-CoV-2 infection (Table). All seven miRNAs were up-regulated in severe SARS-CoV-2 cases. A logistic regression using a single ratio of miR-296-5p/miR-378j yielded 1.0 AUROC for differentiating children with severe infection (Figure). Conclusion: In this interim analysis of salivary miRNA in childhood SARS-CoV-2 infection, we found a differential expression of 7 salivary miRNAs in children with severe infection. Ongoing work will seek to validate these findings and explore the role of miRNA in predicting severe SARS-CoV-2 infection in children. Receiver operating characteristic curve and box plot displaying the complete differentiation of severe and non- severe SARSCoV-2 cases using a ratio of miR-296-5p and miR-378j levels in saliva.
ABSTRACT
Background: The COVID-19 pandemic has been shown to have a compounding effect on families across various social and healthcare needs. However, the impact of social determinants of health (SDOH) on COVID-19 disease severity in children is unknown. Our objectives were to describe the SDOH in children with SARS-CoV-2 infection and determine their association with severity of the infection Methods: This prospective observational study was supported by the National Institutes of Health RADx program and conducted in the emergency department (ED) of two large children's hospitals. Children ≤ 18 years of age with symptoms due to SARS-CoV-2 infection (positive RT PCR test, serology or epidemiological link) were enrolled between 03/29/2021 and 05/30/2021. Data collected from electronic medical records included demographics, clinical features, treatment, disposition, and outcomes. Severe cases were defined as the following within 30 days of test positivity: diagnosis of Multisystem inflammatory syndrome in children or Kawasaki disease, requirement for oxygen > 2L, inotropes, mechanical ventilation, extracorporeal membrane oxygenation (ECMO), or death. Following informed consent, caregivers were surveyed via an electronic device on previously validated PhenX questions. Aligned with the Healthy People 2020 SDOH framework, caregivers reported on economic stability, education, social and community context, health and health care, and neighborhood and built environment. Stata was used to analyze descriptive statistics, and unadjusted comparisons between groups were assessed using two sample t-tests for continuous variables and Fisher's exact test for categorical variables due to small sizes. Results: A total of 107 children (mean age 6.9 (±5.9) years, 44.9% males), with SARS-CoV-2 infection were enrolled, and 85 caregivers (79.4%) completed the survey (71.4% Black). In this sample, 97% of children were RT PCR positive, 3% had an epidemiological link, and 23 (27.1%) were categorized as severe. Almost half of caregivers (47.6%) reported employment or income loss due to COVID-19. The three most common SDOH needs identified were that of childcare (22.0%), housing instability (22.0%), and food insecurity (21.7%). Children with severe COVID-19 were significantly more likely to have a caregiver who was single, including never married, separated/divorced, and widowed (82.6% vs. 52.5%;Table 1). Although not statistically significant, children with severe COVID-19 tended to have higher levels of social needs including housing instability, poor caregiver mental health, and lower levels of social support compared to children with nonsevere infection (Table 2). Conclusion: Our preliminary data on SDOH suggest that among children with SARS-CoV-2 infection, housing instability, food insecurity and childcare needs are particularly prevalent. Children with severe SARS-CoV-2 infection were more likely to have single caregivers. Family structure may influence severe COVID-19 in children and programming and supports for single parent households should be considered. Larger studies in the ED setting will help confirm these findings and to direct resources to address these social needs.