Post-Mortem Diagnosis of Pediatric Dengue Using Minimally Invasive Autopsy during the COVID-19 Pandemic in Brazil.

We report the first pediatric disease in which the use of minimally invasive autopsy (MIA) confirmed severe dengue as the cause of death. During the COVID-19 pandemic, a previously healthy 10-year-old girl living in north-eastern Brazil presented fever, headache, diffuse abdominal pain, diarrhoea, and vomiting. On the fourth day, the clinical symptoms worsened and the patient died. An MIA was performed, and cores of brain, lungs, heart, liver, kidneys, and spleen were collected with 14G biopsy needles. Microscopic examination showed diffuse oedema and congestion, pulmonary intra-alveolar haemorrhage, small foci of midzonal necrosis in the liver, and tubular cell necrosis in the kidneys. Dengue virus RNA and NS1 antigen were detected in blood and cerebrospinal fluid samples. Clinical, pathological, and laboratory findings, in combination with the absence of other lesions and microorganisms, allowed concluding that the patient had died from complications of severe dengue.

COVID-19 beyond DAD: persisting microcirculation thrombosis, hidden infections, and early pulmonary fibrosis as remaining challenges of the disease

Background Despite advances in the management of COVID-19, some events occurring in the course of disease still represent challenges to patient treatment. Case presentation Here we present a case of severe COVID-19 in which the patient received standard treatment including dexamethasone and prophylactic anticoagulation and died on the 24th day of disease. Autopsy showed exudative and proliferative DAD accompanied by remaining microcirculation thrombosis despite anticoagulation treatment, unperceived fungal infection, concomitantly with the presence of dense acellular fibrotic areas amidst organizing lung lesions. Conclusions Although improvements were achieved in COVID-19 therapeutics lung microcirculation coagulopathy, unperceived fungus infection and early developing alveolar fibrosis remain unsolved problems associated to the disease.

SARS-Cov-2 pneumonia phenotyping on imaging exams of patients submitted to minimally invasive autopsy.

Background: Correlation between pathology and imaging of the new SARS-Cov-2 disease (COVID-19) is scarce. This study aimed to characterize SARS-Cov-2 pneumonia on imaging of patients submitted to minimally invasive autopsy (MIA). Methods: This unicentric retrospective observational study included 46 consecutive patients with confirmed COVID-19 who underwent MIA. All clinical chest images were reviewed and classified for the presence and grade of viral pneumonia, as well as disease evolution. On CT, phenotypes were described as consistent with mild, moderate, or severe viral pneumonia, with or without radiological signs of organizing pneumonia (OP). In severe pneumonia, CT could also be classified as diffuse progressive OP or radiological diffuse alveolar damage (DAD). Specific features on CT were noted, including fibroproliferative signs that could indicate potential or initial fibrosis. Results: MIA showed a heterogeneous panel of alterations, with a high prevalence of OP and acute fibrinous and organizing pneumonia (AFOP). Also, signs of interstitial fibrosis corresponded to the most prevalent pathological feature. Initial chest radiography (CXR) findings were mainly consistent with moderate or severe viral pneumonia. Most patients showed stability or improvement (reduction of opacities) on imaging. CTs were performed on 15 patients. Consolidations were found in most patients, frequently showing features consistent with an OP phenotype. Fibroproliferative changes were also prevalent on CT. Conclusions: In this study, SARS-Cov-2 pneumonia showed heterogeneous radiological and pathological patterns. Signs of organization and potential or initial fibrosis were prevalent on both imaging and pathology. Imaging phenotyping may help to predict post-infection fibrosing interstitial pneumonitis in COVID-19.

Ultrasound-Guided Minimally Invasive Tissue Sampling: A Minimally Invasive Autopsy Strategy During the COVID-19 Pandemic in Brazil, 2020.

BACKGROUND: Minimally invasive autopsies, also known as minimally invasive tissue sampling (MITS), have proven to be an alternative to complete diagnostic autopsies (CDAs) in places or situations where this procedure cannot be performed. During the coronavirus disease 2019 (COVID-19) pandemic, CDAs were suspended by March 2020 in Brazil to reduce biohazard. To contribute to the understanding of COVID-19 pathology, we have conducted ultrasound (US)-guided MITS as a strategy. METHODS: This case series study includes 80 autopsies performed in patients with COVID-19 confirmed by laboratorial tests. Different organs were sampled using a standardized MITS protocol. Tissues were submitted to histopathological analysis as well as immunohistochemical and molecular analysis and electron microscopy in selected cases. RESULTS: US-guided MITS proved to be a safe and highly accurate procedure; none of the personnel were infected, and accuracy ranged from 69.1% for kidney, up to 90.1% for lungs, and reaching 98.7% and 97.5% for liver and heart, respectively. US-guided MITS provided a systemic view of the disease, describing the most common pathological findings and identifying viral and other infectious agents using ancillary techniques, and also allowed COVID-19 diagnosis confirmation in 5% of the cases that were negative in premortem and postmortem nasopharyngeal/oropharyngeal swab real-time reverse-transcription polymerase chain reaction. CONCLUSIONS: Our data showed that US-guided MITS has the capacity similar to CDA not only to identify but also to characterize emergent diseases.

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

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.

Minimally Invasive Autopsy Practice in COVID-19 Cases: Biosafety and Findings.

Postmortem studies are crucial for providing insight into emergent diseases. However, a complete autopsy is frequently not feasible in highly transmissible diseases due to biohazard challenges. Minimally invasive autopsy (MIA) is a needle-based approach aimed at collecting samples of key organs without opening the body, which may be a valid alternative in these cases. We aimed to: (a) provide biosafety guidelines for conducting MIAs in COVID-19 cases, (b) compare the performance of MIA versus complete autopsy, and (c) evaluate the safety of the procedure. Between October and December 2020, MIAs were conducted in six deceased patients with PCR-confirmed COVID-19, in a basic autopsy room, with reinforced personal protective equipment. Samples from the lungs and key organs were successfully obtained in all cases. A complete autopsy was performed on the same body immediately after the MIA. The diagnoses of the MIA matched those of the complete autopsy. In four patients, COVID-19 was the main cause of death, being responsible for the different stages of diffuse alveolar damage. No COVID-19 infection was detected in the personnel performing the MIAs or complete autopsies. In conclusion, MIA might be a feasible, adequate and safe alternative for cause of death investigation in COVID-19 cases.

Tracking the time course of pathological patterns of lung injury in severe COVID-19.

BACKGROUND: Pulmonary involvement in COVID-19 is characterized pathologically by diffuse alveolar damage (DAD) and thrombosis, leading to the clinical picture of Acute Respiratory Distress Syndrome. The direct action of SARS-CoV-2 in lung cells and the dysregulated immuno-coagulative pathways activated in ARDS influence pulmonary involvement in severe COVID, that might be modulated by disease duration and individual factors. In this study we assessed the proportions of different lung pathology patterns in severe COVID-19 patients along the disease evolution and individual characteristics. METHODS: We analysed lung tissue from 41 COVID-19 patients that died in the period March-June 2020 and were submitted to a minimally invasive autopsy. Eight pulmonary regions were sampled. Pulmonary pathologists analysed the H&E stained slides, performing semiquantitative scores on the following parameters: exudative, intermediate or advanced DAD, bronchopneumonia, alveolar haemorrhage, infarct (%), arteriolar (number) or capillary thrombosis (yes/no). Histopathological data were correlated with demographic-clinical variables and periods of symptoms-hospital stay. RESULTS: Patient´s age varied from 22 to 88 years (18f/23 m), with hospital admission varying from 0 to 40 days. All patients had different proportions of DAD in their biopsies. Ninety percent of the patients presented pulmonary microthrombosis. The proportion of exudative DAD was higher in the period 0-8 days of hospital admission till death, whereas advanced DAD was higher after 17 days of hospital admission. In the group of patients that died within eight days of hospital admission, elderly patients had less proportion of the exudative pattern and increased proportions of the intermediate patterns. Obese patients had lower proportion of advanced DAD pattern in their biopsies, and lower than patients with overweight. Clustering analysis showed that patterns of vascular lesions (microthrombosis, infarction) clustered together, but not the other patterns. The vascular pattern was not influenced by demographic or clinical parameters, including time of disease progression. CONCLUSION: Patients with severe COVID-19 present different proportions of DAD patterns over time, with advanced DAD being more prevalent after 17 days, which seems to be influenced by age and weight. Vascular involvement is present in a large proportion of patients, occurs early in disease progression, and does not change over time.

Ultrasound assessment of pulmonary fibroproliferative changes in severe COVID-19: a quantitative correlation study with histopathological findings.

PURPOSE: This study was designed to evaluate the usefulness of lung ultrasound (LUS) imaging to characterize the progression and severity of lung damage in cases of COVID-19. METHODS: We employed a set of combined ultrasound parameters and histopathological images obtained simultaneously in 28 patients (15 women, 0.6-83 years) with fatal COVID-19 submitted to minimally invasive autopsies, with different times of disease evolution from initial symptoms to death (3-37 days, median 18 days). For each patient, we analysed eight post-mortem LUS parameters and the proportion of three histological patterns (normal lung, exudative diffuse alveolar damage [DAD] and fibroproliferative DAD) in eight different lung regions. The relationship between histopathological and post-mortem ultrasonographic findings was assessed using various statistical approaches. RESULTS: Statistically significant positive correlations were observed between fibroproliferative DAD and peripheral consolidation (coefficient 0.43, p = 0.02) and pulmonary consolidation (coefficient 0.51, p = 0.005). A model combining age, time of evolution, sex and ultrasound score predicted reasonably well (r = 0.66) the proportion of pulmonary parenchyma with fibroproliferative DAD. CONCLUSION: The present study adds information to previous studies related to the use of LUS as a tool to assess the severity of acute pulmonary damage. We provide a histological background that supports the concept that LUS can be used to characterize the progression and severity of lung damage in severe COVID-19.

Pulmonary and systemic involvement in COVID-19 patients assessed with ultrasound-guided minimally invasive autopsy.

AIMS: Brazil ranks high in the number of coronavirus disease 19 (COVID-19) cases and the COVID-19 mortality rate. In this context, autopsies are important to confirm the disease, determine associated conditions, and study the pathophysiology of this novel disease. The aim of this study was to assess the systemic involvement of COVID-19. In order to follow biosafety recommendations, we used ultrasound-guided minimally invasive autopsy (MIA-US), and we present the results of 10 initial autopsies. METHODS AND RESULTS: We used MIA-US for tissue sampling of the lungs, liver, heart, kidneys, spleen, brain, skin, skeletal muscle and testis for histology, and reverse transcription polymerase chain reaction to detect severe acute respiratory syndrome coronavirus 2 RNA. All patients showed exudative/proliferative diffuse alveolar damage. There were intense pleomorphic cytopathic effects on the respiratory epithelium, including airway and alveolar cells. Fibrinous thrombi in alveolar arterioles were present in eight patients, and all patients showed a high density of alveolar megakaryocytes. Small thrombi were less frequently observed in the glomeruli, spleen, heart, dermis, testis, and liver sinusoids. The main systemic findings were associated with comorbidities, age, and sepsis, in addition to possible tissue damage due to the viral infection, such as myositis, dermatitis, myocarditis, and orchitis. CONCLUSIONS: MIA-US is safe and effective for the study of severe COVID-19. Our findings show that COVID-19 is a systemic disease causing major events in the lungs and with involvement of various organs and tissues. Pulmonary changes result from severe epithelial injury and microthrombotic vascular phenomena. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches.

[Pathological changes of fatal coronavirus disease 2019 (COVID-19) in the lungs: report of 10 cases by postmortem needle autopsy].

Objectives: To observe the pulmonary changes with coronavirus disease 2019 (COVID-19) in postmortem needle specimens, to detect the presence of 2019 novel coronavirus(2019-nCoV) in the lung tissues, and to analyze the clinicopathological characteristics. Methods: For 10 decedents with 2019-nCoV infection in Wuhan, bilateral lungs underwent ultrasound-guided percutaneous multi-point puncture autopsy, and pulmonary pathological changes were described in routine hematoxylin-eosin staining (HE) slides. Electron microscopy was also performed. The reverse transcription polymerase chain reaction (RT-PCR) was employed to detect 2019-nCoV nucleic acid in lung tissue, and the pathological characteristics were demonstrated in combination with clinical data analysis. Results: Of the 10 deaths associated with COVID-19, 7 were male and 3 were female. The average age was 70 (39-87) years. Medical record showed that 7 patients had underlying diseases. The average course of disease was 30 (16-36) days. Nine cases showed fibrinous and suppurative exudation in the alveolar cavity accompanied by the formation of hyaline membrane, and fibroblastic proliferation of alveolar septum. Type Ⅱ alveolar epithelial cells showed reactive hyperplasia and desquamation. Many macrophages accumulated in the alveolar cavity. Capillary hyaline thrombus and intravascular mixed thrombus were noted. In some cases, acute bronchiolitis with mucous membrane exfoliation, accumulation of bronchiolar secretions, and bronchiolar epithelial metaplasia occurred. In the cohort, a large number of bacteria (cocci) were detected in 1 case and a large number of fungi (yeast type) were detected in 1 case. Nine cases were positive for the nucleic acids of 2019-nCoV while one case remained negative by RT-PCR. Coronavirus particles were detected in the cytoplasm of type Ⅱ alveolar epithelium. Conclusions: The pulmonary pathological changes of fatal COVID-19 are diffuse alveolar damage (DAD), mainly in the acute exudative stage and the organic proliferative stage. There are fibrinous exudate aggregation in alveolar cavity with hyaline membrane formation, fibroblastic proliferation in alveolar septum, and alveolar epithelial cell injuries with reactive hyperplasia and desquamation of type Ⅱ alveolar epithelial cells. A large amount of neutrophils and monocytes infiltration is present in most cases and bacteria and fungi are detected in some cases, suggesting a serious bacterial or fungal infection secondary to the DAD.

[A pathological report of three COVID-19 cases by minimal invasive autopsies].

Objective: To investigate the pathological characteristics and the clinical significance of novel coronavirus (2019-nCoV)-infected pneumonia (termed by WHO as coronavirus disease 2019, COVID-19). Methods: Minimally invasive autopsies from lung, heart, kidney, spleen, bone marrow, liver, pancreas, stomach, intestine, thyroid and skin were performed on three patients died of novel coronavirus pneumonia in Chongqing, China. Hematoxylin and eosin staining (HE), transmission electron microcopy, and histochemical staining were performed to investigate the pathological changes of indicated organs or tissues. Immunohistochemical staining was conducted to evaluate the infiltration of immune cells as well as the expression of 2019-nCoV proteins. Real time PCR was carried out to detect the RNA of 2019-nCoV. Results: Various damages were observed in the alveolar structure, with minor serous exudation and fibrin exudation. Hyaline membrane formation was observed in some alveoli. The infiltrated immune cells in alveoli were majorly macrophages and monocytes. Moderate multinucleated giant cells, minimal lymphocytes, eosinophils and neutrophils were also observed. Most of infiltrated lymphocytes were CD4-positive T cells. Significant proliferation of type Ⅱ alveolar epithelia and focal desquamation of alveolar epithelia were also indicated. The blood vessels of alveolar septum were congested, edematous and widened, with modest infiltration of monocytes and lymphocytes. Hyaline thrombi were found in a minority of microvessels. Focal hemorrhage in lung tissue, organization of exudates in some alveolar cavities, and pulmonary interstitial fibrosis were observed. Part of the bronchial epithelia were exfoliated. Coronavirus particles in bronchial mucosal epithelia and type Ⅱ alveolar epithelia were observed under electron microscope. Immunohistochemical staining showed that part of the alveolar epithelia and macrophages were positive for 2019-nCoV antigen. Real time PCR analyses identified positive signals for 2019-nCoV nucleic acid. Decreased numbers of lymphocyte, cell degeneration and necrosis were observed in spleen. Furthermore, degeneration and necrosis of parenchymal cells, formation of hyaline thrombus in small vessels, and pathological changes of chronic diseases were observed in other organs and tissues, while no evidence of coronavirus infection was observed in these organs. Conclusions: The lungs from novel coronavirus pneumonia patients manifest significant pathological lesions, including the alveolar exudative inflammation and interstitial inflammation, alveolar epithelium proliferation and hyaline membrane formation. While the 2019-nCoV is mainly distributed in lung, the infection also involves in the damages of heart, vessels, liver, kidney and other organs. Further studies are warranted to investigate the mechanism underlying pathological changes of this disease.