Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States

Roosecelis B. Martines; Jana M. Ritter; Eduard Matkovic; Joy Gary; Brigid C. Bollweg; Hannah Bullock; Cynthia S. Goldsmith; Luciana Silva-Flannery; Josilene N. Seixas; Sarah Reagan-Steiner; Timothy Uyeki; Amy Denison; Julu Bhatnagar; Wun-Ju Shieh; Sherif R. Zaki


Emerging Infectious Diseases. 2020;26(9):2005-2015. 

In This Article


Clinical Data

Of the 8 case-patients, 7 were residents of a long-term care facility (LTCF) in Washington state (Table 1).[23,24] Seven (87.5%) were White, non-Hispanic. The median age of the 8 case-patients was 73.5 years; 2 were <65 years of age. The median number of days from illness onset until death was 12.5 (range 6–15). Common signs and symptoms reported included fever (75%), cough (62.5%), and shortness of breath (62.5%). All patients had abnormal findings on chest radiographs. Case-patients were all hospitalized for a median of 3.5 days. Six (75%) patients received mechanical ventilation; 2 received comfort care. Underlying medical conditions were identified in all case-patients; hypertension (75%), chronic kidney disease (75%), cardiovascular disease (75%), obesity (62.5%), and diabetes (50%) were the most frequent conditions reported.

Histopathology, Immunohistochemistry, and Electron Microscopy

Histopathologic findings and results of testing performed on FFPE tissues showed mild to moderate tracheobronchitis was consistently present and characterized by mononuclear inflammation, with epithelial denudation and submucosal congestion (Table 2; Figure 1, panels A, B). The predominant lung pathology was diffuse alveolar damage (DAD); acute phases, organizing phases, or both were present in 7 (87.5%) of 8 patients. Desquamation of pneumocytes and the presence of hyaline membranes, alveolar edema and fibrin deposits, type II pneumocyte hyperplasia, and alveolar infiltrates, including increased alveolar macrophages, were seen (Figure 1, panels C, D). Squamous metaplasia and atypical pneumocytes were present in 3 case-patients, and rare multinucleated cells were present in 1 case-patient (patient no. 1) (Figure 1, panel E); no definitive viral inclusions were seen. One case-patient without DAD (patient no. 7) had diffuse bronchopneumonia with filling of alveolar spaces by mixed inflammation with abundant neutrophils (Figure 1, panel F). Three additional case-patients had focal bronchopneumonia and increased pulmonary intravascular leukocytes. Hemosiderin-laden macrophages (4/8), hemorrhage (4/8), mucus aspiration (3/8), emphysema (2/8), and microthrombi (1/8) were seen (Figure 2). Anthracosis, common in elderly persons as a result of chronic carbon accumulation, was present in the lungs and pulmonary hilar lymph nodes in all cases. In 6 cases, lymph nodes also showed sinus histiocytosis and hemophagocytosis in subcapsular sinuses. Notable pathologic findings in extrapulmonary tissues included evidence of chronic renal disease (5/8), acute renal tubular injury (3/8), hepatic steatosis (4/8) and cirrhosis (1/8), and focal myocardial fibrosis (3/8) (Figure 2). No myocarditis or myocardial necrosis and no notable histopathologic changes in the intestine were seen in any case. Brain tissues were not available for histopathologic evaluation or testing.

Figure 1.

Pulmonary histopathology in fatal coronavirus disease cases caused by severe acute respiratory syndrome coronavirus 2 infection. A) Patient no. 5: tracheitis characterized by moderate mononuclear inflammation within the submucosa (original magnification ×10). B) Patient no. 3: extensive denudation of tracheal epithelium; submucosal congestion, mild edema, and mononuclear inflammation (original magnification ×10). C) Patient no. 4: exudative phase of diffuse alveolar damage characterized by abundant hyaline membranes lining alveolar spaces (arrow) (original magnification ×20). D) Patient no. 8: proliferative phase of diffuse alveolar damage characterized by proliferation of type II pneumocytes (arrow) (original magnification ×20). E) Patient no. 1: atypical pneumocytes with enlarged and multiple nuclei, and expanded cytoplasm in a case with proliferative DAD (original magnification ×40). F) Patient no. 7: bronchopneumonia with filling of alveolar spaces by neutrophils and patchy hemorrhage (arrow) (original magnification ×10).

Figure 2.

Histopathologic findings associated with underlying conditions in fatal coronavirus disease. A) Patient no. 2: lung, hemosiderin-laden macrophages (brown pigment, bottom left), and anthracosis (black pigment, top right) in a patient with congestive heart failure (original magnification ×20). B) Patient no. 3: lung, emphysema in a patient with chronic obstructive pulmonary disease (original magnification ×5). C) Patient no. 7: lung, pulmonary microthrombosis (arrow) (original magnification ×20). D) Patient no. 2: kidney, extensive glomerulosclerosis in a patient with renal disease (original magnification ×10). E) Patient no. 3: liver, steatosis in a patient with morbid obesity (original magnification ×20). F) Patient no. 2: heart, myocardial fibrosis and mild cardiomyocyte hypertrophy in a patient with cardiomegaly (original magnification ×5).

We detected SARS-CoV-2 by IHC in the upper airways in 4/8 (50%) case-patients and in the lungs in 7/8 (92%) case-patients. We observed immunostaining of viral antigens in upper airway and bronchiolar epithelium, submucosal gland epithelium, and in type I and type II pneumocytes, alveolar macrophages, and hyaline membranes in the lung (Figure 3, panels A–C, F). Upper airways and lung tissues from all 8 case-patients were positive by SARS-CoV-2 RT-PCR. Double staining with surfactant showed colocalization of SARS-CoV-2 antigen with type II pneumocytes (Figure 3, panel D); double staining with CD-163 showed viral antigen colocalization with macrophages (Figure 3, panel E). We also found viral immunostaining in scattered macrophages in the hilar lymph node from 1 severely immunosuppressed patient with a history of solid-organ transplant (Figure 3, panel G). We did not detect SARS-CoV-2 by IHC in heart, liver, kidney, spleen, or intestine from any patient.

Figure 3.

Immunostaining of severe acute respiratory syndrome coronavirus 2 in pulmonary tissues from fatal coronavirus disease cases. A) Patient no. 5: scattered immunostaining of tracheal epithelial cells (original magnification ×40). B) Patient no. 5: higher magnification shows immunostaining of ciliated cells (original magnification ×63). C) Patient no. 8: immunostaining of desquamated type I pneumocyte in an alveolar lumen (original magnification ×63). D) Patient no. 4: colocalization of SARS-CoV-2 viral antigen (red) with type II pneumocyte stained by surfactant (brown; arrow) (original magnification ×63). E) Patient no. 4: colocalization of SARS-CoV-2 viral antigen (red) with macrophages stained by CD163 (brown; arrows); virus immunostaining within type II pneumocytes is also seen (arrowheads) (original magnification ×40). F) Patient no. 4: extensive immunostaining of hyaline membranes in a region of exudative DAD (original magnification ×20). G) Patient no. 3: scattered immunostaining within macrophage in hilar lymph node; anthracosis is also present (original magnification ×63).

Six (75%) of 8 case-patients had either viral or bacterial co-infections, but not both, identified by IHC, PCR, or both in addition to SARS-CoV-2. Respiratory viral PCR testing detected parainfluenza virus type 3 coinfection in upper airway and lung tissue in 2/8 (25%) case-patients and influenza B virus coinfection in upper airway in 1 case-patient. Three (75%) of the 4 case-patients with SARS-CoV-2 and bronchopneumonia had immunostaining for Streptococcus spp. Two of these patients had nonpneumococcal Streptococcus spp. confirmed by PCR testing.

EM examination of respiratory tissues showed virions with prominent surface projections (spikes) characteristic of the family Coronaviridae. In the lung, extracellular virions free in the alveolar space were, on average, 105 nm in diameter, including surface projections (Figure 4, panel A). In upper airways, virions were seen extracellularly among the cilia and within the cytoplasm of respiratory epithelial cells (Figure 4, panel B; Figure 5). Intracellular virions in type II pneumocytes (Figure 4, panels C, D) and in cytoplasmic vesicles or phagosomes of alveolar macrophages (Figure 4, panel E) were, on average, 75 nm in diameter and lacked prominent spikes. Viral particles were also found associated with fibrin or hyaline membranes within alveolar spaces (Figure 4, panel F).

Figure 4.

Ultrastructural features of severe acute respiratory syndrome coronavirus 2 lung infection in fatal coronavirus disease. A) Top: alveolar space containing extracellular virions (arrows) with prominent surface projections. Bottom: cluster of virions in the alveolar space. Scale bars indicate 200 nm. B) Extracellular virions (arrow) associated with ciliated cells of the upper airway. Scale bar indicates 200 nm. C) Membrane-bound vacuoles (arrows) containing viral particles within the cytoplasm of an infected type II pneumocyte; surfactant (lamellated material) indicted by arrowheads. Scale bar indicates 1 μm. D) Membrane-bound vacuole (double-headed arrow in panel C) containing virus particles (arrows) with the characteristic black dots that are cross-sections through the viral nucleocapsid. Arrowheads indicate vacuolar membrane. Scale bar indicates 200 nm. E) Viral particles (arrow) within a phagosome of an alveolar macrophage. Scale bar: 200 nm. F) Viral particles within a portion of a hyaline membrane. Scale bar indicates 800 nm. Inset: Higher magnification of virus particles indicated by arrow; scale bar indicates 200 nm.

Figure 5.

Ultrastructural features of severe acute respiratory syndrome coronavirus 2 infection within the upper airway of a fatal coronavirus disease case from formalin-fixed paraffin-embedded (FFPE) tissue. Viral particles associated with the cilia of ciliated cells (A, C, and D) and the cytoplasm of respiratory epithelial cells (B) in the upper airway are indicted by arrows. Images in panels A and C were obtained from FFPE tissue removed from a paraffin block using a 2-mm biopsy punch. Images in panels B and D were collected from a 3 μm section of FFPE tissue affixed to a glass slide. Viral particles visualized in FFPE samples were smaller than those observed from fresh tissue; extracellular viral particles in fresh tissue samples were 105 nm in diameter and those from FFPE tissues were 75 nm in diameter. Scale bars indicate 1 μm (panel A), 800 nm (panel B), and 200 nm (panels C and D).