Peculiar Histopathological Alterations of Enterocytes in a Patient Co-infected with Severe Acute Respiratory Syndrome Coronavirus 2 and Mycobacterium Tuberculosis: A Case Study

The ongoing novel coronavirus disease 2019 (COVID-19) is principally dened by its respiratory symptoms. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect the gastrointestinal tract (GIT) and although the pathogenesis of COVID-19 is understood, the exact pathological alterations following infection require further investigation. Here, we report our histopathological ndings from a right hemicolectomy specimen from a patient coinfected with COVID-19 and Mycobacterium tuberculosis. Our observations showed that the novel SARS-CoV-2 can affect the GIT, causing epithelial injury and pathological alterations attributed to its ability to infect absorptive enterocytes by interacting with the angiotensin converting enzyme-2 (ACE2) receptor. These pathological ndings are regarded as viral cytopathic changes and should be considered when evaluating gastrointestinal specimens from COVID-19-infected patients.


Introduction
The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread worldwide. It has infected more than 100 million individuals and led 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. SARS-CoV-2 can also affect the gastrointestinal tract (GIT), but the incidence of COVID-19 in the GIT as the main presentation or as a concurrent infection with the respiratory tract is low. The range of GIT symptoms include 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). There is a good understanding of the pathogenesis of GIT infections caused by SARS-CoV-2 due to continuous investigations. However, the histopathological alterations of the gastrointestinal mucosa have not been well elucidated. We observed incidental peculiar pathological alterations of the terminal ileal mucosa and the mesenteric lymph nodes, which were part of a right hemicolectomy specimen obtained as a result of a bowel perforation event secondary to a GIT infection with Mycobacterium tuberculosis in a COVID-19-positive patient. Our aim was to share these microscopic ndings with practicing pathologists and improve the awareness of what they can expect to see in gastrointestinal biopsies and resections of COVID-19infected patients.

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

Immunohistochemical Evaluation
Our observations of the immunohistochemical stains are summarized in Table 1.

Discussion
The novel SARS-CoV-2 causes 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 the spike (S), small envelope (E), and membrane (M) glycoproteins, and the 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 angiotensin converting enzyme-2 (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. ACE2 expression is approximately 100-fold higher in the GIT than in the respiratory system. This makes the 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 the RNA and protein expression of ACE2 is higher in the small intestine than in the colon (1). Importantly, 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 propensity for the GIT, similar to other members of the coronavirus family, although it affects the GIT less than it affects the respiratory system. Notably, 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). In one study, 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 patients with COVID-19 and suggested that the virus can replicate and exist in the GIT. (7) The pathogenesis of SARS-CoV-2-associated GIT infections mainly relies on the entry of the virus into the cytoplasm of absorptive enterocytes through its interaction with ACE2. Successful virus entry depends on the ACE2 receptor and endogenous serine proteases, such as furin and cellular transmembrane protease serine 2 (TMPRSS2) which cleaves the S protein 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 the viral genomic material (RNA). Subsequently, 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 represents the newly formed SARS-CoV-2 structural glycoproteins. 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 mechanistic target of rapamycin 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 was 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 is best evaluated by performing CD10 immunohistochemical staining. CD10 is a membrane-associated neutral peptidase that 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). Another alteration was the reduced expression of Ecadherins which are a major constituent of adherens junctions and play an essential role in intestinal homeostasis by providing mechanical integrity and maintaining cell polarization. Additionally, Ecadherins are necessary for the proper maturation and differentiation of secretory cell lineages, including Paneth and goblet cells. Downregulation of E-cadherin function due to decreased expression occurs secondary to the effect of in ammatory cytokines that lead to the activation of signaling pathway mediators, such as GTPases. This in turn, destabilizes adherens junctions and disrupts cell contacts, thus, facilitating pathogen invasion. Loss of E-cadherin function has been reported in in ammatory conditions of the intestine, such as Crohn's disease and ulcerative colitis (9). This explains the obvious detachment and "hobnailing" appearance of the enterocytes as well as the diminished immunostaining for E-cadherin seen in our patient. Similarly, possible alterations in the expression of EpCAM, which is a transmembrane glycoprotein, may occur secondary to SARS-CoV-2 infection. EpCAM plays an important role in intercellular adhesion, cell signaling, proliferation, differentiation, and cell polarity. Furthermore, EpCAM is enriched in the basolateral membrane of intestinal absorptive cells (10). Diminished or loss of immunostaining for this adhesion molecule was also noted. This suggests that signi cant subcellular alterations are induced by SARS-CoV-2 infection. In addition, M. tuberculosis coinfection certainly played a signi cant synergistic role in causing the overwhelming SARS-CoV-2 infection in this case.

Review of Literature
Few studies describing the pathological ndings of SARS-CoV-2 in the intestine have been reported to date. Liu et al. described an autopsy nding from a patient with COVID-19 who developed alternating segmental dilation and stenosis of the small intestine (11). Xiao et al. performed an endoscopy of the GIT in a patient with COVID-19. They observed damage to the esophageal mucosa, numerous plasma cells, and lymphocytes in the lamina propria of the stomach, duodenum, and rectum. Furthermore, viral nucleocapsid proteins were detected in the cytoplasm of the infected cells (12). A reported case of acute hemorrhagic colitis showed a slight expansion of the lamina propria due to edema with normal cellularity, and intact crypts were found (13). A study of 67 autopsies performed on patients who died due to COVID-19 infection revealed no gross or microscopic abnormalities of 16 small intestines and 17 colons examined. However, hemophagocytic histiocytes (with engulfment of red blood cells) were noted in the spleen (9/22 cases), bone marrow (4/6 cases), thoracic lymph nodes (9/11 cases), liver Kupffer cells and epicardial in ammation (1 case). According to this observation, the study suggested that SARS-CoV-2 may induce a macrophage activation syndrome-like state, which might occur secondary to the cytokine storm. (14) Conclusion COVID-19 is a threatening disease that causes systemic illness which primarily affects the respiratory system but may affect the GIT. While the pathogenesis of this disease becomes more evident, it appears that the pathological alterations of the small intestinal epithelium and the mesenteric lymph nodes are associated with the direct injury induced by the virus on the host cells, as well as the effect of the aberrant in ammatory response produced by the host's immune system. We suggest that SARS-CoV-2 might cause cellular and subcellular alterations that induce certain microscopic changes in the affected tissue. The pathological alterations of the intestine associated with SARS-CoV-2 infection are important to be known by practicing anatomic pathologists; thus, they deserve close attention and further investigation.  Tables   Table 1 Immunohistochemical  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.
Competing interests: The authors declare that they have no competing interests.

Funding: Nil
Authors' contributions: RZ performed the histopathological examination of the specimen and noticed the presence of the peculiar pathological alterations described in the manuscript and preformed the appropriate stains. RZ is a major contributor in writing and preparing the manuscript. NN was a contributor in evaluating the histological sections and concurred the presence of such novel ndings. NN contributed in the literature search as well as in writing the manuscript. AH was contributed in the literature search and in revising the manuscript. All authors read and approved the nal manuscript.

Figure 1
Section from the terminal ileum shows mucosal erosion with overlying brino-purulent exudate, loss of villous architecture, and crypt elongation. An intervening uninvolved mucosa at the center of the image is noted (hematoxylin-eosin, original magni cation x40).

Figure 3
Villous blunting, erosion with brino-purulent exudate, lamina propria edema, and regenerated crypts compared to normal mucosa on the left side of the image (hematoxylin-eosin, original magni cation x100).