BACKGROUND. Weeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date. METHODS. Here, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient’s clinical response. RESULTS. We showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments. CONCLUSION. These mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19–related disease in children.

The COVID-19 pandemic continues to infect millions of people worldwide. In order to curb its spread and reduce morbidity and mortality, it is essential to develop sensitive and quantitative methods that identify infected individuals and enable accurate population-wide screening of both past and present infection. Here we show that Single Molecule Array assays detect seroconversion in COVID-19 patients as soon as one day after symptom onset using less than a microliter of blood. This multiplexed assay format allows us to quantitate IgG, IgM and IgA immunoglobulins against four SARS-CoV-2 targets, thereby interrogating 12 antibody isotype-viral protein interactions to give a high resolution profile of the immune response. Using a cohort of samples collected prior to the outbreak as well as samples collected during the pandemic, we demonstrate a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter. This assay should become the gold standard for COVID19 serological profiling and will be a valuable tool for answering important questions about the heterogeneity of clinical presentation seen in the ongoing pandemic.

Background: Seroepidemiology is an important tool to characterize the epidemiology and immunobiology of SARS-CoV-2 but many immunoassays have not been externally validated raising questions about reliability of study findings. To ensure meaningful data, particularly in a low seroprevalence population, assays need to be rigorously characterized with high specificity. Methods: We evaluated two commercial (Roche Diagnostics and Epitope Diagnostics IgM/IgG) and two non-commercial (Simoa and Ragon/MGH IgG) immunoassays against 68 confirmed positive and 232 pre-pandemic negative controls. Sensitivity was stratified by time from symptom onset. The Simoa multiplex assay applied three pre-defined algorithm models to determine sample result. Results: The Roche and Ragon/MGH IgG assays each registered 1/232 false positive, the primary Simoa model registered 2/232 false positives, and the Epitope registered 2/230 and 3/230 false positives for the IgG and IgM assays respectively. Sensitivity >21 days post symptom-onset was 100% for all assays except Epitope IgM, but lower and/or with greater variability between assays for samples collected 9-14 days (67-100%) and 15-21 days (69-100%) post-symptom onset. The Simoa and Epitope IgG assays demonstrated excellent sensitivity earlier in the disease course. The Roche and Ragon/MGH assays were less sensitive during early disease, particularly among immunosuppressed individuals. Conclusions: The Epitope IgG demonstrated good sensitivity and specificity. The Roche and Ragon/MGH IgG assays registered rare false positives with lower early sensitivity. The Simoa assay primary model had excellent sensitivity and few false positives. Summary: SARS-CoV-2 immunoassays can be valuable tools for informing the global response, but many currently available assays have not been independently validated. We conducted a performance assessment of four assays including the Roche Diagnostics and Epitope Diagnostics immunoassays.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide. PCR tests are currently the gold standard for diagnosis of the current coronavirus disease (COVID-19) and serology tests are used to detect seroconversion in infected patients. However, there is a lack of quantitative and ultra-sensitive viral antigen tests for COVID-19. Here we show that Single Molecule Array (Simoa) assays can quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of COVID-19 patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we show correlation between production of antibodies and clearance of viral antigens from serial plasma samples from COVID-19 patients. Furthermore, we demonstrate the presence of viral antigens in blood correlates with disease severity in hospitalized COVID-19 patients. These data suggest that SARS-CoV-2 viral antigens in the blood could be a marker for severe COVID-19 cases. One Sentence Summary: SARS-CoV-2 antigens S1, spike, and nucleocapsid and anti-SARS-Cov-2 antibodies were measured in longitudinal plasma samples from COVID-19 patients using Single Molecule Array (Simoa) assays.