Peculiar Histopathological Alterations of Enterocytes in A Coronavirus Disease 2019 Patient with Mycobacterial Tuberculosis Co-Infection

Background: The ongoing novel Coronavirus Disease 2019 (COVID-19) pandemic is principally dened by its respiratory symptoms. While it is clear that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can affect the gastrointestinal tract (GIT) and the pathogenesis of coronavirus disease 2019 is better understood, the exact pathological alterations following infection require further investigation. The purpose of this paper is to report and share our histopathological ndings from a right hemicolectomy specimen of a conrmed COVID-19 positive case, which exhibited a Mycobacterium Tuberculosis co-infection. Methods: Microscopic sections from right hemicolectomy specimen were appropriately stained and studied by two anatomical pathologists. Additionally, we searched PubMed and Google Scholar databases for reports/observations regarding pathological alterations of the intestine following COVID-19 infection. Results: Histological sections showed novel peculiar pathological alterations in the terminal ileal mucosa involving principally absorptive enterocytes with evidence of striking cellular injury as well as prominent erythrophagocytosis in the mesenteric lymph nodes. No specic pathological alterations were observed in the appendix or colon. The characteristic pathological features of Mycobacterium Tuberculosis infection were also observed throughout the specimen. Our showed that interacting with the Converting Enzyme-2 receptor. These pathological ndings could be regarded as viral cytopathic changes and should be considered when evaluating gastrointestinal specimens from COVID-19 infected


Introduction
The ongoing global pandemic of COVID-19, caused by the novel SARS-CoV-2, has rapidly spread worldwide. It has infected more than 100 million individuals, leading to more than 2 million deaths. SARS-CoV-2 is associated with the development of a systemic disease that typically affects the respiratory tract. The most common symptoms are fever, dry cough, fatigue, headache, and myalgia. The gastrointestinal tract has a lower incidence, either as a concurrent infection with the respiratory tract or as the initial presentation of COVID-19 infection. The range of GIT symptoms included diarrhea, abdominal pain, nausea, vomiting, and anorexia [1]. While the virus is highly contagious and spreads predominantly by respiratory droplets and aerosols, SARS-CoV-2 has been isolated from stool samples, suggesting that the virus could be transmitted via the fecal-oral route [2]. Continuous investigation of the pathogenesis of GIT infections caused by SARS-CoV-2 led to a better understanding. However, the histopathological alterations of the gastrointestinal mucosa have not been well elucidated. We observed peculiar pathological alterations of the terminal ileal mucosa and the mesenteric lymph nodes, which were part of a right hemicolectomy specimen performed for bowel perforation secondary to GIT

Pathological Findings
Microscopic examination of the terminal ileum, cecum, ascending colon, and appendix revealed multiple caseating epithelioid granulomas scattered throughout the wall. The overlying mucosa was ulcerated, with granulation tissue formation. Numerous acid-fast bacilli within the epithelioid granulomas were observed following Ziehl-Neelsen staining. The reminder of the terminal ileum mucosa showed patchy peculiar architectural alterations with normal intervening mucosa (Fig. 1). The villi of the affected mucosa showed shortening, widening, and blunting (Fig. 2). There was a striking "hobnail" appearance with focal detachment/denudation of enterocytes, erosions, and brinopurulent exudate (Fig. 3). Loss of microvilli and goblet cells were also observed (Fig. 4). The underlying lamina propria was expanded by marked edema and congestion with occasional thrombosis of the blood vessels (Fig. 5). Crypt regeneration and elongation was observed. The high-power view showed striking cytological alterations of the absorptive enterocytes including cuboidal to rounded appearance with striking nuclear enlargement, coarse chromatin pattern, prominent nucleoli, and thickening of the nuclear membrane (Fig. 6). A subset of absorptive cells showed binucleation. Predominantly subnuclear intracytoplasmic vacuoles with a signet ring-like appearance were apparent (Fig. 7). Periodic acid-Schiff staining showed positive intracytoplasmic material in scattered enterocytes, which might represent viral glycoproteins ( Fig. 8). Sections from the mesenteric lymph nodes showed involvement by caseating granulomatous in ammation. In addition, the nodal trabecular and medullary sinuses were expanded with histocytes that showed prominent erythrophagocytosis (Fig. 9).

Immunohistochemical Evaluation
We observed a diminished to complete loss of the normal linear brush-border staining pattern of CD10, in contrast to the adjacent normal mucosa (Fig. 10). Furthermore, diminished to complete loss of the normal basolateral immunohistochemical staining pattern of Ep-CAM and the normal diffuse pattern of E-cadherin staining was noted in the infected cells (Figs. 11 and 12, respectively). We observed aberrant cytoplasmic expression of EMA in a subset of infected cells, in contrast to the limited staining of the luminal surface of the normal crypts (Fig. 13). The Ki-67 labeling index was noted to extend upward toward the surface of the villi, in contrast to the limited staining pattern in the base of normal crypts ( Fig. 14).

Discussion
The novel SARS-CoV-2 causes the COVID-19, which has rapidly spread worldwide, threatening global health. SARS-CoV-2 is a single-stranded, enveloped RNA virus that belongs to the betacoronavirus 2b lineage [1,3]. Its diameter is approximately 65-125 nm. Structurally, it has four main proteins, including spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. The S glycoprotein is a transmembrane protein that protrudes from the viral surface and facilitates binding and entry of the virus into host cells by interacting with the ACE2 receptor [4]. According to the Human Protein Atlas database (proteinatlas.org), ACE2 is widely expressed in various human organs and tissues, including the oral and nasal mucosa, nasopharynx, lung, small intestine, colon, liver, spleen, kidney, and brain. It has been shown that the ACE2 expression is approximately 100fold higher in the GIT than in the respiratory system. This makes GIT highly susceptible to SARS-CoV-2 infections. ACE2 is expressed on the luminal surface of the absorptive enterocytes of the small intestine and colon, with lower expression in crypt epithelial cells [5]. A previous study showed that both RNA and protein expression of ACE2 is higher in the small intestine than that in the colon [1]. ACE2 plays an important role in the regulation of dietary amino acid homeostasis, innate immunity, microbial ecology, and susceptibility to colitis [5].
SARS-CoV-2 has a tropism to the GIT, similar other members of the coronavirus family, although it affects the GIT at a lower frequency than the respiratory system. Up to 30% of patients with pulmonary infection complain of GIT symptoms, mostly in association with respiratory symptoms; however, only 4% of patients complain of GIT symptoms alone [6]. The most common GIT symptoms were nausea and vomiting (41.6%), diarrhea (17.2%), abdominal pain, and anorexia [1]. Several studies have documented the presence of SARS-CoV-2 RNA in stool or anal/rectal swabs in COVID-19 patients and suggested that the virus can replicate and exist in the GIT [7].
The pathogenesis associated with the GIT infection with SARS-CoV-2 mainly relies on the entry of the virus into the cytoplasm of absorptive enterocytes through its interaction with ACE2. Successful virus entry depends not only on the ACE2 receptor, but also on endogenous serine proteases such as furin and cellular transmembrane protease serine 2 (TMPRSS2), which cleaves the S protein of SARS-CoV-2 into two segments (S1 and S2) [4,7]. Both furin and TMPRSS2 are widely distributed in the small bowel mucosa. This cleavage is critical for the attachment of the virus to both the ACE2 receptor and the cellular membrane. This attachment is followed by endocytosis of viral genomic material (RNA). Next, the viral mRNA is translated into new structural proteins with subsequent insulation in the endoplasmic reticulum-Golgi intermediate compartment, from which they form small vesicles that eventually undergo exocytosis [4,7]. This explains the presence of intracytoplasmic vesicles/vacuoles that are noted in the affected enterocytes from our samples, some of which contain periodic acid-Schiff positive material that represent the newly formed structural glycoproteins of SARS-CoV-2. Furthermore, the partial or complete blockage of the ACE2 receptor by a large viral load impairs the host cell nourishment supply and capabilities necessary to ensure a balanced immune response. The transport of amino acids, particularly tryptophan, is also impaired. This results in aberrant mTOR activation and impaired expression of antimicrobial peptides from Paneth cells, which eventually leads to alterations in the gut microbial environment [5,7]. The infected cells probably undergo subcellular alterations that might lead to certain pathological changes, as seen in our case. One of these alterations represents the loss of brush borders of the enterocytes, which is known to occur in the setting of active enteritis and is the leading cause of diarrhea. This alteration was best evaluated by performing CD10 immunohistochemical staining. CD10 is a membrane-associated neutral peptidase which is normally observed as a linear brush-border staining pattern of the small intestinal mucosa. Variable loss of brush-border immunostaining for CD10 is usually observed in the setting of active enteritis [8]. Declarations Ethics approval and consent to participate: Not applicable.
Consent for publication: written informed consent for publication was obtained.
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 14. Bryce C, Grimes Z, Pujadas E, Ahuja S, Beasley M, Albrecht R, et al. Pathophysiology of SARS-CoV-2: Pathophysiology of SARS-CoV-2: targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. The Mount Sinai COVID-19 autopsy experience. Preprint at medRxiv. 2020. Figure 1 Section from the terminal ileum shows mucosal erosion with overlying brino-purulent exudate, loss of villous architecture, and crypts elongation. An intervening un-involved mucosa at the center of the image is noted (hematoxylin-eosin, original magni cation x40).