Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality (preprint)

The precise molecular mechanisms behind severe life-threatening lung abnormalities during severe SARS-CoV-2 infections are still unclear. To address this challenge, we performed whole transcriptome sequencing of lung autopsies from 31 patients suffering from severe COVID-19 related complications and 10 uninfected controls. Using a metatranscriptome analysis of lung tissue samples we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant "classical" signature (n=23) showed upregulation of unfolded protein response, steroid biosynthesis and complement activation supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) potentially representing "Cytokine Release Syndrome" (CRS) showed upregulation of IL1 cytokines such CCL19 but absence of complement activation and muted inflammation. Further, dissecting expression of individual genes within enriched pathways for patient signature suggests heterogeneity in host response to the primary infection. We found that the majority of patients cleared the SARS-CoV-2 infection, but all suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Gordonia bronchialis in "classical" patients and Staphylococcus warneri in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients that can be identified through the status of the complement activation, presence of specific cytokines and characteristic microbiome. This information can be used to design personalized therapy to treat COVID-19 related complications corresponding to patient signature such as using the identified drug molecules or mitigating specific secondary infections.

ACE2 Expression in Lungs of Severe COVID-19 Infection: A Study on Minimally Invasive Post- mortem Tissue Samples (preprint)

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COVID-19 (COrona VIrus Disease 2019). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-9 patients for other indications. IHC for CD61 and CD163 were performed for assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. Quantification of immunostaining, random sampling, and correlation analysis was used to substantiate the morphologic findings. Our results show that among a total of 44 COVID-19 post-mortem lung tissues and 15 lung biopsies in non-COVID-19 patients included, ACE2 protein expression was significantly higher in COVID-19 patients than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

Clinico-pathological features in fatal Covid-19 Infection: A Preliminary Experience of a Tertiary Care Centre in North India using Post-Mortem Minimally Invasive Tissue Biopsies (preprint)

Background: The Covid-19 pandemic began in China in December 2019. India is the second most affected country, as of November 2020 with more than a 8.5million cases. Covid-19 infection primarily involves the lung with the severity of illness varying from influenza-like illness to acute respiratory distress syndrome. Other organs have also found to be variably affected. Studies evaluating the histopathological changes of Covid-19 are critical in providing a better understanding of the disease pathophysiology and guiding treatment. Minimally invasive biopsy techniques (MITS/B) provide an easy and suitable alternative to complete autopsies. In this prospective single-center study we present the histopathological examination of 37 patients who died with complications of Covid-19. Methods: This was an observational study conducted in the Intensive Care Unit of JPN Trauma Centre AIIMS. A total of 37 patients who died of Covid-19 were enrolled in the study. Post-mortem percutaneous biopsies were taken with the help of surface landmarking/ultrasonography guidance from lung, heart, liver, and kidneys; after obtaining ethical consent. The biopsy samples were then stained with haematoxylin and eosin stain. Immunohistochemistry (IHC) was performed using CD61 and CD163 in all lung cores. SARS-CoV-2 virus was detected using IHC with primary antibodies in selected samples. Details regarding demographics, clinical parameters, hospital course, treatment details, and laboratory investigations were also collected for clinical correlation. Results: A total of 37 patients underwent post-mortem minimally invasive tissue sampling. Mean age of the patients was 48.7years and 59.5% of them were males. Respiratory failure was the most common complication seen in 97.3%. Lung histopathology showed acute lung injury and diffuse alveolar damage in 78% of patients. Associated bronchopneumonia was seen in 37.5% of patients and scattered microthrombi were visualized in 21% of patients. Immunostaining with CD61 and CD163 highlighted megakaryocytes and increased macrophages in all samples. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of the renal biopsies but none of them showed evidence of microvascular thrombosis. 71% of the liver tissue cores showed evidence of Kupfer cell hyperplasia. 27.5% had evidence of submassive hepatic necrosis and 14% had features of acute on chronic liver failure. All the heart biopsies showed non-specific features such as hypertrophy with nucleomegaly with no evidence of myocardial necrosis in any of the samples. Conclusions The most common finding in this cohort is the diffuse alveolar damage with demonstration of SARS-CoV-2 protein in the acute phase of DAD. Microvascular thrombi were rarely identified in the lung, liver and kidney. Substantial hepatocyte necrosis, hepatocyte degeneration, Kupffer cell hypertrophy, micro, and macrovesicular steatosis unrelated to microvascular thrombi suggests that liver might be a primary target of Covid-19. This study highlights the importance of MITS/B in better understanding the pathological changes associated with Covid-19.