An autopsy study of the spectrum of severe COVID-19 in children: From SARS to different phenotypes of MIS-C

Abstract

Background: COVID-19 in children is usually mild or asymptomatic, but severe and fatal paediatric cases have been described. The pathology of COVID-19 in children is not known; the proposed pathogenesis for severe cases includes immune-mediated mechanisms or the direct effect of SARS-CoV-2 on tissues. We describe the autopsy findings in five cases of paediatric COVID-19 and provide mechanistic insight into the mechanisms involved in the pathogenesis of the disease.

Methods: Children and adolescents who died with COVID-19 between March 18 and August 15, 2020 were autopsied with a minimally invasive method. Tissue samples from all vital organs were analysed by histology, electron microscopy (EM), reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC).

Findings: Five patients were included, one male and four female, aged 7 months to 15 years. Two patients had severe diseases before SARS-CoV-2 infection: adrenal carcinoma and Edwards syndrome. Three patients were previously healthy and had multisystem inflammatory syndrome in children (MIS-C) with distinct clinical presentations: myocarditis, colitis, and acute encephalopathy with status epilepticus. Autopsy findings varied amongst patients and included mild to severe COVID-19 pneumonia, pulmonary microthrombosis, cerebral oedema with reactive gliosis, myocarditis, intestinal inflammation, and haemophagocytosis. SARS-CoV-2 was detected in all patients in lungs, heart and kidneys by at least one method (RT-PCR, IHC or EM), and in endothelial cells from heart and brain in two patients with MIS-C (IHC). In addition, we show for the first time the presence of SARS-CoV-2 in the brain tissue of a child with MIS-C with acute encephalopathy, and in the intestinal tissue of a child with acute colitis. Interpretation: SARS-CoV-2 can infect several cell and tissue types in paediatric patients, and the target organ for the clinical manifestation varies amongst individuals. Two major patterns of severe COVID-19 were observed: a primarily pulmonary disease, with severe acute respiratory disease and diffuse alveolar damage, or a multisystem inflammatory syndrome with the involvement of several organs. The presence of SARS-CoV-2 in several organs, associated with cellular ultrastructural changes, reinforces the hypothesis that a direct effect of SARS-CoV-2 on tissues is involved in the pathogenesis of MIS-C.

Funding: Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Bill and Melinda Gates Foundation.

Keywords: Autopsy; Children; Covid-19; MIS-C, multisystem inflammatory syndrome in children; Minimally invasive autopsy; Pathology; Sars-cov-2.

Fig. 1

Pathology of COVID-19 in patient 1. (A) Exudative diffuse alveolar damage with alveolar haemorrhage and hyaline membranes; H&E. (B) Thrombus in a medium sized pulmonary artery (arrow); H&E. (C-D) Detection of SARS-CoV-2 N antigen by IHC in the bronchiolar epithelium (dots in red, C), and perivascular astrocyte (arrow in D). (E) Thrombus in a centrilobular vein (arrow), associated with hepatic ischaemic necrosis and sinusoidal congestion; H&E. (F) Detection of SARS-CoV-2 N antigen by IHC in hepatocytes, in a granular cytoplasmic pattern (arrows). (G) Low magnification electron micrograph of the alveolar wall showing two typical ultrastructural features of SARS-CoV-2 lung infection: congestion of a red blood cell (rc) within a capillary lumen and desquamation of epithelium (arrow) into the alveolar lumen (al). The inset shows viral particles (arrowheads) inside a type 1 pneumocyte, and also in the alveolar lumen. Bar: 2 µm; Inset bar: 500 nm. (H) Low magnification electron micrograph of the myocardium showing several longitudinally cut myofibrils in which the regular striation pattern of the sarcomeres is visible. Some sarcomeres are ruptured (arrows). The asterisks highlight focal areas with more advanced lesions, characterized by massive fragmentation and loss of myofibrils. Also note a capillary with an injured endothelial cell in two points (arrowheads), resulting in direct contact of the myocardial cells with the vascular lumen, filled with red blood cells (rc) and fibrin (f); The inset shows a virus particle (black arrow) in a focal area of ruptured myofilaments at the Z line level (Z). Bar: 2 µm; Inset bar: 200 nm.

Fig. 2

Pathology of fatal COVID-19 in patient 2. (A) Extensive and bilateral COVID-19-related pneumonia on the chest tomography, with ground-glass opacities, consolidation and air-bronchograms. (B) Typical COVID-19 pneumonia, with type 2 pneumocyte atypia and exudative diffuse alveolar damage; H&E. (C-D) Detection of SARS-CoV-2 N antigen by IHC in the bronchiolar epithelium (arrows in C), and endothelial coronary artery (D, arrow and inset). (E) Giant and multinucleated hepatocytes (arrows), containing bile pigment, and with cytoplasmic detection of SARS-CoV-2 N antigen by IHC (inset). (F) Reactive bone marrow with megakaryocytic emperipolesis, haemophagocytosis by macrophages and positive SARS-CoV-2 N antigen by IHC in mononuclear cells (insets, from top to bottom respectively). (G) Electron micrograph of type 2 pneumocytes in a area of pneumocyte hyperplasia (n, nucleus). Numerous microvilli (arrowhead) are seen on the apical surface of the cells. The alveolar space (al) is filled with red blood cells (rc), clusters of fibrin (f) and cell debris (arrow). Bar: 2 µm. (H) Electron micrograph of a capillary vessel in the alveolar septum. The endothelial cell is ruptured (arrowheads), and the capillary lumen is filled with a fine, dense, granular material (asterisk) and fibrin filaments (arrow). Bar: 500 nm. (I) High magnification electron micrograph showing parts of two cardiomyocytes with viral particles (arrows); mf: myofibrils. Bar: 200 nm.

Fig. 3

Pathology of fatal MIS-C related to COVID-19 in patient 3. (A) Inflammatory infiltration on epicardium, surrounding coronary arteries, and myocardium; H&E. (B-H) Detection of SARS-CoV-2 N antigen by IHC; (B) heart: cardiomyocytes and cardiac endothelium (arrows and inset); (C) lungs: bronchiolar epithelium and pulmonary megakaryocytes (inset); (D) brain: endothelial cells (arrows and inset); (E) spleen: mononuclear cells in a “red dot pattern”; (F) skin: sweat glands and subcutaneous nerves (inset); (G) kidneys: epithelial tubular cells in the centre; (H) liver: main biliary ducts. (I) Electron micrograph shows a capillary inside an alveolar septum. A mononuclear inflammatory cell (nu, nucleus) and red blood cell (rc) are seen in the capillary lumen (cl). The arrowheads point to areas where endothelial cells have been denuded. Note the absence of pneumocytes in the alveolar surface (arrow); dotted lines correspond to Fig. 3J. Bar: 1 µm; (J) The image corresponds to a higher magnification of the area surrounded by dotted lines in Fig. 3I. Ultrastructural examination reveals the presence of viral particles inside the endothelial cell (arrows) and in the septal interstitium (arrowheads); al: alveolar lumen; cl: capillary lumen. Bar: 200 nm.

Fig. 4

Pathology of fatal MIS-C related to COVID-19 in patient 4. (A) Chest CT with ground-glass opacities and consolidations, mainly in posterior areas of the left lower lobe. (B) Focal exudative diffuse alveolar damage; H&E. (C-D) Acute colitis (C, H&E) with detection of SARS-CoV-2 N antigen by IHC in the intestinal glands (D). (E) Surgical specimen of coecal appendix showing inflammatory infiltrate in the lamina propria and expansion of Peyer's patch. In red, immunolabelling of SARS-CoV-2 N antigen in the epithelial cells and in some macrophages in the germinative centres (inset). (F) High magnification electron micrograph of a fragment of the intestinal wall showing part of a smooth muscle cell (nu, nucleus) with the typical bundles of cytoplasmic myofilaments (asterisks). Note the presence of several coronavirus particles within the cell (some of them are indicated by arrows). Bar: 200 nm. (G) Ultrastructural aspect of a high endothelial venule in the intestinal wall. Confirming the findings of light microscopy, the image shows a lymphocyte (ly) passing between plump endothelial cells (asterisks). The lumen is filled by red blood cells (rc) and fibrin (arrow). Bar: 2 µm. (H) Contraction band necrosis in the myocardium, with positive detection of C4d by immunohistochemistry (inset). (I) Electron micrograph of myocardium presenting the ultrastructural feature of necrosis with areas devoid of myofibrils (asterisks) in infected cardiomyocytes. An abnormal vessel can be seen, presenting endothelial rupture (arrowheads) and large membrane-bound vacuoles that fill the lumen (lu). The inset shows two viral particles (arrows) near a degenerating myofibril. Bar: 1 µm; Inset bar: 200 nm. (J) Microthrombi in glomerular capillaries and detection of SARS-CoV-2 N antigen by IHC in epithelial ductular cells. (K) Fibrinoid thrombus in a cerebral arteriole (arrow); H&E. (L) Intense lymphoid hypoplasia in the spleen and red pulp haemorrhage; H&E.

Fig. 5

Pathology of fatal MIS-C related to COVID-19 in patient 5 (lung and heart). (A) Chest tomography showing sparse and small areas of ground-glass infiltrate (arrow). (B) Pulmonary thrombus in a medium sized artery; H&E. The inset shows an alveolar cell (cytoplasm in red) with positive expression of SARS-CoV-2 N antigen by IHC. (C) Electron micrograph showing a congested capillary within an alveolar septum. Note the ultrastructural feature of the disruption of the blood–air barrier (arrow) allowing the extravasation of red blood cells (rc) into the alveolar lumen (al). Inset: Several viral particles on the alveolar surface. Bar: 2 µm; Inset Bar: 200 nm. (D) High magnification electron micrograph of an immunolabelled section from the lung (antibody: SARS-CoV-2 anti-N protein, visualized with 10-nm protein A-gold). Several gold particles mark a virus (arrow) in the cytoplasm of a pneumocyte type I (pn); notice the presence of cellular debris in the alveolar lumen (al). Bar: 200 nm. (E) The echocardiography parasternal short axis view shows hyperechogenic left coronary artery, suggesting coronaritis. (F) Cardiac endothelial cell expression of SARS-CoV-2 N antigen by IHC (blue square and inset). (G) High magnification electron micrograph of the myocardium showing a viral particle (arrow) within a membrane-bound vacuole (arrowhead) adjacent to a sarcomeric Z line (Z); mf: ruptured myofilaments. Bar: 200 nm. (H) High magnification electron micrograph of an immunolabelled section from the heart (antibody: SARS-CoV-2 anti-N protein). A virus particle (arrow) labelled with colloidal gold can be seen within the cytoplasm of a cardiomyocyte; spike-like projections (thin arrow) are visible; the arrowheads indicate membranes invaginations of the intercalated disc.

Fig. 6

Pathology of fatal MIS-C related to COVID-19 in patient 5 (brain). (A) Brain with reactive microglia, and perivascular and parenchymal oedema. Inset: GFAP immunodetection showing vacuolar neuropil. (B) Detection of SARS-CoV-2 N antigen by IHC in intracerebral perivascular astrocytes (arrow). (C) High magnification electron micrograph showing a brain capillary whose lumen is filled with fibrin (f). Viral particles are found inside the endothelial cell (arrows) as well as in perivascular astrocytes (arrowheads). Note that the basal lamina (bl) organization is missing where the endothelial cell is ruptured (curved arrows). Bar: 200 nm. (D) High magnification electron micrograph of an immunolabelled section from the brain (antibody: SARS-CoV-2 anti-S2 protein, visualized with 10-nm protein A-gold). Gold particles are highlighted with thin arrows in two virions localized within an endothelial cell (en) of a brain capillary vessel; arrowheads indicate the plasma membrane of the cell facing the capillary lumen (cl). Bar: 200 nm. (E) Electron micrograph showing a cell body of an oligodendrocyte surrounded by myelinated axons (asterisks). Note an extensive disorganization of the cytoplasm with vesiculation, which is typical of coronavirus infection. Some vesicles (arrows) contain virus-like particles. Inset: High magnification of an intracellular vesicle showing its membrane boundary (black arrowhead) and viral particles consistent in size and shape with the Coronaviridae family (white arrows point to nucleocapsids). Bar: 200 nm; Inset bar: 100 nm. (F) Electron micrograph of an axon surrounded by a myelin (my) sheath in the brain. Virus-like particles (arrows) are found inside a vesicle in the axoplasm (axe). Inset: higher magnification of a virus, showing electron dense dots (white arrow) resembling nucleocapsids, and spikes (white arrowhead) projecting from the surface of the viral envelope; the black arrowhead highlights the membrane of the vesicle. Bar: 200 nm; Inset bar: 100 nm. We show for the first time the presence of SARS-CoV-2 in the brain tissue of a child with MIS-C with acute encephalopathy.

Fig. 7

Summary of pathological findings in five paediatric patients who died with COVID-19.