SARS-CoV-2 nucleocapsid-protein and ultrastructural modifications in small bowel of a four week negative COVID-19 patient

Dear Editor, HumanCoVs (HCoVs) account for up to 30% of infections of the upper respiratory tract and 8.1% of enteritis.1,2 However, related to the wide distribution of ACE2 in human tissues, several reports already hypothesized that SARS-CoV-2 may directly infect multiple organs, including liver, stomach, ileum and colon.3 Our patient, a man in his fifties with no prior medical history, had a positive to nop-swab for SARS-CoV-2 RNA four days after the start of symptoms. He was treated at home with hydroxychloroquine and azithromycin for seven days. The patient recovered and had a repeated two nop-swab, both with negative results. After two weeks he was admitted to the Emergency Department complaining of stomach pain followed by a syncopal episode and intestinal bleeding. Asymptomatic bilateral interstitial pneumonia was documented by CT scan. A colonscopy detected an ileocecal valve proximal ulceration (supplementary Fig 1), without active bleeding. The patient underwent emergency surgery due to an hypotensive episode associated with haematic stools. Terminal ileum and cecum were resected as well as Meckel diverticulum. At the histological examination, terminal ileum wall showed chronic inflammatory infiltrates with prevalence of lymphocytes, focal ulceration of both mucosa (Fig. 1d) and submucosa (Fig. 1e). Abnormally enlarged and tortuous thick-walled veins were seen in the submucosa. IHC analysis with anti-SARS-CoV-2 nucleocapsid-protein revealed the presence of viral protein expression in epithelial cell of ulcerated intestinal mucosa (cytoplasmic staining) and in a minority of lymphocytes (Fig. 1d); no staining in the submucosa (Fig. 1e). We also analyzed the expression of a non-classical MHC-I molecule, Human Leukocyte Antigen-G (HLA-G), that blocks endothelial cell proliferation and vessel formation.4 We observed HLA-G in epithelial cells of the intestinal mucosa and in some lymphocytes (Fig. 1d), in correspondence of SARS-CoV-2 positive sites. In the submucosa, HLA-G expression was detectable only in few lymphocytes (Fig. 1e). Transmission Electron Microscopy (TEM) analysis revealed a significantly different morphological microvilli profile,the intestinal tract of a normal ileum from a subject pre-COVID-19 appearance, showed well organized and aligned microvilli, with a regular distribution protruding from the apical cell membrane (acm) and an homogeneous glycocalyx (gc) (Fig. 1f and g). The tissue, examined in the area without focal ulceration showed a morphology similar to the control (Fig. 1h and i). Regarding the ulceration area, microvilli appeared shorter than those in the not ulcerated area, partially depeneed beyond the acm, and gc appeared disorganized and almost absent showing a relevant cytopatic effect (Fig. 1j and k).