Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies

Abstract

Introduction: We describe post-mortem pulmonary histopathologic findings of COVID-19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation.

Methods and results: Histopathologic findings in post-mortem lung tissue from eight patients who died from COVID-19 pneumonia were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS-CoV-2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium- and large-calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi.

Conclusions: Presence of SARS-CoV-2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID-19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.

Keywords: COVID-19; SARS-CoV-2; diffuse alveolar damage; immunohistochemistry; lung histopathology; next-generation sequencing; thrombi; viral pneumonia.

Figure 1

Spectrum of diffuse alveolar damage (DAD) seen in COVID‐19 pneumonia. A, All cases demonstrated DAD, seven of eight of which showed a component of acute phase DAD. Note the demarcation between affected lung parenchyma with thickened alveolar septa and hyaline membranes (lower left) and the relatively preserved lung parenchyma (upper right) separated by a thickened oedematous interlobular septum. B, Oedema of variable severity was seen in all cases and is marked in this example. Note also the conspicuous hyaline membranes. C, The presence of interstitial neutrophilic infiltrates was focally seen in areas with acute DAD. D, Reactive type II pneumocytes within alveolar spaces showed abundant eosinophilic cytoplasm, irregular nuclear contours, occasional binucleation, vesicular condensed chromatin and prominent macronucleoli. Intracapillary megakaryocytes (arrows) were also present within alveolar capillaries and were highlighted by CD61 immunohistochemistry (inset). E, Lungs from six patients showed organising phase DAD alone or in combination with acute phase DAD, with characteristic type II pneumocyte and fibroblastic proliferation within alveolar walls and focal intraluminal plugs of loose connective tissue. F, Some areas within organising DAD showed interstitial chronic inflammation.

Figure 2

Viral antigen detection by immunohistochemistry (IHC) against SARS‐CoV nucleoprotein [monoclonal antibody (mAb) 001]. A, In this example (case 3) both acute and organising phases of DAD are present in a single section. Areas with organising DAD (upper right) that lack hyaline membranes failed to demonstrate the presence of viral antigen by IHC, while areas with acute DAD (lower left) showed staining in hyaline membranes but not in pneumocytes (B). C, In all IHC‐positive cases, viral antigen was detected in hyaline membranes (thick arrows) within areas of acute phase DAD, and in two cases diffusely within regenerating and reactive type II pneumocytes (arrowheads). Viral antigen was also present in alveolar macrophages in these latter two cases (D). In case 1, the case with the most viral antigen detected by IHC, weak cytoplasmic staining was observed in endothelial cells of scattered venules and alveolar capillaries (E).