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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
Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, SpainInstituto de Investigación Sanitaria del Principado de Asturias, Oviedo, SpainCentro de Investigación Biomédica En Red–Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, SpainCentro de Investigación Biomédica En Red–Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, SpainInstituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain
Department of Pediatrics, Hospital Universitario Central de Asturias, Oviedo, SpainInstituto de Investigación Sanitaria del Principado de Asturias, Oviedo, SpainDepartment of Pediatrics, University of Oviedo, Oviedo, SpainMaternal and Child Health and Development Research Network, Institute of Health Carlos III, Madrid, Spain
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.
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.
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
Respiratory system: Lung
Severe lung congestion, with petechiae on the external surface; no thrombi in the pulmonary artery or main branches
Enlarged 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
Edematous and congested vessels and hyaline thrombi in microvessels; no deposits of complement C4d in the microvasculature noted
No 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 pyelonephritis
Ischemic changes, podocyte vacuolation, and accumulation of plasma in Bowman space
Nonisometric vacuolar degeneration and edematous epithelial cells; focal interstitial hemorrhage
Erythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material, fibrin thrombus, and shrinkage of capillary loops in glomeruli
Edematous cecum mucosa. A 9 × 2.5 cm lymphadenopathy found next to the cecum, which on cut sections revealed extensive areas of hemorrhage
Numerous 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 congestion
Focal 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 system
No significant changes
Partial 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)
No significant changes
Nondilated heart showing a 4 × 1 cm thrombus in the right ventricle, encircling a papillary muscle; patent coronary arteries
Significant 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
No significant changes
No significant changes
Macular rash mainly in the dorsum, with occasional petechiae
Vessels: Perivascular inflammatory cells, intraluminal thrombi; significant deposits of complement C4d in the microvasculature Epidermis: no significant alterations
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).
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.
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.
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.
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).
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.
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.
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.
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.
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,
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.
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.
C.L.-M. is funded by the Ministerio de Universidades, Spain (Grant FPU18/02965 ), which had no role in the study design, data analysis, manuscript preparation, or the decision to submit the manuscript. The authors declare no conflicts of interest.
Portions of this study were presented at the European Society of Pediatric and Neonatal Intensive Care, June 15-18, 2021 (virtual).