PMC8287416_01
Timeline showing the disease course of the patient from positive molecular testing for SARS-CoV-2 up to death.
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On the 20th day, the patient died of severe respiratory failure ( Figure 1 ). The non-COVID-19 ulcer was totally negative at immunohistochemistry for SARS-CoV-2 Spike Subunit 1 ( Supplementary Figures 1A, B ). The lung histopathology ( Supplementary Figures 1C-E ) was characterized by diffuse alveolar damage, vascular congestion, platelet/fibrin thromboses in small- to medium-sized vessels, alveolar and subpleural haemorrhagic infiltrations, and lymphomonocytic infiltrations. Figure 1F ). Focal lympho-monocytic infiltrations were also found in liver and adrenal glands.
Macroscopic examination of fresh (A) and fixed (B) hypopharynx and esophagus, with evidence of two necrotic ulcers [white arrows in (A)], with raised sharply demarcated borders, in the anterior and posterior hypopharyngeal walls. Note the hemorrhagic infiltration of the external surface of the posterior pharyngeal wall [asterisk in (B)]. No lesions were detected in the esophagus. Histopathologic examination of transverse section of the posterior ulcer, with thinning of the pharyngeal wall at the level of the ulcer center and full-thickness inflammatory infiltration extending beyond the lesion margins (C) (scale bar 800 microm). Higher magnification of 
(C)
 insert, showing inflammatory infiltration of the muscle layer with necrosis and degeneration of the skeletal muscle fibers (D) (scale bar 200 microm).
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At autopsy, two necrotic ulcerations with overlying fibrin clot (about 1.5 cm in diameter) were found at the level of the anterior and posterior walls of the hypopharynx ( Figures 2A, B ). Figure 2B ). Full-thickness samples of the above lesions were collected. Histopathologic analysis of the ulcer showed thinning of the pharyngeal wall, due to necrosis/disruption of the mucosal layer and deep skeletal muscle fibers ( Figures 2C, D ).
Characterization of lympho-monocytic infiltration (A-C) through anti-CD68 (A), -CD3 (B) and -CD20 (C) immunostaining (scale bars 60 microm). Anti-CD61 immunostaining with evidence of platelet microthrombus (D) (scale bars 60 microm). Immunohistochemistry for SARS-CoV-2 Spike Subunit 1 (E-G), showing positive cells in perivascular infiltration (E) (scale bar 30 microm), among necrotic skeletal muscle fibers (F) (scale bar 30 microm) and in the deep lamina propria (G) (scale bar 30 microm), with detail of another positive cell in insert (scale bar 20 microm). Immunohistochemistry for HSV1 (H), showing general negativity of inflammatory infiltration of the muscle layer, except for some rare positive cells (scale bar 120 microm).
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Lympho-monocytic component was mainly composed of CD68-positive elements ( Figure 3A ); CD3- ( Figure 3B ) and CD20- ( Figure 3C ) positive lymphocytes were also detected. Some small vessels were occluded by fibrin and platelet microthrombi, the presence of the latter being confirmed by anti-CD61 immunostaining ( Figure 3D ). SARS-CoV-2-positive cells were also recognized throughout the thickness of the pharyngeal wall, from the ulcerated mucosa to the deep muscle layer ( Figures 3E-G ). Immunohistochemistry for HSV1 showed general negativity for inflammatory infiltration, although in the presence of some positive cells ( Figure 3H ).
Esophageal sample at distance from the hypopharyngeal ulcer, showing moderate lympho-monocytic infiltration (A, anti-CD68; B, anti-CD3; C, anti-CD20) and some cells immunostained for SARS-CoV-2 Spike Subunit 1 (D, arrows) (scale bars 120 microm).
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