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      SARS-CoV-2 in cardiac tissue of a child with COVID-19-related multisystem inflammatory syndrome

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          Abstract

          We report the case of an 11-year-old child with multisystem inflammatory syndrome in children (MIS-C) related to COVID-19 who developed cardiac failure and died after 1 day of admission to hospital for treatment. An otherwise healthy female of African descent, the patient was admitted to the paediatric intensive care unit (ICU) with cardiovascular shock and persistent fever. Her initial symptoms were fever for 7 days, odynophagia, myalgia, and abdominal pain. On admission to the ICU, the patient presented with respiratory distress, comprising tachypnoea (respiratory rate 70 breaths per min) and hypoxia, and signs of congestive heart failure, including jugular vein distention, crackles at the base of the lungs, displaced liver, hypotension (blood pressure 80/36 mm Hg), tachycardia (134 beats per min [bpm]), and cold extremities with filiform pulses. Non-exudative conjunctivitis and cracked lips were present on physical examination. The patient was promptly intubated and antibiotic treatment was started with ceftriaxone and azithromycin. Peripheral epinephrine was initiated in the emergency room before the patient was moved to paediatric ICU. A point-of-care echocardiogram showed diffuse left-ventricular hypokinesia with no segmental wall motion abnormalities. Left-ventricular ejection fraction was estimated with the M-mode Teichholz method in the parasternal short axis view, at the level of the papillary muscles of the mitral valve; substantial myocardial dysfunction was noted, with decreased left-ventricular ejection fraction (31%) and no respiratory collapsibility of the inferior vena cava. The patient received furosemide, and central line and invasive arterial monitoring were established. Initial radiography showed an enlarged cardiac area and bilateral lung opacities (appendix p 1). Chest CT showed multiple ground-glass pulmonary opacities associated with thickening of interlobular septa and sparse bilateral foci of consolidation, predominantly in the peripheral and posterior areas of lower lobes (appendix p 1). Laboratory results showed high concentrations of markers of systemic inflammation and myocardial injury, including C-reactive protein, interleukin-6, ferritin, triglycerides, D-dimer, troponin, and creatine kinase myocardial band. Moreover, a left-shifted white-blood-cell count and substantial lymphopenia were seen. Blood gas analysis showed hypoxia and acidosis (table ). Table Laboratory results at various timepoints after presentation 0 h 7 h 14 h 17 h 24 h Normal range Haemoglobin, g/dL 10·0 11·8 12·1 11·4 11·0 12·7–14·7 Hematocrit, % 28·8% 34·3% 36·4% 34·3% 33·0% 38·0–44·0% Platelets, ×103 cells per μL 167 .. 191 .. 145 150–450 White blood cell count, ×103 cells per mm3 25·73 24·28 35·90 40·30 38·22 4·50–14·40 Lymphocytes, % 1·03% 0·73% 0·36% 0·40% 3·44% 38·00–42·00% Urea, mg/dL 67 73 78 78 93 11–38 Creatinine, mg/dL 1·27 1·31 1·56 1·73 2·19 0·53–0·79 D-dimer, ng/mL 11 495 .. 54 153 .. .. <500 Troponin, ng/dL 0·281 .. 0·290 0·342 .. <0·014 Creatine kinase myocardial band, ng/dL 5·76 .. 28·50 15·66 .. 0·10–2·88 Interleukin-6, pg/mL 4105·0 .. .. .. .. 0·2–7·8 Creatine kinase, U/L 96 .. .. .. .. <167 Blood pH 7·21 7·30 .. 7·28 7·31 7·35–7·45 Bicarbonate, mEq/L 15·7 16·4 .. 17·6 17·2 21·0–28·0 PaCO2, mm Hg 41 32 .. 31 .. 35–45 PaO2, mm Hg 60 270 .. 133 .. 80–90 ScvO2, % 87·2% 97·3% .. 82·0% 82·2% 60·0–85·0% Lactate, mg/dL 38·0 39·0 .. 27·0 .. 4·5–14·4 C-reactive protein, mg/dL 266·6 .. 309·5 .. .. <500 Total protein, g/dL 5·0 .. .. .. .. 6·0–8·0 Albumin, g/dL 2·6 .. .. .. .. 3·8–5·4 Aspartate aminotransferase, U/L 61 .. 67 .. .. 13–35 Alanine aminotransferase, U/L 67 .. 67 .. .. 7–35 Oxygenation index .. 3·1 .. 4·2 .. <4·0 International normalised ratio .. .. 1·4 .. .. 0·9–1·2 Fibrinogen, mg/dL .. .. 513 .. .. 200–393 Ferritin, ng/mL .. .. .. 1501 .. 20–200 Triglycerides, mg/dL .. .. .. 162 .. <100 PaCO2=partial pressure of carbon dioxide in arterial blood. PaO2=partial pressure of oxygen in arterial blood. ScvO2=central venous saturation of oxygen. Mechanical ventilation was implemented during the first hour in the ICU and ventilatory parameters reached a maximum positive end-expiratory pressure of 8 cm H2O and peak inspiratory pressure of 25 cm H2O, with an initial fraction of inspired oxygen of 60%. After initiation of mechanical ventilation and use of diuretics, ventilatory parameters could be reduced and less opacification was seen on chest radiography. The patient had sinus tachycardia throughout the hospital stay (heart rate >200 bpm); the initial electrocardiogram is shown in figure 1 . The patient progressed to hyperdynamic vasoplegic shock refractory to volume resuscitation and vasoactive agents. After 28 h of hospital admission, she developed ventricular fibrillation and died. Figure 1 Electrocardiogram showing sinus tachycardia on admission An ultrasound-guided minimally invasive autopsy was done, with tissue sampling of the heart, lungs, liver, spleen, kidneys, brain, inguinal lymph node, quadriceps muscle, and skin. 1 Post-mortem CT angiography was done before tissue collection and did not show any signs of coronary artery alterations (appendix p 2). Post-mortem ultrasound examination of the heart showed a hyperechogenic and diffusely thickened endocardium (mean thickness 10 mm), a thickened myocardium (18 mm thick in the left ventricle), and a small pericardial effusion. Histopathological examination showed myocarditis, pericarditis, and endocarditis characterised by inflammatory cell infiltration (figure 2A ). Inflammation was mainly interstitial and perivascular, associated with foci of cardiomyocyte necrosis (figure 2B, C), and was mainly composed of CD68+ macrophages (figure 2E), a few CD45+ lymphocytes (figure 2F), and a few neutrophils and eosinophils. C4d immunostaining was used for detection of cardiomyocyte necrosis (figure 2D). Analysis of cardiac tissue by electron microscopy identified spherical viral particles of 70–100 nm in diameter, consistent in size and shape with the Coronaviridae family, in the extracellular compartment and within several cell types—cardiomyocytes, capillary endothelial cells, endocardium endothelial cells, macrophages, neutrophils, and fibroblasts (figure 3 ). Microthrombi in the pulmonary arterioles (appendix p 3) and renal glomerular capillaries were also noted at autopsy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia was mild, with patchy exudative changes in alveolar spaces and mild pneumocyte hyperplasia (appendix p 3). Lymphoid depletion and signs of haemophagocytosis were noted in the spleen and lymph nodes, indicating secondary haemophagocytic lymphohistiocytosis associated with systemic inflammation. Acute tubular necrosis in the kidneys and hepatic centrilobular necrosis, secondary to shock, were also seen. Brain tissue showed microglial reactivity. Figure 2 Post-mortem histological findings (A) Diffuse myocardial interstitial inflammation. (B, C) Interstitial and perivascular myocardial inflammation containing lymphocytes, macrophages, a few neutrophils and eosinophils, and foci of cardiomyocyte necrosis. (D) Myocardial necrosis indicated by C4d staining. (E, F) Myocardial interstitial inflammation containing CD68+ (E) and CD45+ (F) cells. Figure 3 Post-mortem electron microscopy findings (A) Part of a cardiomyocyte, with viral particles (arrows) within a cytoplasmic area close to the nucleus. (B) Part of a fibroblast; arrow points to a viral particle inside a ruptured fragment of the rough endoplasmic reticulum. Inset in (B) corresponds to a higher magnification of the virus. (C) Endothelial lining (endocardium and subendocardium) of the left ventricular lumen; two viral particles (arrows) are present inside the endocardial endothelial cell. (D) Neutrophil in late stages of NETosis; asterisks indicate neutrophil extracellular traps (decondensed and dispersed chromatin); arrow points to a viral particle inside a cytoplasmic vesicle. Inset in (D) shows the viral particle at high magnification. cm=cardiomyocyte. col=collagen fibrils. end=endothelial cell. mf=myofibrils. NET=neutrophil extracellular trap. nu=nucleus. se=subendocardial fibroblast. SARS-CoV-2 RNA was detected on a post-mortem nasopharyngeal swab and in cardiac and pulmonary tissues by real time RT-PCR using primers and probes set for E (envelope) gene. 2 Cycle threshold values for lung and heart samples were 35·6 and 36·0, respectively, suggesting a low viral load in both organs. To investigate a primary immunodeficiency, whole-exome sequencing from genomic DNA extracted from whole blood was done, using a customised Twist Human Core Exome kit (Twist Bioscience, San Francisco, CA, USA) for exon capture, and sequenced in an Illumina NovaSeq platform (Illumina, San Diego, CA, USA). Sequence reads were aligned to the reference human genome (GrCh38/hg38 in the University of California Santa Cruz [UCSC] Genome Browser) with Burrows-Wheeler Aligner software. Genotyping was done using the Genome Analysis Toolkit (Broad Institute, Cambridge, MA, USA). 3 No pathogenic, likely pathogenic, or variant of unknown significance was found associated with inborn errors of immunity. MIS-C is a severe clinical condition that has been described in several paediatric patients diagnosed with COVID-19 and that might be associated with cardiac dysfunction.4, 5, 6, 7, 8, 9, 10 Since the disorder shares similarities with Kawasaki disease, it has also been reported as Kawasaki-like disease or Kawasaki-like multisystem inflammatory syndrome.4, 5, 6, 7, 8 A substantial increase in the incidence of Kawasaki-like disease has been described in several countries with high incidence of COVID-19.4, 5, 7 In Italy, the first European country to be affected by the COVID-19 pandemic, Verdoni and colleagues 4 found that, over a period of 1 month, the spread of SARS-CoV-2 was associated with a 30-fold increase in the incidence of Kawasaki-like disease. Compared with classic Kawasaki disease, children with MIS-C are older, have respiratory, gastrointestinal, neurological, and cardiovascular involvement, substantial lymphopenia, thrombocytopenia, and markers of myocarditis.4, 7, 8 Although previous studies have reported low mortality among children with MIS-C (<2%), patients presented with cardiogenic shock, acute left-ventricular dysfunction, and signs of myocarditis, indicating a potential risk of a life-threatening condition.4, 5, 6, 7, 8, 9, 10 The mechanism of heart failure in these patients and its relation to SARS-CoV-2 infection is not understood. Possible mechanisms involved in cardiac dysfunction in children with COVID-19 include myocardial stunning or oedema associated with a severe systemic inflammatory state, direct myocardial injury by SARS-CoV-2, and hypoxia secondary to viral pneumonia.4, 5, 6, 7, 8, 9, 10, 11 Reports of substantial numbers of children presenting with MIS-C or Kawasaki-like disease during the COVID-19 pandemic indicate that SARS-CoV-2 is probably a trigger of this clinical condition, either by eliciting a severe systemic immune response or by direct tissue damage, or both.4, 5, 6, 7, 8, 9, 10 Our case report shows inflammatory changes in the cardiac tissue of a child with MIS-C related to COVID-19, which led to cardiac failure and death. SARS-CoV-2 could be detected in cardiac tissue by RT-PCR and electron microscopy. Despite the evident systemic inflammation and final progression to multiorgan failure, clinical, echocardiographic, and laboratory findings strongly indicated that heart failure was the main determinant of the fatal outcome. Further, the autopsy showed myocarditis, pericarditis, and endocarditis, with intense and diffuse tissue inflammation, and necrosis of cardiomyocytes. Moreover, the finding of SARS-CoV-2 in heart tissue indicates that myocardial inflammation was probably a primary response to the virus-induced injury to cardiac cells. The presence of SARS-CoV-2 in different cell types of cardiac tissue suggests potential mechanisms for heart damage. First, infection of cardiomyocytes probably leads to local inflammation in response to cell injury; both the virus-induced injury and the inflammatory response could lead to necrosis of cardiomyocytes. The finding of viral particles in neutrophils supports the idea of virus-induced inflammation. Also, infection of endothelial cells in the endocardium could result in haematogenous spread of SARS-CoV-2 to other organs and tissues. Detection of both SARS-CoV-2 RNA by RT-PCR and viral particles by electron microscopy in cardiac tissue has been reported in endomyocardial biopsy specimens from adults with COVID-19.12, 13 Tavazzi and colleagues 13 detected viral particles in cardiac macrophages in an adult patient with acute cardiac injury associated with COVID-19; no viral particles were seen in cardiomyocytes or endothelial cells. Our case report is the first to our knowledge to document the presence of viral particles in the cardiac tissue of a child affected by MIS-C. Moreover, viral particles were identified in different cell lineages of the heart, including cardiomyocytes, endothelial cells, mesenchymal cells, and inflammatory cells. Two other reports in adolescents with COVID-19 detected myocarditis by MRI or at autopsy.14, 15 In the report from Craven and colleagues, 15 histological analysis of the heart of a 17-year-old boy showed diffuse myocarditis with mixed inflammatory infiltrate, with a predominance of eosinophils. In our case report, cardiac inflammation also included a small number of eosinophils. In these two previous reports,14, 15 common symptoms of COVID-19 were absent, except for fever; pulmonary changes were absent or mild, and there was no multiorgan involvement. The pulmonary involvement noted in our case report was probably the result of mild pneumonia, cardiogenic oedema, and microthrombi in the pulmonary arteriolar bed, which—associated with the finding of microthrombi in the kidney and the presence of virus in the cardiac capillary endothelium—suggest a SARS-CoV-2-induced endothelial dysfunction that probably involved several organs. Whole-exome sequencing could not identify any inborn error of immunity in our patient. It is still unclear which host factors could predispose children to MIS-C; further investigation of potential genetic determinants is important to understand the pathogenesis of this syndrome. 10 In conclusion, our pathological observations support the hypothesis that the direct effect of SARS-CoV-2 infection on cardiac tissue was a major contributor to myocarditis and heart failure in our patient. Hopefully, our findings could help to shed light on the understanding of the complex interaction between SARS-CoV-2 infection, MIS-C, and cardiac dysfunction in children and adolescents with COVID-19. For the UCSC Genome Browser see http://genome.ucsc.edu

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          Acute myocarditis and multisystem inflammatory emerging disease following SARS-CoV-2 infection in critically ill children

          Background A recent increase in children admitted with hypotensive shock and fever in the context of the COVID-19 outbreak requires an urgent characterization and assessment of the involvement of SARS-CoV-2 infection. This is a case series performed at 4 academic tertiary care centers in Paris of all the children admitted to the pediatric intensive care unit (PICU) with shock, fever and suspected SARS-CoV-2 infection between April 15th and April 27th, 2020. Results 20 critically ill children admitted for shock had an acute myocarditis (left ventricular ejection fraction, 35% (25–55); troponin, 269 ng/mL (31–4607)), and arterial hypotension with mainly vasoplegic clinical presentation. The first symptoms before PICU admission were intense abdominal pain and fever for 6 days (1–10). All children had highly elevated C-reactive protein (> 94 mg/L) and procalcitonin (> 1.6 ng/mL) without microbial cause. At least one feature of Kawasaki disease was found in all children (fever, n = 20, skin rash, n = 10; conjunctivitis, n = 6; cheilitis, n = 5; adenitis, n = 2), but none had the typical form. SARS-CoV-2 PCR and serology were positive for 10 and 15 children, respectively. One child had both negative SARS-CoV-2 PCR and serology, but had a typical SARS-CoV-2 chest tomography scan. All children but one needed an inotropic/vasoactive drug support (epinephrine, n = 12; milrinone, n = 10; dobutamine, n = 6, norepinephrine, n = 4) and 8 were intubated. All children received intravenous immunoglobulin (2 g per kilogram) with adjuvant corticosteroids (n = 2), IL 1 receptor antagonist (n = 1) or a monoclonal antibody against IL-6 receptor (n = 1). All children survived and were afebrile with a full left ventricular function recovery at PICU discharge. Conclusions Acute myocarditis with intense systemic inflammation and atypical Kawasaki disease is an emerging severe pediatric disease following SARS-CoV-2 infection. Early recognition of this disease is needed and referral to an expert center is recommended. A delayed and inappropriate host immunological response is suspected. While underlying mechanisms remain unclear, further investigations are required to target an optimal treatment.
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            Is Open Access

            Human Inborn Errors of Immunity: 2019 Update of the IUIS Phenotypical Classification

            Since 2013, the International Union of Immunological Societies (IUIS) expert committee (EC) on Inborn Errors of Immunity (IEI) has published an updated phenotypic classification of IEI, which accompanies and complements their genotypic classification into ten tables. This phenotypic classification is user-friendly and serves as a resource for clinicians at the bedside. There are now 430 single-gene IEI underlying phenotypes as diverse as infection, malignancy, allergy, autoimmunity, and autoinflammation. We herein report the 2019 phenotypic classification, including the 65 new conditions. The diagnostic algorithms are based on clinical and laboratory phenotypes for each of the ten broad categories of IEI.
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              Emergence of Kawasaki disease related to SARS-CoV-2 infection in an epicentre of the French COVID-19 epidemic: a time-series analysis

              Summary Background Kawasaki disease is an acute febrile systemic childhood vasculitis, which is suspected to be triggered by respiratory viral infections. We aimed to examine whether the ongoing COVID-19 epidemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with an increase in the incidence of Kawasaki disease. Methods We did a quasi-experimental interrupted time series analysis over the past 15 years in a tertiary paediatric centre in the Paris region, a French epicentre of the COVID-19 outbreak. The main outcome was the number of Kawasaki disease cases over time, estimated by quasi-Poisson regression. In the same centre, we recorded the number of hospital admissions from the emergency department (2005–2020) and the results of nasopharyngeal multiplex PCR to identify respiratory pathogens (2017–2020). These data were compared with daily hospital admissions due to confirmed COVID-19 in the same region, recorded by Public Health France. Findings Between Dec 1, 2005, and May 20, 2020, we included 230 patients with Kawasaki disease. The median number of Kawasaki disease hospitalisations estimated by the quasi-Poisson model was 1·2 per month (IQR 1·1–1·3). In April, 2020, we identified a rapid increase of Kawasaki disease that was related to SARS-CoV-2 (six cases per month; 497% increase [95% CI 72–1082]; p=0·0011), starting 2 weeks after the peak of the COVID-19 epidemic. SARS-CoV-2 was the only virus circulating intensely during this period, and was found in eight (80%) of ten patients with Kawasaki disease since April 15 (SARS-CoV-2-positive PCR or serology). A second peak of hospital admissions due to Kawasaki disease was observed in December, 2009 (six cases per month; 365% increase ([31–719]; p=0.0053), concomitant with the influenza A H1N1 pandemic. Interpretation Our study further suggests that viral respiratory infections, including SAR-CoV-2, could be triggers for Kawasaki disease and indicates the potential timing of an increase in incidence of the disease in COVID-19 epidemics. Health-care providers should be prepared to manage an influx of patients with severe Kawasaki disease, particularly in countries where the peak of COVID-19 has recently been reached. Funding French National Research Agency.
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                Author and article information

                Contributors
                Journal
                Lancet Child Adolesc Health
                Lancet Child Adolesc Health
                The Lancet. Child & Adolescent Health
                Elsevier Ltd.
                2352-4642
                2352-4650
                20 August 2020
                20 August 2020
                Affiliations
                [a ]Departamento de Patologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
                [b ]Instituto da Criança, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
                [c ]Departamento de Gastroenterologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
                [d ]Serviço de Verificação de Óbitos da Capital, Universidade de São Paulo, São Paulo, Brazil
                Author notes
                [* ]Correspondence to: Dr Marisa Dolhnikoff, Faculdade de Medicina da Universidade de São Paulo, Departamento de Patologia, Sao Paulo 01246-903, Brazil maridol@ 123456usp.br
                [†]

                Contributed equally

                Article
                S2352-4642(20)30257-1
                10.1016/S2352-4642(20)30257-1
                7440866
                © 2020 Elsevier Ltd. All rights reserved.

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