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Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host-pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period., (© 2022. The Author(s).)

From March through December 2020, 100 autopsies were performed (Semmelweis University, Budapest, Hungary), with chart review, of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection demonstrated by real-time reverse-transcription polymerase chain reaction testing (mean age, 74.73 years, range 40-102 years; 50 males, mean age 71.96 years, and 50 females, mean age 77.5 years). Classified by the date of death, 21 cases were from the pandemic's "first wave" (March through July) and 79 from the "second wave" (August through December). Three mortality categories were defined by relevance of SARS-CoV-2 infection: (1) "strong" association (n=57), in which COVID-19 was primary responsible for death; (2) "contributive" association (n=27), in which a pre-existing condition independent of COVID-19 was primary responsible for death, albeit with substantial COVID-19 co-morbidity; (3) "weak" association (n=16), in which COVID-19 was minimally or not at all responsible for death. Distributions among categories differed between the first wave, in which the "contributive" association cases dominated (strong: 24%, contributive: 48%, weak: 28%), and the second wave, in which the "strong" association cases dominated (strong: 66%, contributive: 21%, weak: 13%). Charted co-morbidities included hypertension (85 %), cardiovascular diseases (71 %), diabetes (40 %), cerebrovascular diseases (31 %), chronic respiratory diseases (30 %), malignant tumors (20 %), renal diseases (19 %), diseases of the central nervous system (15 %), and liver diseases (6 %). Autopsy evaluation analyzed alterations on macroscopy as well as findings on microscopy of scanned and scored sections of formalin-fixed, paraffin-embedded tissue samples (50-80 blocks/case). Severity of histological abnormalities in the lung differed significantly between "strong" and "contributive" (p<0.0001) and between "strong" and "weak" categories (p<0.0001). Abnormalities included diffuse alveolar damage, macrophage infiltration, and vascular and alveolar fibrin aggregates (lung), with macro- and microvascular thrombi and thromboemboli (lung, kidney, liver). In conclusion, autopsies clarified in what extent COVID-19 was responsible for death, demonstrated the pathological background of clinical signs and symptoms, and identified organ alterations that led to the death. Clinicopathologic correlation, with conference discussions of severity of co-morbidities and of direct pathological signs of disease, permitted accurate categorization of cause of death and COVID-19 association as "strong," "contributive," or "weak." Lung involvement, with reduced ventilatory capacity, was the primary cause of death in the "strong" and "contributive" categories. Shifts in distribution among categories, with "strong" association between COVID-19 and death dominating in the second wave, may reflect improved clinical management of COVID-19 as expertise grew., (© 2021. The Author(s).)