key: cord-277855-pfmslxy6
authors: Nunes Duarte‐Neto, Amaro; de Almeida Monteiro, Renata Aparecida; da Silva, Luiz Fernando Ferraz; Malheiros, Denise Maria Avancini Costa; de Oliveira, Ellen Pierre; Theodoro Filho, Jair; Pinho, João Renato Rebello; Soares Gomes‐Gouvêa, Michele; Salles, Ana Paula Moreira; de Oliveira, Ilka Regina Souza; Mauad, Thais; do Nascimento Saldiva, Paulo Hilário; Dolhnikoff, Marisa
title: Pulmonary and systemic involvement of COVID‐19 assessed by ultrasound‐guided minimally invasive autopsy
date: 2020-05-22
journal: Histopathology
DOI: 10.1111/his.14160
sha: 
doc_id: 277855
cord_uid: pfmslxy6

AIMS: Brazil ranks high in the number of COVID‐19 cases and COVID‐19’s mortality rate. In this context, autopsies are important to confirm the disease, determine associated conditions, and study the pathophysiology of this novel disease. In order to follow biosafety recommendations, we used Ultrasound‐Guided Minimally Invasive Autopsy (MIA‐US) to assess the systemic involvement of COVID‐19 and present the results of ten initial autopsies. METHODS AND RESULTS: We used MIA‐US for tissue sampling of lungs, liver, heart, kidneys, spleen, brain, skin, skeletal muscle and testis for histology and RT‐PCR to detect SARS‐COV‐2‐RNA. All patients presented 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 presented a high density of alveolar megakaryocytes. Small thrombi were less frequently observed in 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 with major events in the lungs and involvement of various organs and tissues. Pulmonary changes are the result of severe epithelial injury and microthrombotic vascular phenomena. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches.

The beginning of 2020 has been marked by the expansion of the SARS-COV-2 infection pandemic , which started in China in late 2019. 1, 2 In Brazil, the first case was diagnosed on February 25, 2020, and the first death was recorded on March 17, 2020. Currently, the COVID-19 epidemic in Brazil is in its accelerating phase, with 61,888 confirmed cases and 4,205 deaths (mortality rate 6.8%) as of April 26, 2020. The State of Sao Paulo is the most affected region in the country, with the largest number of cases (20,715) and deaths (1, 700) . 3 Clinically, severe COVID-19 presents as respiratory failure due to diffuse and bilateral interstitial pneumonia. 1,2 Those with advanced age, obesity, diabetes mellitus, and chronic cardiovascular, renal, and pulmonary diseases are at higher risk for severe disease. 1, 2, 3, 4 Other causes of respiratory failure may be associated with or simulate COVID-19 and a non-invasive diagnostic approach, such as naso/oropharyngeal RT-PCR, can generate a large number of false negatives. 5 This issue is even more critical in cases of death, where the actual mortality rate can be underestimated.

In this context, autopsies of fatal COVID-19 allow pathologists to effectively confirm or exclude the disease and determine the associated conditions. Post-mortem tissue sampling is also of paramount importance for conducting in-situ and molecular studies and for a detailed investigation of the pathophysiology of this new disease. However, the number of autopsies reported up to now is small, and tissue sampling is often limited to few organs, mostly lungs, liver, and heart. [6] [7] [8] [9] [10] [11] [12] [13] The scarce literature on COVID-19 autopsies markedly contrasts with the high number of deaths, probably due to the risk of contagion. In addition, strict protection procedures were recommended for autopsies of COVID-19, restricting autopsies to a limited number of institutions. 14, 15 As a rapid response and preparedness for this new epidemic, our University Clinical Hospital (HC-FMUSP) was assigned to treat the patients with severe COVID-19 in the city of Sao Paulo and we developed a procedure of ultrasound-based minimally invasive autopsy (MIA-US) to study the fatal cases. MIA-US had already been used during the recent 2018 yellow fever epidemic in Sao Paulo and showed a full diagnostic agreement with the conventional autopsy. 16 MIA-US is a suitable alternative to conduct autopsies on COVID-19 cases because it considerably reduces its costs; the risk of producing aerosols is low and, therefore, it can be performed in services

This article is protected by copyright. All rights reserved without negative pressure autopsy rooms; the images obtained by ultrasound are good enough to localise and orient the sampling in several organs; it provides information within a time window fast enough to orient the management of critically ill patients. 17, 18 In the present pandemic scenario, we describe our MIA-US procedure considering that its low cost and portability, combined with a significant reduction in the risk of contagion from a closedbody autopsy, could be a way to increase the rate of COVID-19 autopsies, contributing to a better understanding of the mechanisms of tissue injury and possibly adding useful information for the development of new therapeutic procedures. Here we describe the MIA-US procedure and present the results of ten COVID-19 autopsies.

This is a case series study based on the autopsy of the first ten fatal cases of COVID-19 who died at HC-FMUSP, from March 18 to 30, 2020, in the city of Sao Paulo, Brazil. This protocol was approved by the HC-FMUSP Ethical Committee (protocol #3951.904). The procedures were performed at the "Image Platform in the Autopsy Room", a research centre in the University of Sao Paulo Medical School, located next to the Autopsy Service of Sao Paulo University (https://pisa.hc.fm.usp.br/). All the autopsies were performed after informed consent from the next-of-kin.

The case definition for confirmed cases of COVID-19 from the World Health Organization is for patients with laboratory confirmation of SARS-COV-2 infection, regardless of clinical signs and symptoms. 1 As the WHO has not yet incorporated the autopsy result in the case definition, considering the possibility of false-negative molecular tests and the absence of an alternative diagnosis for the cause of death, we extended the case definition to one patient with acute and fatal respiratory distress, with typical radiological and histological pulmonary changes, even with negative laboratory results. 1, 5 Epidemiological, clinical and laboratorial data were obtained from the medical charts.

The deceased bodies were transported in a plastic safety bag from the hospital to the morgue by nurses with appropriate personal protective equipment (PPE). Two trained technicians prepared the body for the procedure, covering it with an additional plastic bag, sliding it through a Accepted Article stretcher with pulleys. Access to the autopsy room was limited to two people, the US examiner and the supporting technician, who wore PPE following standard protocols (surgical clothing protected by two aprons, rubber boot, plastic sleeve, 3 layers of gloves, rubber cap, N95 mask under a surgical mask and eye protection). 14,15 After the procedure and release of the body to the morgue, the personnel involved directly with the MIA-US went to a support room to take out the PPE with the aid of protected technicians. The removal of garbage and a terminal cleaning protocol of the autopsy room was performed by trained personnel. 14,15 Plastic vials containing the sampled material had their external surfaces cleaned with 70% isopropyl alcohol solution.

The personnel involved directly or indirectly with MIA-US were tested once a week for COVID-19

(naso/oropharyngeal RT-PCR). 19 After 15 days of procedures, all team members tested negative.

We used a portable SonoSite M-Turbo R (Fujifilm, Bothell, WA, USA) ultrasound equipment with C60x (5-2 MHz Convex) multi-frequency broadband transducers and DICOM R standard images.

We chose to employ this transducer due to its lower frequency ultrasound waves, which allows a deeper visualisation of all organs, including pulmonary parenchyma. The images obtained by ultrasound were used to localise and orient the sampling in several organs, as well as to select the most affected areas within each organ. Examples of those images are presented in Figure 1 .

After packing the body with resistant plastic, small 10 cm openings were made at appropriate sites on the body surface. Initially, we accessed the heart and the left lung through an incision adjacent to the sternum bone. After completing the tissue sampling at this site we accessed the right lung through an incision at the right parasternal area. The opening in the plastic wrap was closed, and subsequent incisions were done at the right subcostal space (liver and right kidney) and left subcostal space (spleen and left kidney). Tissue sampling was performed using Tru-Cut R semi-automatic coaxial needles of 14G, 20 cm long. The following tissue samples were collected under ultrasound guidance: lungs (8 samples from each lung for histology and two lung samples for molecular analysis), liver (at least two samples), both kidneys (one sample each), spleen (one sample), and heart (one sample). Other tissues were sampled without direct image guidance: skeletal muscle (femoral quadriceps), skin (in the left thigh, with punch needle 5 mm), and brain (transsphenoidal puncture).

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Tissue samples were fixed in buffered 10% formalin, embedded in paraffin and stained with haematoxylin and eosin (H&E). A semiquantitative analysis was performed to grade pulmonary changes, where 0 = absence, 1 = mild and 2 = intense.

Immunohistochemistry was performed in the lung tissue for detecting the antigens TTF-1 (Abcam, 8G7G3/1 clone, 1:500 dilution), p63 (Biocare Medical, 4A4 clone, 1:100 dilution), Ki67 (Dako, MIB-1 clone, 1:400 dilution), CD4 (Leica Biosystems-Novocastra, 4B12 clone, 1:50 dilution), CD8 (Dako, C8/144B clone, 1:400 dilution), CD20 (Dako, L26 clone, 1:2500 dilution), CD57 (Dako, TB01 clone, 1:200 dilution) and CD68 (Dako, PG-M1 clone, 1:2500 dilution). The antigens retrieval was done with citrate at pH 6.0, except for p63 and CD4 that was performed with TRIS-EDTA, at pH 9.0. The reactions followed standard protocols validated in our laboratories. 16

Lung tissue samples measuring 1 cm 3 were stored at −80°C. Tissue samples were macerated, and nucleic acid extraction was performed using the TRIzol® reagent (Invitrogen). Molecular detection of SARS-COV-2 was performed with the use of the SuperScriptTM III PlatinumTM One-

Step qRT-PCR Kit (Invitrogen) with primers/probes for E and RdRp genes amplification. qRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) and

consisted of a step of reverse transcription at 55°C for 10 min for reverse transcription, 95°C for 3 min and 45 cycles at 95°C for 15 s and 58°C for 30s. 20 Table 1 presents clinical data of the ten patients. The patients comprised 5 women and 5 men, with a median age of 69 (33-83) years. The median time span between the occurrence of symptoms and hospital admission was 4.5 days (1-10), and the median total duration of disease was 10 days (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . The most frequent associated clinical conditions were systemic arterial hypertension, diabetes mellitus and chronic cardiopathy. All patients presented with severe respiratory distress. Three patients died within 24 hours of hospitalisation, the other 7 were

This article is protected by copyright. All rights reserved admitted to the ICU under mechanical ventilation. All but one patient had a positive RT-PCR result on the naso/oropharyngeal swab and/or lung tissue. Table 2 shows the semiquantitative analysis of the main pulmonary histological changes. All ten patients presented a histological picture of exudative/proliferative Diffuse Alveolar Damage 

This work presents the pathological features observed in ten initial autopsies of fatal cases of COVID-19 in Brazil. It represents a response by local pathologists to deal with a pandemic disease with a large number of deaths, which has caused an unprecedented social and economic disruption and severe repercussions on health systems worldwide. When we started this study knowledge of the pathology of COVID-19 was scarce, with few reports of isolated cases. 6, 7, 8 The post-mortem examination became necessary to understand the pathophysiology of the disease and to contribute to therapeutic decisions. We adopted the MIA-US approach as we do not have in our institution an autopsy room with the appropriate biosafety recommendations. 14,15,16 MIA-US provides fast, precise responses to investigate the pathogenesis of new infectious agents and, thus, represents a valuable alternative to conventional autopsies in situations of high contagiousness. Also, it has great portability and considerably reduces autopsy costs, something increasingly important as the world faces financial distress; the risk of producing aerosols is low and, therefore, it can be performed in areas without negative pressure autopsy rooms; the images obtained by ultrasound are good enough to localise and orient the sampling in several organs, and to select the most affected areas within each organ; it provides information within a time window fast enough to orient the management of critically ill patients. 16 21 We have previously reported the pathology and lung immuneprofile of H1N1pdm cases, which presented a different histopathological picture and possibly another immuneprofile compared to COVID-19. In H1N1pdm lungs extensive necrosis and haemorrhage were present, which are much less prominent or even absent in COVID-19 cases. In addition, a cytotoxic type of inflammation predominated in H1N1pdm cases, with CD8+T cells, CD57 NK cells and granzyme B+ cells. 23 In COVID-19, we and others describe a paucity of CD8 and CD57 NK cells Accepted Article in the alveolar septa, indicating a status of CD8 T cell lymphocyte depletion also in the lungs, as seen in the lymphoid organs (spleen and lymph nodes) and peripheral blood. 13, 24 In our present series, distal bronchioles showed extensive areas of metaplastic epithelium with Previous reports of autopsy series of COVID-19 have also shown that exudative DAD is a major finding. 10,11,12,13 A mixed pattern of exudative/proliferative DAD, observed in most of our patients and also by others, indicates the progression of the lung injury, probably related to both the temporal evolution of the injury and the effects of mechanical ventilation. 7,10 Three of our patients presented a mixed pattern of DAD, even with a short period from symptoms onset to

This article is protected by copyright. All rights reserved death. Although there is a direct relationship between the duration of DAD and fibroproliferation, fibroproliferative changes may start early in the course of the disease.

Secondary bacterial pneumonia, a frequent finding in our series, has also been described in other reports. 7, 10 The main extrapulmonary findings are related to comorbidities, such as diabetes mellitus and hypertension, and also reflect changes associated with septic shock (splenitis, neutrophilic infiltration in the hepatic sinusoids and lymphoid hypoplasia). 2, 6, 33 However, it is likely that some changes in other organs can be attributed to viral disease -superficial perivascular dermatitis, myositis, orchitis, myocarditis, alterations in the renal glomeruli, endothelium of small vessels and cerebral cortex. Further analysis, with the help of immunohistochemistry and electron microscopy, is needed to define the role of SARS-COV-2 in their pathogenesis, as well as to validate results found by other authors. 6, 34, 35 We conclude that MIA-US is a safe, rapid and effective procedure to represent the main organs and tissues for the study of severe COVID-19. Our findings show that COVID-19 is a systemic disease with major events in the lungs and involvement of various organs and tissues such as kidneys, spleen, lymph nodes, brain, testicles and skin. Pulmonary changes in fatal cases are the result of severe epithelial injury and microthrombotic vascular phenomena, which cause ventilation-perfusion mismatch and hypoxemia, leading to respiratory failure. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches. 

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World Health Organization. Coronavirus disease 2019 (COVID-19) Situation Report -101

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Boletim Epidemiológico Especial-14 | SE 18 -26 de abril de

pii: ciaa415. Accepted Article This article is protected by copyright. All rights reserved 5

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

A pathological report of three COVID-19 cases by minimally invasive autopsies. Zhonghua Bing Li Xue Za Zhi

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

Endothelial cell infection and endotheliitis in COVID-19

Pulmonary Pathology of Early-Phase 2019 Novel Coronavirus (COVID-19) Pneumonia in Two Patients With Lung Cancer

The authors wish to thank Mrs. Kely Cristina Soares Bispo, Mrs. Angela B G dos Santos, Ms. 

ANDN: designed the research study, performed the tissue sampling, analysed the data and wrote the paper; RAAM: designed the research study, performed the tissue sampling and wrote the paper; LFFS: designed the research study and analysed the data; DMACM: analysed the data; EPO: performed the clinical data collection; JTF: performed the tissue sampling; JRRP: performed PCR analysis, MSGG: performed PCR analysis, APMS: performed PCR analysis; IRSO: designed the research study; TM: designed the research study, analysed the data and wrote the paper; PHNS: designed the research study, performed the tissue sampling, analysed the data and wrote the paper; MD: designed the research study, analysed the data and wrote the paper.