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SARS-CoV-2 Spike Protein in Intestinal Cells of a Patient with Coronavirus Disease 2019 Multisystem Inflammatory Syndrome

  • Juan Mayordomo-Colunga
    Correspondence
    Reprint requests: Juan Mayordomo-Colunga, MD, PhD, Sección de Cuidados Intensivos Pediátricos, Área de Gestión Clínica de Pediatría, Hospital Universitario Central de Asturias, Avda, de Roma s/n 33011, Oviedo, Asturias, Spain
    Affiliations
    Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, Spain

    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain

    Centro de Investigación Biomédica En Red–Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
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  • Ana Vivanco-Allende
    Affiliations
    Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, Spain

    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain
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  • Inés López-Alonso
    Affiliations
    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain

    Centro de Investigación Biomédica En Red–Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain

    Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain
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  • Cecilia López-Martínez
    Affiliations
    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain

    Centro de Investigación Biomédica En Red–Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
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  • Iván Fernández-Vega
    Affiliations
    Department of Pathology, Hospital Universitario Central de Asturias, Oviedo, Spain
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  • Helena Gil-Peña
    Affiliations
    Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, Spain

    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain
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  • Corsino Rey
    Affiliations
    Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, Spain

    Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain

    Department of Pediatrics, University of Oviedo, Oviedo, Spain

    Maternal and Child Health and Development Research Network, Institute of Health Carlos III, Madrid, Spain
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Published:November 26, 2021DOI:https://doi.org/10.1016/j.jpeds.2021.11.058
      A previously healthy 12-year-old boy had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related multisystem inflammatory syndrome (MIS-C) that was rapidly fatal. Autopsy revealed the presence of a large intracardiac thrombus. SARS-CoV-2 spike protein was detected in intestinal cells, supporting the hypothesis that viral presence in the gut may be related to the immunologic response of MIS-C.

      Keywords

      Abbreviations:

      MIS-C (Multisystem inflammatory syndrome associated with SARS-CoV-2 infection), PICU (Pediatric intensive care unit), SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)
      Multisystem inflammatory syndrome associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (MIS-C) was described during the first SARS-CoV-2 pandemic wave in Europe.
      Royal College of Paediatrics and Child Health (RCPCH)
      Guidance: Paediatric multisystem inflammatory syndrome temporally associated with COVID-19 (PIMS): guidance for clinicians.
      Days or weeks after SARS-CoV-2 infection or exposure, some children (predominantly school-aged) develop this life-threatening disease.
      • Bautista-Rodriguez C.
      • Sanchez-de-Toledo J.
      • Clark B.C.
      • Herberg J.
      • Bajolle F.
      • Randanne P.C.
      • et al.
      Multisystem inflammatory syndrome in children: an international survey.
      Patients present with fever, gastrointestinal symptoms, high levels of inflammatory markers, and cardiogenic shock.
      • Bautista-Rodriguez C.
      • Sanchez-de-Toledo J.
      • Clark B.C.
      • Herberg J.
      • Bajolle F.
      • Randanne P.C.
      • et al.
      Multisystem inflammatory syndrome in children: an international survey.
      • Feldstein L.R.
      • Tenforde M.W.
      • Friedman K.G.
      • Newhams M.
      • Rose E.B.
      • Dapul H.
      • et al.
      Characteristics and outcomes of US children and adolescents with multisystem inflammatory syndrome in children (MIS-C) compared with severe acute COVID-19.
      • Carter M.J.
      • Fish M.
      • Jennings A.
      • Doores K.J.
      • Wellman P.
      • Seow J.
      • et al.
      Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection.
      Although the pathophysiology remains unclear, MIS-C encompasses cytokine-mediated hyperinflammation.
      • Gruber C.N.
      • Patel R.S.
      • Trachtman R.
      • Lepow L.
      • Amanat F.
      • Krammer F.
      • et al.
      Mapping systemic inflammation and antibody responses in multisystem inflammatory syndrome in children (MIS-C).
      A requirement for intensive care is reported in 50%-80% of cases of MIS-C, and the fatality rate is 0-2%.
      • Bautista-Rodriguez C.
      • Sanchez-de-Toledo J.
      • Clark B.C.
      • Herberg J.
      • Bajolle F.
      • Randanne P.C.
      • et al.
      Multisystem inflammatory syndrome in children: an international survey.
      ,
      • Carter M.J.
      • Fish M.
      • Jennings A.
      • Doores K.J.
      • Wellman P.
      • Seow J.
      • et al.
      Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection.
      • Gruber C.N.
      • Patel R.S.
      • Trachtman R.
      • Lepow L.
      • Amanat F.
      • Krammer F.
      • et al.
      Mapping systemic inflammation and antibody responses in multisystem inflammatory syndrome in children (MIS-C).
      • Feldstein L.R.
      • Rose E.B.
      • Horwitz S.
      • Collins J.P.
      • Newhams M.M.
      • Son M.B.F.
      • et al.
      Multisystem inflammatory syndrome in US children and adolescents.
      • Valverde I.
      • Singh Y.
      • Sanchez-de-Toledo J.
      • Theocharis P.
      • Chikermane A.
      • Di Filippo S.
      • et al.
      Acute cardiovascular manifestations in 286 children with multisystem inflammatory syndrome associated with COVID-19 infection in Europe.
      • García-Salido A.
      • de Carlos Vicente J.C.
      • Belda Hofheinz S.
      • Balcells Ramírez J.
      • Slöcker Barrio M.
      • Leóz Gordillo I.
      • et al.
      Severe manifestations of SARS-CoV-2 in children and adolescents: from COVID-19 pneumonia to multisystem inflammatory syndrome: a multicentre study in pediatric intensive care units in Spain.
      • Whittaker E.
      • Bamford A.
      • Kenny J.
      • Kaforou M.
      • Jones C.E.
      • Shah P.
      • et al.
      Clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2.
      In the 3 reported pediatric autopsies, SARS-CoV-2 was found in several tissues, and in intestinal cells in 1 child.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      Here we present a case of overwhelming MIS-C in a previously healthy child, providing relevant information about autopsy findings and histologic, immunohistochemical, and immunofluorescent analysis for SARS-CoV-2.

      Case Presentation

      A 12-year-old boy with no significant past medical history presented with symptoms that began 3 days before admission consisting of malaise, fever, maculopapular rash, and diffuse abdominal pain, with diarrhea. Two family members had tested positive for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction on nasopharyngeal swab 6 weeks earlier. At presentation, the patient tested negative for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction on nasopharyngeal swab but positive for serum SARS-CoV-2 immunoglobulin G.
      Abdominal ultrasound showed inflammation of the terminal ileum and peritoneal free fluid inconsistent with appendicitis. Amoxicillin-clavulanate acid and intravenous fluids were started. Microscopic urinalysis showed no leukocytes or nitrates, although a sample sent for culture revealed the presence of 50 000 CFU/mL of Providencia stuartii. Blood cultures and subsequent tracheal aspirate cultures were negative.
      Several hours after admission, the patient developed fluid-refractory shock and was transferred to a pediatric intensive care unit (PICU). Initial tests revealed lymphopenia and increased levels of inflammatory markers (Table I; available at www.jpeds.com). Dopamine was begun, and the patient was intubated and subsequently treated with adrenaline and milrinone. Antibiotic coverage was changed to cefotaxime and clindamycin. MIS-C was considered a possible diagnosis, and he was given corticosteroids (methyl-prednisolone 50 mg/12 hours, ∼1.7 mg/kg/day). Echocardiography performed after intubation in the PICU showed a slight reduction in left ventricular ejection fraction (∼60%), with no other obvious abnormalities. Blood tests showed progressive increases in levels of inflammatory markers (Table I).
      The next day, the child developed a fever up to 42.1°C and was unresponsive to intravenous immunoglobulin and a 1-g methyl-prednisolone pulse, necessitating the addition of noradrenaline to maintain blood pressure. Low molecular weight heparin was started, and antibiotic therapy was changed to vancomycin and meropenem. Levels of serum creatinine, procalcitonin, D-dimer, lactate dehydrogenase, and cardiac markers showed abrupt elevations (Table I). The echocardiogram was repeated, demonstrating a left ventricular ejection fraction of ∼50%, without any other anomalies. At 8 hours after this second echocardiogram (45 hours after PICU admission), the child developed pulseless ventricular tachycardia and did not respond to cardiopulmonary resuscitation.
      At autopsy, macroscopically the heart was nondilated and had a 4 × 1 cm thrombus in the right ventricle, encircling a papillary muscle (Figure 1, A; available at www.jpeds.com). Other macroscopic findings are shown in Figure 1 and described in Table II. Disseminated intravascular coagulopathy was identified in most organs. Severe inflammation was found in the intestine and the heart, and hemophagocytosis was prevalent in the bone marrow (Figure 2; available at www.jpeds.com). Complement C4d deposits were found in microvessels of the intestine, skin, liver, and heart (Figure 3; available at www.jpeds.com), but not in other organs.
      Table IIMain macroscopic and histopathologic findings of systems/organs observed
      Macroscopic findingsMicroscopic findings
      Respiratory system: Lung
       Severe lung congestion, with petechiae on the external surface; no thrombi in the pulmonary artery or main branchesAlveoliEnlarged pneumocytes with large nuclei, type II pneumocyte hyperplasia, focal sloughing, intra-alveolar hemorrhage; no evidence of intra-alveolar neutrophil infiltration, amphophilic granular cytoplasm, or viral cytopathic-like changes; no hyaline membranes identified
      VesselsEdematous and congested vessels and hyaline thrombi in microvessels; no deposits of complement C4d in the microvasculature noted
      Cellular componentsNo presence of syncytial giant cells or infiltration of immune and inflammatory (lymphocytes and monocytes); mild increase in stromal cells
      Urinary system: Kidney
       Visible clots in calices; no signs consistent with pyelonephritisGlomerulusIschemic changes, podocyte vacuolation, and accumulation of plasma in Bowman space
      Renal tubulesNonisometric vacuolar degeneration and edematous epithelial cells; focal interstitial hemorrhage
      VesselsErythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material, fibrin thrombus, and shrinkage of capillary loops in glomeruli
      Gastrointestinal system
       Edematous cecum mucosa. A 9 × 2.5 cm lymphadenopathy found next to the cecum, which on cut sections revealed extensive areas of hemorrhageColonNumerous infiltrating plasma cells and lymphocytes with interstitial edema in the lamina propria; intense edema of submucosa with mild infiltration of immune cells; significant deposits of complement C4d in the microvasculature; arteriolar microthrombi
       Severe liver congestionLiverFocal macrovesicular steatosis, nuclear glycogen accumulation in hepatocytes, moderate zone 3 sinusoidal dilatation with extensive centrilobular necrosis (submassive hepatic necrosis)
       No other significant macroscopic findings in other parts of the gastrointestinal systemEsophagusNo significant changes
      StomachPartial epithelial degeneration, necrosis, and shedding of the gastric mucosa. Dilatation and congestion of small blood vessels; moderate edema of submucosa with mild infiltration of immune cells (as lymphocytes, monocytes, and plasma cells)
      PancreasNo significant changes
      Cardiovascular system
       Nondilated heart showing a 4 × 1 cm thrombus in the right ventricle, encircling a papillary muscle; patent coronary arteriesHeartSignificant interstitial edema and presence of inflammatory cells; foci of lymphocytic inflammation CD3+; presence of diffuse mobilization and infiltration by CD68+ macrophages in the myocardium; significant deposits of complement C4d in the microvasculature; no signs of vasculitis in coronary arteries; microscopy normal
      Reproductive system
       No significant changes
      Nervous system
       No significant changes
      Skin
       Macular rash mainly in the dorsum, with occasional petechiaeVessels: Perivascular inflammatory cells, intraluminal thrombi; significant deposits of complement C4d in the microvasculature

      Epidermis: no significant alterations
      Bone marrow
      Intense hemophagocytosis
      Skeletal muscle
      Focal myonecrosis
      To perform immunofluorescence, tissue slices were deparaffinated and antigens retrieved in citrate buffer 0.1 M (pH 6). Autofluorescence was diminished using Sudan black saturated solution for 30 minutes. Slides were then washed with phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 1% bovine serum albumin in PBS. Slides were then incubated overnight at 4°C with a rabbit monoclonal antibody against SARS-CoV-2 spike protein (Invitrogen) at 1:100 dilution. The next day, slides were incubated with the corresponding secondary fluorescent antibody (donkey IgG Alexa Fluor 594-conjugated anti-rabbit; Thermo Fisher Scientific) at room temperature for 45 minutes. Slides were coverslipped with SlowFade Diamond Mountant with DAPI (Invitrogen) for nuclear visualization. Immunofluorescence detected the presence of protein S (spike) in the intestine (Figure 4) and in a positive control sample (Figure 5; available at www.jpeds.com). Spike protein could not be identified in the heart, lungs, or enlarged pericecal lymph node (Figure 6; available at www.jpeds.com).
      Figure thumbnail gr1
      Figure 4Immunofluorescence of cecum cells. The first column shows cell nuclei using DAPI staining. In the second and third columns, SARS-CoV-2 S (spike) protein is shown in red, demonstrating a perinuclear pattern. Images were obtained by confocal microscopy (Leica SP8) at 400 × and 630 × and processed using ImageJ.

      Discussion

      The most relevant observation in the present case is the presence of spike protein in intestinal cells in a child with MIS-C. In reports of autopsies of children with MIS-C, intestinal cells were studied in only 1 subject, revealing the presence of SARS-CoV-2 components in the intestinal epithelium.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      Our case has some unique features, mainly the overwhelming evolution in <48 hours, refractory hyperpyrexia, and cardiac thrombus. The child met all the current criteria for MIS-C, but because many clinical features are common with bacterial septic shock, broad-spectrum antibiotics were used. The formation of a large cardiac thrombus is noteworthy, as this is an uncommon complication in MIS-C.
      • Bigdelian H.
      • Sedighi M.
      • Sabri M.R.
      • Dehghan B.
      • Mahdavi C.
      • Ahmadi A.
      • et al.
      Case report: Acute intracardiac thrombosis in children with coronavirus disease 2019 (COVID-19).
      Arrythmia has been one of the most frequently reported cardiovascular complications in MIS-C,
      • Valverde I.
      • Singh Y.
      • Sanchez-de-Toledo J.
      • Theocharis P.
      • Chikermane A.
      • Di Filippo S.
      • et al.
      Acute cardiovascular manifestations in 286 children with multisystem inflammatory syndrome associated with COVID-19 infection in Europe.
      and ventricular tachycardia has been reported as well.
      • Whittaker E.
      • Bamford A.
      • Kenny J.
      • Kaforou M.
      • Jones C.E.
      • Shah P.
      • et al.
      Clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2.
      Histologic examination revealed frequent extensive thrombosis and areas of hemorrhage in most organs, findings indistinguishable from other causes of disseminated intravascular coagulopathy. Duarte-Neto et al also reported thrombi in most organs of patients with MIS-C; inflammation was present mainly in the heart and the intestine, and inflammation was described in most organs in their case series.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      We cannot exclude the possibility that the short time from our patient's onset of symptoms to death could be related to limited sites of inflammation. However, given that immune activation may be primarily responsible for tissue damage, there could be a discrepancy between the presence of virus and tissue inflammation, as has been described in adults with fatal coronavirus disease 2019 (COVID-19).
      • Dorward D.A.
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      • Um I.H.
      • Elshani M.
      • Armstrong S.D.
      • Penrice-Randal R.
      • et al.
      Tissue specific immunopathology in fatal COVID-19.
      MIS-C has some clinical similarities with Kawasaki disease, although they appear to be distinct entities.
      • Vella L.A.
      • Rowley A.H.
      Current insights into the pathophysiology of multisystem inflammatory syndrome in children.
      Histologically, systemic vasculitis is the main feature in Kawasaki disease, affecting the coronary arteries in most patients. Furthermore, inflammation is present in most organs and tissues in fatal Kawasaki disease.
      • Amano S.
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      • Kubagawa H.
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      • Hamashima Y.
      General pathology of Kawasaki disease. On the morphological alterations corresponding to the clinical manifestations.
      In contrast, in our case, inflammation was confined to the heart and the gut, and vasculitis was not found. Moreover, infiltration of coronary arteries was not found in our case or in other reported fatal cases of MIS-C.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      ,
      • Fox S.E.
      • Lameira F.S.
      • Rinker E.B.
      • Vander Heide R.S.
      Cardiac endotheliitis and multisystem inflammatory syndrome after COVID-19.
      Neither diffuse alveolar damage nor hyaline membrane formation was present in our case or in other reported cases of MIS-C, except for some foci of exudative diffuse alveolar damage in 1 child.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      These findings are consistent with the lack of respiratory symptoms at presentation of our patient, as well as the relative paucity of respiratory involvement described in MIS-C.
      • Carter M.J.
      • Shankar-Hari M.
      • Tibby S.M.
      Paediatric inflammatory multisystem syndrome temporally-associated with SARS-CoV-2 infection: an overview.
      The lungs in our case and in previously reported cases showed congestion, edema, and foci of hemorrhage and thrombi.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      Conversely, the lungs in 2 patients with a pulmonary form of COVID-19 exhibited pneumonia and diffuse alveolar damage, which is a hallmark in adults with COVID-19.
      • Vella L.A.
      • Rowley A.H.
      Current insights into the pathophysiology of multisystem inflammatory syndrome in children.
      ,
      • Fox S.E.
      • Akmatbekov A.
      • Harbert J.L.
      • Li G.
      • Brown J.Q.
      • Vander Heide R.S.
      Pulmonary and cardiac pathology in African American patients with COVID-19: an autopsy series from New Orleans.
      In our patient, the heart showed myocarditis with significant lymphocyte and macrophage infiltrate. Autopsies of patients with MIS-C showed significant cardiac involvement, with areas of necrosis. In contrast, only myocardial interstitial edema was seen on autopsies of 2 patients with acute COVID-19 infection.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      Gruber et al observed a reduction of peripheral blood nonclassical monocytes and subsets of T lymphocytes and natural killer cells, compatible with extravasation to affected tissues,
      • Gruber C.N.
      • Patel R.S.
      • Trachtman R.
      • Lepow L.
      • Amanat F.
      • Krammer F.
      • et al.
      Mapping systemic inflammation and antibody responses in multisystem inflammatory syndrome in children (MIS-C).
      which is in accordance with the microscopic findings in our patient's heart.
      The presence of complement C4d in several tissues, mainly the intestine, heart, liver, and skin is consistent with an underlying immunologic process. Duarte-Neto et al also described positive C4d immunohistochemistry in the heart.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      The existence of hemophagocytosis in the bone marrow in our case, and in 2 of the 3 reported MIS-C cases in the spleen as well as in the liver in 1 case, also indicates significant immune activation.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      SARS-CoV-2 spike protein was identified in colonic cells of our patient. This finding is in accordance with a report describing that SARS-CoV-2 RNA remains in the gastrointestinal tract for weeks in children with MIS-C,
      • Yonker L.M.
      • Gilboa T.
      • Ogata A.F.
      • Senussi Y.
      • Lazarovits R.
      • Boribong B.P.
      • et al.
      Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
      the severe gut inflammation in our case and in 1 child in the series of Duarte-Neto et al
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      could be related to increased permeability of the gastrointestinal mucosal barrier. The breakdown of mucosal barrier function coincides with SARS-CoV-2 antigenemia, likely instigating and driving the aberrant immune activation defining MIS-C.
      • Yonker L.M.
      • Gilboa T.
      • Ogata A.F.
      • Senussi Y.
      • Lazarovits R.
      • Boribong B.P.
      • et al.
      Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
      Along with persistent antigen leakage from the gut,
      • Vella L.A.
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      • Kuri-Cervantes L.
      • et al.
      Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19.
      poor antigen clearance also could play a role in MIS-C pathogenesis.
      • Yonker L.M.
      • Gilboa T.
      • Ogata A.F.
      • Senussi Y.
      • Lazarovits R.
      • Boribong B.P.
      • et al.
      Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
      Carter et al reported impaired antigen presentation in this condition.
      • Carter M.J.
      • Fish M.
      • Jennings A.
      • Doores K.J.
      • Wellman P.
      • Seow J.
      • et al.
      Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection.
      Furthermore, the spike protein has been hypothesized to have superantigen-like properties.
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      • Rivas M.N.
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      • et al.
      HLA class I–associated expansion of TRBV11-2 T cells in multisystem inflammatory syndrome in children.
      Gastrointestinal symptoms are the most frequent manifestation of MIS-C along with fever.
      • Bautista-Rodriguez C.
      • Sanchez-de-Toledo J.
      • Clark B.C.
      • Herberg J.
      • Bajolle F.
      • Randanne P.C.
      • et al.
      Multisystem inflammatory syndrome in children: an international survey.
      ,
      • Carter M.J.
      • Shankar-Hari M.
      • Tibby S.M.
      Paediatric inflammatory multisystem syndrome temporally-associated with SARS-CoV-2 infection: an overview.
      ,
      • Yonker L.M.
      • Gilboa T.
      • Ogata A.F.
      • Senussi Y.
      • Lazarovits R.
      • Boribong B.P.
      • et al.
      Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
      The predominance of this clinical presentation, along with the finding of viral components in gut cells,
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      may suggest that the viral antigenic source is in the gut.
      • Yonker L.M.
      • Gilboa T.
      • Ogata A.F.
      • Senussi Y.
      • Lazarovits R.
      • Boribong B.P.
      • et al.
      Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
      Similarly, it has been described that the gut serves as a nidus for SARS-CoV-2 in adults.
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      In our child, SARS-CoV-2 protein S was not observed in the lungs or heart, and in the previous report, SARS-CoV-2 particles were found in the heart and lungs of all 3 children as well as in the liver, kidney, spleen, brain, and sweat glands.
      • Duarte-Neto A.N.
      • Caldini E.G.
      • Gomes-Gouvêa M.S.
      • Kanamura C.T.
      • de Almeida Monteiro R.A.
      • Ferranti J.F.
      • et al.
      An autopsy study of the spectrum of severe COVID-19 in children: from SARS to different phenotypes of MIS-C.
      Our demonstration of SARS-CoV-2 spike protein in intestinal cells by immunofluorescence supports the hypothesis that the gut is important in the pathogenesis of MIS-C.
      We thank the parents who provided consent to include their children in the medical literature. We also thank Drs Santiago Melón and Marta Elena Argüelles for their help in interpreting viral findings.

      Appendix

      Table IHematologic and biochemical markers obtained during admission
      MarkersHospital admission (−15 h)PICU admission (0 h)+10 h+18 h+28 h+40 h
      Hematologic measures
       Hemoglobin, g/dL12.211.111.410.310---
       Platelets, 103/μL133131233242143---
       Leukocytes/μL854010 36031 11017 1104290---
       Neutrophils/μL7450942028 70015 5003700---
       Lymphocytes/μL51044010101110470---
       D-dimer, ng/mL---28183045253811 35818 219
       Fibrinogen, mg/dL1224799840779532340
      Biochemical parameters
       C-reactive protein, mg/dL28.731.633.427.722.416.8
       Procalcitonin, ng/mL6.756.1823.183.8225>800
       IL-6, pg/mL515459486121---43
       Ferritin, ng/mL---871------------
       NTproBNP, pg/mL---14 39831 78835 965--->70 000
       Cardiac troponin T, ng/L---1613603704951059
       Sodium, mmol/L134136139142142143
       Potassium, mmol/L3.9---3.22.83.14.9
       Urea, mg/dL2342464971102
       Creatinine, mg/dL0.570.71.081.011.763.25
       Albumin, g/L---3031312624
       AST, U/L124876414319085220
       ALT, U/L138104921078441913
       LDH, U/L372------54939806180
       Lactate, mmol/L---2.79.32.42.83.5
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; IL-6, interleukin 6; LDH, lactate dehydrogenase; NTproBNP, N-terminal pro B-type natriuretic peptide.
      Figure thumbnail fx1
      Figure 1The most relevant macroscopic findings. A, Nondilated heart showing a 4 × 1 cm thrombus in the right ventricle (white arrow), encircling a papillary muscle. Coronary arteries were patent. B and C, Severely congested lungs, showing petechiae on the external surface. No thrombi were found in the pulmonary artery and main branches. D, Dorsum showing a rash. E, Congested liver. F, Edematous cecum mucosa. G, A 9 × 2.5 cm lymphadenopathy (asterisks) found next to the cecum, showing extensive areas of hemorrhage on cut sections. H, Kidneys showing multiple clots in calices (black arrows).
      Figure thumbnail fx2
      Figure 2The most relevant microscopic findings. Formalin-fixed, paraffin-embedded tissue blocks were processed and hematoxylin and eosin–stained after a standardized process in the hospital pathology laboratory. A, Organized cardiac thrombus, adhered to the ventricular wall (arrow). B, Intense pulmonary hemorrhage. C, Thrombi in pulmonary microvasculature (arrows). D, Perivascular inflammatory cells (arrow) in the skin (rash area on the dorsum). E, Centrilobular necrosis in the liver (30%-40% of the liver exhibited this pattern). F, Pericecal adenopathy showing intense necrosis secondary to thrombi (arrows). G, Transmural chronic inflammation in the cecum. H, Thrombi in glomerular microcirculation; the square shows a glomerulus in detail, with microthrombi (arrows). I, Hemophagocytosis (arrows) in bone marrow aspirate.
      Figure thumbnail fx3
      Figure 3Immunohistochemistry of several organs. Infiltration of the heart with A, CD3+ T lymphocytes and B, CD68+ macrophages. C, Complement C4d deposits in heart arterioles. D and E, Complement C4d deposits in the liver, mainly in zone 3 of the lobule. F, Complement C4d deposits in cecum subserous fat tissue arterioles.
      Figure thumbnail fx4
      Figure 5Immunofluorescence of pharyngeal cells inoculated with SARS-CoV-2. Images were obtained by confocal microscopy (Leica SP8) at 400 × and 630 × and processed using ImageJ. The first photograph shows cell nuclei using DAPI staining. In the second and third photos, SARS-CoV-2 S (spike) protein are shown in red, demonstrating a perinuclear pattern. The same protocol described in was used to perform immunofluorescence. In this case, pharyngeal cells inoculated with SARS-CoV-2 were smeared and fixed with acetone.
      Figure thumbnail fx5
      Figure 6Immunofluorescence of the child's other organs. The first column shows cells nuclei using DAPI staining. SARS-CoV-2 S (spike) protein was not observed, as shown in the second and third columns. Images were obtained by confocal microscopy (Leica SP8) at 400 × and 630 × and processed using ImageJ. The same protocol described in was used to perform immunofluorescence.

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