Clinical and pathological investigation of patients with severe COVID-19

Abstract

Background: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a pandemic. This study addresses the clinical and immunopathological characteristics of severe COVID-19.

Methods: Sixty-nine patients with COVID-19 were classified into severe and nonsevere groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from 2 deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations.

Results: Circulating cytokines, including IL-8, IL-6, TNF-α, IP10, MCP1, and RANTES, were significantly elevated in patients with severe COVID-19. Dynamic IL-6 and IL-8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II and type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues.

Conclusions: A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both cytopathy caused by SARS-CoV-2 and immunopathologic damage. Strategies that prohibit pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of patients with severe COVID-19.

Keywords: Apoptosis; COVID-19; Infectious disease; Macrophages; Medical statistics; Pulmonology.

Figure 1. CT images of 2 patients with severe COVID-19 and 2 patients with nonsevere COVID-19.

(A) CT image of a 33-year-old man showing bilateral ground-glass opacities (GGO) partially fused into consolidation at onset of hospitalization and (B) mild GGO in the bilateral lungs. (C) CT image of a 51-year-old man showing bilateral GGO at onset of hospitalization and (D) multiple slightly high dense shadows in the bilateral lungs. (E) CT image of a 53-year-old woman showing bilateral GGO at the onset of hospitalization and (F) absorption after recovery. (G) CT image of a 17-year-old woman showing mild consolidation at the onset of hospitalization and (H) absorption after recovery.

Figure 2. Dynamic characteristics of laboratory variants between severe patients and nonsevere patients.

Dynamics of blood (A), neutrophils, (B) lymphocytes, (C) monocytes, (D) glucose, (E) C-reactive protein, (F) D-dimer, (G) IL-6, and (H) IL-8 between severe patients (n = 21) and nonsevere patients (n = 25). Data are represented as median (IQR). A generalized linear mixed model was used to compare repeated measures (nonnormal distribution). Data are presented as the mean of triplicate measurements.

Figure 3. Postmortem lung biopsy specimens from case 1.

(A) Lung tissue showing pulmonary edema with hyaline membranes and desquamation of alveolar epithelial cells (H&E staining; original magnification, ×200). (B) Ultrastructural image showing cytoplasmic viral particles in a type II pneumocyte (arrow), with swollen mitochondria and dilated endoplasmic reticulum (original magnification, ×20,000). (C) CK7⁺ cells reflecting pneumocytes (original magnification, ×200). (D) SPB⁺ cells indicating type II pneumocytes with marked vacuolation and mild hyperplasia (original magnification, ×200). (E) Increased CD68⁺ macrophages with cytomegaly mainly in alveolar spaces (original magnification, ×200). (F) Immunohistochemical staining for MPO, indicating a large number of interstitial infiltrated polymorphonuclear cells (original magnification, ×200). (G) CD4⁺ and (H) CD8⁺ T cells distributed in the alveolar septal walls and interstitial areas (original magnification, ×200). (I) Gasdermin D positivity indicating cell pyroptosis (original magnification, ×200). (J and K) TUNEL staining showing apoptotic cells (original magnification, ×200). (L) RNAscope in situ hybridization indicating SARS-CoV-2 nucleic acids, which manifest as spotted brown particles (original magnification, ×200).

Figure 4. Postmortem lung biopsy from case 2.

(A) H&E and (B) Masson’s trichrome staining showing features of early interstitial and alveolar fibrosis and mild pneumocyte hyperplasia, with focal exudative edema and hyaline membranes (arrows) in alveolar spaces. (C) Ultrastructural image showing cytoplasmic viral particles (arrow) characterized by spherical and spike-like projections in type II pneumocytes, with depleted laminar bodies, swollen mitochondria, and dilated endoplasmic reticulum. (D) CK7⁺ cells showing pneumocytes (original magnification, ×200). (E) SPB⁺ cells reflecting type II pneumocytes (original magnification, ×200). (F) Abundantly increased CD68⁺ macrophages in alveolar spaces. (G) Immunohistochemical staining of MPO, indicating numerous polymorphonuclear cells, aggregated in focal areas of bronchiolitis (original magnification, ×200). (H) A few CD4⁺ and (I) CD8⁺ T cells were distributed in the alveolar septal walls and interstitial areas (original magnification, ×200). (J) Gasdermin D⁺ cells representing cell pyroptosis (original magnification, ×200). (K and L) TUNEL staining showing apoptotic cells (original magnification, ×200). (M) RNAscope in situ hybridization indicating SARS-CoV-2 nucleic acids, which manifest as spotted brown particles (original magnification, ×200).