The authors reply

We have carefully read and considered the letter from Prof. Miller and Dr. Brealey, distinguished experts of electron microscopy (EM), and appreciate that they pointed out the limitations of our study.

Liver Histopathological Analysis of 24 Postmortem Findings of Patients With COVID-19 in China.

Although the pathologic investigation of liver injury was observed in a couple of cases in China, the detailed description of liver histopathologic and ultrastructural changes in a relatively larger series of liver tissues from COVID-19 patients is lacking. Samples from the liver were obtained from 24 COVID-19 cases from February 1 to April 1, 2020. Light microscopy showed that all liver sections had different degrees of liver injury manifested as swelling of the hepatocytes, hepatocellular necrosis, steatosis, lobular inflammation, portal inflammation, dilatation of sinusoids, and so on. SARS-CoV-2 induced liver injury might be independent of pre-existing Schistosoma infection or obstructive cholestasis. Patients combined with respiratory failure had more severe hepatocellular necrosis and male patients were more susceptible to liver injury. Although coronavirus particles or viral inclusions were not detected in the liver tissues for all cases, vacuolar degenerations in hepatocytes, edematous of mitochondria with the disruption of cristae, and expansions of the endoplasmic reticulum were observed. In conclusion, pathologic changes of liver tissues provide us a further understanding of liver injury in COVID-19 patients. Changes in the liver seem to be related to the underlying diseases/conditions.

Follow-up Study on the Outcomes of Recovered Pregnant Women with a History of COVID-19 in the First and Second Trimesters: A Case Series from China.

OBJECTIVE: To determine the pregnancy and neonatal outcomes of women who recovered from coronavirus disease 2019 (COVID-19) that developed in early pregnancy. METHODS: This case series analyzed five pregnant women (26-33 years) whom recovered from COVID-19 which were developed in early pregnancy (6-27 weeks) and admitted at the Wuhan Union Hospital from January 15, 2020 to April 30, 2020. The clinical manifestation, laboratory examinations, treatment, pregnancy outcomes, maternal and neonatal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throat swab reverse transcription polymerase chain reaction test results, and SARS-CoV-2 antibody test results in neonates were reviewed. The placental pathology, placental angiotensin-converting enzyme 2 expression were studied by hematoxylin-eosin and immunohistochemistry staining, SARS-CoV-2 presence was examined by QT-PCR. We also followed up the infants at 3-6 months. RESULTS: Three pregnant women were diagnosed with COVID-19 in early pregnancy (Cases 1-3), and two were serum immunoglobulin G positive asymptomatic cases (Cases 4 and 5). Cases 1-3 showed complete recovery after severe COVID-19. Case 3 was infected at 6 weeks of gestation during the first trimester and had induced medical abortion at 12 weeks of gestation. All neonates had no pneumonia, SARS-CoV-2 mRNA reverse transcription polymerase chain reaction and serum immunoglobulin M were negative, and immunoglobulin G were positive. All placental samples were negative for SARS-CoV-2 in the nucleic acid test. Placental pathology showed chronic ischemia changes. ACE-2 expressed in both placenta and decidua. The follow-up showed that the infants were healthy and asymptomatic at 3-6 months. CONCLUSION: No adverse outcomes was observed in our case series. However, systemic inflammatory responses to SARS-CoV-2 infection may cause placental injury. At the time of delivery after recovery from COVID-19, no SARS-CoV-2 positive results was found in the placenta in this case series.

Incremental prognostic value of biventricular longitudinal strain and high-sensitivity troponin I in COVID-19 patients.

BACKGROUND: Whether the combination of ventricular strain with high-sensitivity troponin I (hs-TNI) has an incremental prognostic value in coronavirus disease 2019 (COVID-19) patients has not been evaluated. The study aimed to evaluate the prognostic value of biventricular longitudinal strain and its combination with hs-TNI in COVID-19 patients. METHODS: A total of 160 COVID-19 patients who underwent both echocardiography and hs-TNI testing were enrolled in our study. COVID-19 patients were divided into two groups (critical and non-critical) according to severity-of-illness. The clinical characteristics, cardiac structure and function were compared between the two groups. The prognostic value of biventricular longitudinal strain and its combination with hs-TNI were evaluated by logistic regression analyses and receiver operating characteristic curves. Left ventricular longitudinal strain (LV LS) and right ventricular free wall longitudinal strain (RVFWLS) were determined by 2D speckle-tracking echocardiography. RESULTS: The LV LS and RVFWLS both were significantly lower in critical patients than non-critical patients (LV LS: -16.6±2.4 vs -17.9±3.0, P = .003; RVFWLS :-18.8±3.6 vs -23.9±4.4, P<.001). During a median follow-up of 60 days, 23 (14.4%) patients died. The multivariant analysis revealed that LV LS and RVFWLS [Odd ratio (95% confidence interval): 1.533 (1.131-2.079), P = .006; 1.267 (1.036-1.551), P = .021, respectively] were the independent predictors of higher mortality. Further, receiver-operating characteristic analysis revealed that the accuracy for predicting death was greater for the combination of hs-TNI levels with LV LS than separate LV LS (AUC: .91 vs .77, P = .001), and the combination of hs-TNI levels with RVFWLS than RVFWLS alone (AUC: .89 vs .83, P = .041). CONCLUSIONS: Our study highlights that the combination of ventricular longitudinal strain with hs-TNI can provide higher accuracy for predicting mortality in COVID-19 patients, which may enhance risk stratification in COVID-19 patients.

Multi-organ proteomic landscape of COVID-19 autopsies.

The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.

Progression to fibrosing diffuse alveolar damage in a series of 30 minimally invasive autopsies with COVID-19 pneumonia in Wuhan, China.

AIMS: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), infection has been deemed as a global pandemic by the World Health Organisation. While diffuse alveolar damage (DAD) is recognised to be the primary manifestation of COVID-19 pneumonia, there has been little emphasis on the progression to the fibrosing phase of DAD. This topic is of great interest, due to growing concerns regarding the potential long-term complications in prolonged survivors. METHODS AND RESULTS: Here we report a detailed histopathological study of 30 autopsy cases with COVID-19 virus infection, based on minimally invasive autopsies performed between February and March, 2020. The mean age was 69 years, with 20 (67%) males and 10 (33%) females and frequent (70.0%) underlying comorbidities. The duration of illness ranged from 16 to 82 (median = 42) days. Histologically, the most common manifestation was diffuse alveolar damage (DAD) in 28 (93.3%) cases which showed predominantly acute (32%), organising (25%) and/or fibrosing (43%) patterns. Patients with fibrosing DAD were one decade younger (P = 0.034) and they had a longer duration of illness (P = 0.033), hospitalisation (P = 0.037) and mechanical ventilation (P = 0.014) compared to those with acute DAD. Patients with organising DAD had a longer duration of illness (P = 0.032) and hospitalisation (P = 0.023) compared to those with acute DAD. CONCLUSIONS: COVID-19 pneumonia patients who develop DAD can progress to the fibrosing pattern. While we observed fibrosing DAD in fatal cases, whether or not surviving patients are at risk for developing pulmonary fibrosis and the frequency of this complication will require further clinical and radiological follow-up studies.

Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients.

BACKGROUND: The novel coronavirus pneumonia COVID-19 caused by SARS-CoV-2 infection could lead to a series of clinical symptoms and severe illnesses, including acute respiratory distress syndrome (ARDS) and fatal organ failure. We report the fundamental pathological investigation in the lungs and other organs of fatal cases for the mechanistic understanding of severe COVID-19 and the development of specific therapy in these cases. METHODS: The autopsy and pathological investigations of specimens were performed on bodies of two deceased cases with COVID-19. Gross anatomy and histological investigation by Hematoxylin and eosin (HE) stained were reviewed on each patient. Alcian blue/periodic acid-Schiff (AB-PAS) staining and Masson staining were performed for the examinations of mucus, fibrin and collagen fiber in lung tissues. Immunohistochemical staining was performed on the slides of lung tissues from two patients. Real-time PCR was performed to detect the infection of SARS-CoV-2. Flow cytometry analyses were performed to detect the direct binding of S protein and the expression of ACE2 on the cell surface of macrophages. FINDINGS: The main pathological features in lungs included extensive impairment of type I alveolar epithelial cells and atypical hyperplasia of type II alveolar cells, with formation of hyaline membrane, focal hemorrhage, exudation and pulmonary edema, and pulmonary consolidation. The mucous plug with fibrinous exudate in the alveoli and the dysfunction of alveolar macrophages were characteristic abnormalities. The type II alveolar epithelial cells and macrophages in alveoli and pulmonary hilum lymphoid tissue were infected by SARS-CoV-2. S protein of SARS-CoV-2 directly bound to the macrophage via the S-protein-ACE2 interaction. INTERPRETATION: Infection of alveolar macrophage by SARS-CoV-2 might be drivers of the "cytokine storm", which might result in damages in pulmonary tissues, heart and lung, and lead to the failure of multiple organs . FUNDING: Shanghai Guangci Translational Medical Research Development Foundation, Shanghai, China.

Pathological Findings in the Testes of COVID-19 Patients: Clinical Implications.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involves multiple organs. Testicular involvement is largely unknown. OBJECTIVE: To determine the pathological changes and whether SARS-CoV-2 can be detected in the testes of deceased COVID-19 patients. DESIGN, SETTING, AND PARTICIPANTS: Postmortem examination of the testes from 12 COVID-19 patients was performed using light and electron microscopy, and immunohistochemistry for lymphocytic and histiocytic markers. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the virus in testicular tissue. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Seminiferous tubular injury was assessed as none, mild, moderate, or severe according to the extent of tubular damage. Leydig cells in the interstitium were counted in ten 400× microscopy fields. RESULTS AND LIMITATIONS: Microscopically, Sertoli cells showed swelling, vacuolation and cytoplasmic rarefaction, detachment from tubular basement membranes, and loss and sloughing into lumens of the intratubular cell mass. Two, five, and four of 11 cases showed mild, moderate, and severe injury, respectively. The mean number of Leydig cells in COVID-19 testes was significantly lower than in the control group (2.2 vs 7.8, p < 0.001). In the interstitium there was edema and mild inflammatory infiltrates composed of T lymphocytes and histiocytes. Transmission EM did not identify viral particles in three cases. RT-PCR detected the virus in one of 12 cases. CONCLUSIONS: Testes from COVID-19 patients exhibited significant seminiferous tubular injury, reduced Leydig cells, and mild lymphocytic inflammation. We found no evidence of SARS-CoV-2 virus in the testes in the majority (90%) of the cases by RT-PCR, and in none by electron microscopy. These findings can provide evidence-based guidance for sperm donation and inform management strategies to mitigate the risk of testicular injury during the COVID-19 disease course. PATIENT SUMMARY: We examined the testes of deceased COVID-19 patients. We found significant damage to the testicular parenchyma. However, virus was not detected in testes in the majority of cases.

Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China.

Although the respiratory and immune systems are the major targets of Coronavirus Disease 2019 (COVID-19), acute kidney injury and proteinuria have also been observed. Currently, detailed pathologic examination of kidney damage in critically ill patients with COVID-19 has been lacking. To help define this we analyzed kidney abnormalities in 26 autopsies of patients with COVID-19 by light microscopy, ultrastructural observation and immunostaining. Patients were on average 69 years (19 male and 7 female) with respiratory failure associated with multiple organ dysfunction syndrome as the cause of death. Nine of the 26 showed clinical signs of kidney injury that included increased serum creatinine and/or new-onset proteinuria. By light microscopy, diffuse proximal tubule injury with the loss of brush border, non-isometric vacuolar degeneration, and even frank necrosis was observed. Occasional hemosiderin granules and pigmented casts were identified. There were prominent erythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material. Evidence of vasculitis, interstitial inflammation or hemorrhage was absent. Electron microscopic examination showed clusters of coronavirus-like particles with distinctive spikes in the tubular epithelium and podocytes. Furthermore, the receptor of SARS-CoV-2, ACE2 was found to be upregulated in patients with COVID-19, and immunostaining with SARS-CoV nucleoprotein antibody was positive in tubules. In addition to the direct virulence of SARS-CoV-2, factors contributing to acute kidney injury included systemic hypoxia, abnormal coagulation, and possible drug or hyperventilation-relevant rhabdomyolysis. Thus, our studies provide direct evidence of the invasion of SARSCoV-2 into kidney tissue. These findings will greatly add to the current understanding of SARS-CoV-2 infection.

Renal histopathological analysis of 26 postmortem fi ndings of patients with COVID-19 in China

Although the respiratory and immune systems are the major targets of Coronavirus Disease 2019 (COVID-19), acute kidney injury and proteinuria have also been observed. Currently, detailed pathologic examination of kidney damage in critically ill patients with COVID-19 has been lacking. To help de fine this we analyzed kidney abnormalities in 26 autopsies of patients with COVID-19 by light microscopy, ultrastructural observation and immunostaining. Patients were on average 69 years (19 male and 7 female) with respiratory failure associated with multiple organ dysfunction syndrome as the cause of death. Nine of the 26 showed clinical signs of kidney injury that included increased serum creatinine and/or new -onset proteinuria. By light microscopy, diffuse proximal tubule injury with the loss of brush border, non -isometric vacuolar degeneration, and even frank necrosis was observed. Occasional hemosiderin granules and pigmented casts were identi fied. There were prominent erythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material. Evidence of vasculitis, interstitial in flammation or hemorrhage was absent. Electron microscopic examination showed clusters of coronavirus-like particles with distinctive spikes in the tubular epithelium and podocytes. Furthermore, the receptor of SARS-CoV-2, ACE2 was found to be upregulated in patients with COVID-19, and immunostaining with SARS-CoV nucleoprotein antibody was positive in tubules. In addition to the direct virulence of SARS-CoV-2, factors contributing to acute kidney injury included systemic hypoxia, abnormal coagulation, and possible drug or hyperventilation -relevant rhabdomyolysis. Thus, our studies provide direct evidence of the invasion of SARSCoV-2 into kidney tissue. These findings will greatly add to the current understanding of SARS-CoV-2 infection.