Post-mortem Histopathologic Findings of Vital Organs in Critically Ill Patients with COVID-19

Background: The scientific evidence concerning pathogenesis and immunopathology of the coronavirus disease 2019 (COVID-19) is rapidly evolving in the literature. To evaluate the different tissues obtained by biopsy and autopsy from five patients who expired from severe COVID-19 in our medical center. Methods: This retrospective study reviewed five patients with severe COVID-19, confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and imaging, to determine the potential correlations between histologic findings with patient outcome. Results: Diffuse alveolar damage (DAD) and micro-thrombosis were the most common histologic finding in the lung tissues (4 of 5 cases), and immunohistochemical (IHC) findings (3 of 4 cases) suggested perivascular aggregation and diffuse infiltration of alveolar walls by CD4+ and CD8+ T lymphocytes. Two of five cases had mild predominantly perivascular lymphocytic infiltration, single cell myocardial necrosis and variable interstitial edema in myocardial samples. Hypertrophic cardiac myocytes, representing hypertensive cardiomyopathy was seen in one patient and CD4+ and CD8+ T lymphocytes were detected on IHC in two cases. In renal samples, acute tubular necrosis was observed in 3 of 5 cases, while chronic tubulointerstitial nephritis, crescent formation and small vessel fibrin thrombi were observed in 1 of 5 samples. Sinusoidal dilation, mild to moderate chronic portal inflammation and mild mixed macro- and micro-vesicular steatosis were detected in all liver samples. Conclusion: Our observations suggest that clinical pathology findings on autopsy tissue samples could shed more light on the pathogenesis, and consequently the management, of patients with severe COVID-19.


Introduction
As of May 20, 2020, the coronavirus disease 2019 (COVID-19), a pandemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has accounted for 318 789 deaths from 4 789 205 confirmed cases globally. 1 Although the majority of COVID-19 patients develop only mild to moderate symptoms, progressive pneumonia and subsequent acute respiratory distress syndrome (ARDS) and multi-organ failure occur in a significant number of patients. 2 Evidence concerning the pathogenesis and other aspects of COVID-19 immunopathology was limited at the onset of the outbreak. Gradually, some preliminary studies were able to identify new characteristics of the disease in seriously ill hospitalized patients. However, similarities between COVID-19 and severe acute respiratory syndrome (SARS), in terms of pathological, clinical, and pathogenic characteristics, have aided us to gain a better understanding of the pathogenesis of this disease. 3 The SARS-CoV-2 infection can trigger both the innate and the adaptive immune pathways. Nevertheless, uninhibited innate reactions along with a disrupted adaptive immune response can ultimately trigger devastating tissue damage. In patients with severe COVID-19, the expression of the CD94/NK group 2 member A (NKG2A), an exhaustion marker on NK cells, and CD8 + T cells are increased and their normalization is associated with improved CD4 + T cells, CD8 + T cells, B cells and NK cells as well as fatigue markers, during the recovery and improvement phase. 2 In addition to elevated levels of pro-inflammatory cytokines, plasma concentrations of interleukin (IL)-2, IL-7, IL-10, granulocyte-colony stimulating factor, interferon gamma-induced protein 10, monocyte chemo-attractant protein-1, macrophage inflammatory protein 1-a and tumor necrosis factor-alpha in critically ill COVID-19 patients display greater levels compared to those with mild to moderate disease. 4 Also, it has been established that there is a direct relationship between high levels of pro-inflammatory cytokines and end-organ damage and failure of the heart, liver, kidneys and lungs. 2 Recently, a few studies on histopathologic aspects of fatal COVID-19 have been published in the literature. [5][6][7] In addition to the pathological findings compatible with ARDS, other investigators have reported some histological features which tend to be different from the classic cases of ARDS. 5 In addition, endothelial disruption observed in tissues such as the kidneys, intestines and the scalp, indicate the impact of virus-induced vascular impairment during disease progression. 6 Despite the recent efforts in understanding the pathogenesis of the COVID-19, we are still at the early stages of elucidating the various aspects of this disease and studies carried out so far suggest significant clinical and histopathologic variability exhibited by COVID-19. 7 Therefore, there is an urgent need to acquire precise and robust data to explain the immunopathogenesis of the COVID-19 and its impact on the human body.
In this investigation, we aimed to examine a variety of organs, including the lung, kidney, heart and liver, obtained from several fatal cases of COVID-19, in order to have a more clear understanding of the pathogenesis of this disease.

Materials and Methods
Five patients who died of severe COVID-19 in the intensive care unit (ICU) were selected by an infectious disease specialist (IAD) and critical care specialist (MA) for the study. The patients had diffuse pulmonary infiltrates compatible with COVID-19 pneumonitis on their computed tomography (CT) scans, oxygen saturation (SpO 2 ) <93% in ambient air and/or respiratory rate >24/ minute with confirmation of their disease by reverse transcription-polymerase chain reaction (RT-PCR).
All necropsies were performed immediately after the declaration of death by one forensic medicine specialist (BM). For optimal targeting of the organs, tissue sample were acquired under ultrasound-guidance (MyLab, Esaote, Italy) by a radiologist (HBM) with more than 8 years of experience in ultrasound-guided intervention, utilizing an automatic 14-gauge core biopsy needle (Acecut, TSK Laboratory, Japan). All postmortem core needle biopsy samples were preserved in 10% neutral buffered formalin and fixed for 72 hours before processing based on the Centers for Disease Control and Prevention recommendations available at: (https:// www.cdc.gov/ncezid/dhcpp/idpb/specimen-submission/ general-unexplained.html) All hematoxylin and eosin (H&E), cluster differentiation CD3, CD20, CD4 and CD8 stained biopsy sections and cytomegalovirus (CMV) stained lung and liver biopsy slides, related to cases number 4 and 5, respectively, were examined separately by two pathologists (FBZ, SS). This study was undertaken at Loghman Hakim hospital, a major affiliate of the Shahid Beheshti University of Medical Sciences in Tehran, Iran between April 2, 2020 and May 10, 2020. Informed consent for the procedure was obtained from the legal guardians of all deceased patients.

Case Presentations
Case 1: A 78-year-old woman with a history of hypertension and hypothyroidism presented to the emergency department (ED) with increasing shortness of breath and respiratory distress. The patient was recently hospitalized with a SARS-CoV-2 RT-PCR positive COVID-19 diagnosis and had received lopinavir/ritonavir. On admission, arterial oxygen saturation on ambient air was 80% and chest CT findings showed bilateral pleural effusion (more so on the right side), right-sided pneumothorax, and diffused patchy ground-glass opacities. A right-sided chest tube was placed, and amikacin and meropenem were started in the ED. She initially responded to supportive care and oxygen therapy but had to be intubated and mechanically ventilated on day 4 of her admission. She developed organ hypoperfusion and severe hypoxia which was unresponsive to the escalation of mechanical ventilation support and vasoactive agents and expired on the same day.
Case 2: A 48-year-old man presented with dyspnea, purulent cough, fever, and chills for the last 7 days. On admission to the ED, he was in respiratory distress, tachycardic, hypotensive, afebrile with SpO 2 of 70% on ambient air. Chest CT findings showed bilateral diffuse patchy ground-glass opacities. Bi-level positive airway pressure (BIPAP) was initiated for the patient. Oxygen saturation increased to 79% after one hour on BIPAP and then he was intubated immediately. Remdesivir, hydroxychloroquine, ceftriaxone, and azithromycin were started in the ED. On days 5 and 7, the patient received hemoperfusion. On day 8, the patient was intubated and tocilizumab was started on day 9. On day 10, umifenovir and atazanavir were added to his treatment regimen. The patient continued to deteriorate and expired on day 11.
Case 3: A 75-year-old woman with a history of hypertension, ischemic heart disease, cerebrovascular accident, diabetes mellitus, and dyslipidemia presented with fatigue for 3 days before presentation to the ED followed by dyspnea, aphasia and diminished level of consciousness. On admission to the ED, her respiratory rate was 18 per minute, temperature was 36°C, heart rate was 80 per minute and SpO 2 was 50% on ambient air. The CT of the chest and non-contrast head findings were diffused patchy ground-glass opacities and periventricular hypodensities, respectively. Lopinavir/ritonavir, interferon beta-1a and hydroxychloroquine were started in the ED along with supportive care and stepwise oxygen therapy. The patient expired 12 hours after admission.
Case 4: A 71-year-old man presented with dyspnea and fever for the last 14 days, and worsening of symptoms 4 days prior to presentation to the ED. Dyspnea was his first symptom which was followed by fever a week later. He had been admitted to another hospital with confirmed COVID-19 diagnosis on RT-PCR and had received hydroxychloroquine 200 mg twice daily for 72 hours before admission. On admission to the ED, his heart rate was 98 beats per minute, respiratory rate was 18 per minute, the temperature was 38°C, and SpO 2 was 70%. CT scan of the chest illustrated bilateral diffuse patchy groundglass opacities. Lopinavir/ritonavir, interferon beta-1a and one dose of hydroxychloroquine were administered on admission. On day 3 of his ICU admission, vitamin C 1 gram four times daily was started and non-invasive ventilation was initiated for persistent hypoxia. On day 4, the patient had to be intubated and mechanically ventilated with lung protective strategy and FiO 2 of 100%. The patient continued to deteriorate despite critical care support and administration of tocilizumab and expired on day 7 of his admission.
Case 5: A 47-year-old woman presented to the ED with dyspnea, fever, cough, headache, and dizziness. She had previously been hospitalized with an RT-PCR confirmed diagnosis of COVID-19. On admission to the ED, her temperature was 39.5°C and SpO 2 on ambient air was 70%. Lopinavir/ritonavir, hydroxychloroquine and azithromycin were started on admission day. On day 2, ribavirin was added to the treatment regimen. On day 5, azithromycin was discontinued and interferon beta-1a was started. On day 8, she was mechanically ventilated with a lung-protective strategy. On day 10, lopinavir/ritonavir was omitted and teicoplanin and atazanavir were started. Chest CT on day 13 revealed worsening bilateral diffused patchy ground-glass opacities. She continued to deteriorate till day 22 when she received 3 sessions of hemoperfusion to no avail and died on the same day.
Detailed clinical and laboratory findings of all patients are shown in Table 1.

Histologic Findings Lung
All samples of lung tissue revealed different stages of diffuse alveolar damage (DAD) in addition to microvascular changes and micro-hemorrhage, detailed below: Case 1: DAD accompanied by acute exudative pneumonia, likely representing bacterial superinfection.
Case 2: Sample, was not adequate for histopathologic evaluation.
Case 4: Severe DAD, with the same features as specimen 5, as well as atypical pneumocytes with large vesicular nuclei, prominent nucleoli and vacuolated cytoplasm (resembling CMV cytopathic effect), focal squamous metaplasia and early fibrosis associated with focal Masson body formation, the latter two adding a fibrotic component to the DAD.
Case 5: Severe mixed exudative-proliferative DAD with severe inflammation, predominantly lymphocytic, admixed with few neutrophils and histiocytes, prominent hyaline membrane formation, fibrin deposition and hemorrhage in alveolar walls and spaces, frequent microthrombi in alveolar capillaries, thickening of small vessel walls, pneumocyte desquamation and diffuse hyperplasia of pneumocyte type 2 ( Table 2).

Immunohistochemical (IHC) Findings
Cases 3, 4, and 5: Perivascular aggregation and diffuse infiltration of alveolar walls by CD4+ and CD8+ T lymphocytes and smaller amount of CD20+ B lymphocytes. Case 4 was CMV negative and multinucleated giant cells were stained with TTF1 as it was expected in pneumocytes ( Figure 1).

Heart
Cases 1 and 4: Predominantly perivascular mild lymphocytic infiltration, single cell myocardial necrosis and variable interstitial edema.

Kidney
Case 1: Chronic tubulointerstitial nephritis with tubular atrophy (thyroidization), moderate interstitial lymphocytic infiltration and interstitial fibrosis; also, glomerular obsolescence involving nearly 20% of examined glomeruli. Case 2: Normal renal histology. Case 3: Acute tubular necrosis superimposed on chronic changes, including focal tubular atrophy, medullary interstitial fibrosis, and focal glomerular obsolescence Case 4: Acute tubular necrosis and crescent formation in 2 out of 11 glomeruli.
Case 5: Acute tubular necrosis and small vessel fibrin thrombi, mostly in the renal medulla as well as frequent glomerular capillary thrombosis (Figure 3).

Liver
All cases showed sinusoidal dilation, predominantly portal mild to moderate chronic inflammation, sinusoidal congestion and mild mixed macro-, micro-vesicular

COVID-19 Necropsy
steatosis. The additional findings shared by cases 1, 2, and 3 are detailed as follows: Case 1: Interface hepatitis and portal-portal bridging fibrosis, consistent with chronic hepatitis of grade 3/18, and stage 3/6 based on Ishak classification; these were mainly chronic findings.
Case 2: Lobular multifocal neutrophilic micro-abscess formation like CMV hepatitis. No viral cytopathic effect was identified.
Case 3: Centrilobular necrosis, which may be due to cardiac failure or viral hepatotoxicity (Figure 4).

Discussion
This study is a clinicopathologic correlation of five fatal cases of COVID-19 cared for at Loghman Hakim hospital, Tehran, Iran, based on postmortem examination from 4 organs (lung, heart, kidney, and liver). As we do not have any safe autopsy suites to handle highly infective cadavers, we decided to obtain a tissue sample by the ultrasoundguided core needle biopsies rather than a formal autopsy.
The observations in our study contribute further to what we have learned about the pathogenesis of COVID-19 which can improve our clinical approach to the care of these patients. In our opinion, the pathological findings of COVID-19 in our study resemble those of influenza (H1N1), SARS-CoV, and MERS-CoV. 2,3 As indicated in prior studies, the gross and microscopic characteristics of COVID-19, which initiate mainly in the lungs, lead to pulmonary edema, pulmonary consolidation, increased lung weight, pericarditis and pleuritis. Secondary and co-infections may be superimposed on the viral   infection, contributing to purulent inflammation and secretion, which is a characteristic feature of bacterial and/ or fungal infections. 4,5 The remarkable histological findings of lung biopsies were coexistence of all DAD phases, including exudative, proliferative, and fibrotic, suggesting ongoing cycles of lung damage, likely arising from persistent viral replication in the lung tissue and subsequent T cell over-activation and cytokine storms. The presence of micro-thrombi, seen in COVID-19, in the pulmonary microvasculature should be highlighted, which might have resulted from a systemic hypercoagulable state besides being a component of DAD. 6 However, not all DADs lead to a hypercoagulable state.
Furthermore, acute pneumonia which was detected in one of our cases may be presumably representative of superimposed bacterial infection on an underlying SARS-CoV-2 infection.
It should be kept in mind that SARS and COVID-19 infections exhibit common pathogenetic, clinical and pathological findings. Clinically, cough and fever have been the most prominent manifestations of both diseases, while myalgia and diarrhea have been less frequent in COVID-19 cases. Clinically, ARDS has been the most serious pulmonary adverse event that has caused high mortality rates. Histologically, the most common pathology of the non-survivors of either SARS or COVID-19 has been DAD. In previous studies, it was indicated that SARS patients who died with a disease duration under 10-14 days demonstrated acute DAD, while cases with a disease duration surpassing 10-14 days showed the proliferative phase of DAD . 7 Data is still emerging regarding the routine histopathology of major organs in the autopsies of patients with severe COVID-19. Pathological findings are primarily focused on lung injury and DAD as the cause of death, but there are significant changes in other organs, such as the heart, liver, and kidneys, which require more extensive investigation. 8 In two of five myocardial samples, perivascular infiltration of T lymphocytes might have played a role in myocardial injury by disrupting microvascular perfusion and/or T lymphocyte cytotoxic effects. Other studies have demonstrated that a small but noticeable percentage of hospitalized patients develop Acute COVID-19 Cardiovascular Syndrome which can occur in the form of several clinical manifestations, such as cardiomyopathy, acute congestive heart failure, ventricular arrhythmias and hemodynamic instability in the absence of significant obstructive coronary artery disease.
The etiological reasons for these defects remain unknown; however, they are believed to be consistent with cardiomyopathy associated with stress, systemic cytokine-mediated microvascular injuries and myocarditis. Although histologically unproven, direct cytopathic effects of SARS-CoV-2 inside cardiomyocytes and pericytes may cause viral myocarditis.
The presence of platelet/fibrin microthrombi in alveolar small vessels, as we noticed in our study and in several newly published articles, has created ambiguity about the significance of this finding, whether this is part of what usually happens in the context of DAD or a systemic coagulopathy dominates in COVID-19 infected patients. 9 This probable coagulopathic pathogenesis became stronger when we found fibrin thrombi in renal small vessels and some glomeruli. Although definite vasculitis was not observed in our examination, it did raise suspicions about viral induced endotheliopathy as mentioned by Varga et al. 10 Prior experience with SARS has helped to investigate possible therapeutic options such as convalescent serum, interleukin-6 inhibitors, and anti-viral drugs for the management of COVID-19. 11 The central perivenular necrosis and neutrophilic lobular micro-abscess formation, along with sinusoidal lymphocytosis which were identified in the liver biopsy samples, resemble the liver pathology findings in hepatitis provoked by influenza H1N1, CMV or EBV or druginduced hepatitis. The related micro-vesicular steatosis, arising from acquired cellular oxidative defects, can also be a detrimental prognostic feature. 4,12 Accordingly, other related studies have observed micro-vesicular steatosis with moderate inflammation within the liver; it has been deemed uncertain if this is due to the virus itself or caused by iatrogenic factors. These characteristics are remarkably close to those seen in SARS and MERScoronavirus infection. The crescent seen on the renal biopsy samples of our patient may have been the upshot of viral infection or accompanying cytokine storm. The majority of renal biopsies in our study displayed signs of acute tubular injury with necrosis and flattening of tubular epithelium, multiple tubular casts and interstitial edema in some patients superimposed on chronic changes such as interstitial nephritis and tubular atrophy (thyroidization). These findings are similar to other studies and in favor of susceptibility of patients with chronic kidney disease to severe COVID19 infections. 13 However, ischemic ATN may have been the result of systemic coagulopathy or fatal systemic hypotension, compounded by the chronic pathological findings of chronic tubulointerstitial nephritis, tubular atrophy, and glomerular obsolescence which might have contributed to the severe renal injury and patient demise. 1,14 In conclusion, regarding the histopathological autopsy findings of fatal COVID-19 patients in our small case series, the lung samples indicated that the three DAD phases (exudative, proliferative and fibrotic) can be present simultaneously, delineating ongoing cycles of lung damage, which could be explained by tenacious viral replication and T cells over-activation, along with cytokine storms. Also, superimposed bacterial infection could be a possibility, according to one incidence of acute pneumonia in our study. Additionally, the perivascular infiltration of T lymphocytes which we observed in the myocardial tissue showed T lymphocyte cytotoxic effects and disruption of microvascular perfusion which could be a significant underlying cause of myocardial failure. Moreover, findings from other organs like the liver and kidneys were suggestive of acquired cellular oxidative defects, necrosis, micro-abscess formation, tissue atrophy, and coagulopathy. Our pathological findings which are associated with poor prognosis highlight the importance of prognostic significance drawn from the histopathology in patients with COVID-19.

Authors' Contribution
FBZ and SS making pathologic photos and drafted the manuscript. HBM, MA and MH have participated in sampling. IAD Participated