Abstract

Gastrointestinal (GI) symptoms are not uncommon in COVID-19 infection. The varied symptomatology and severity of GI symptoms are related to the difference in host viral interaction. We have reviewed the GI manifestations of these patients and noted that three subgroups exist.A group with self-limited GI disease, a group with predominant GI manifestations and group with severe GI disease. A review of the immune response in the different groups with GI manifestations has been done and illustrated by a cat and mouse game theory. In self-limited GI disease, there is early active innate immunity with type I interferon response and followed by an efficient adaptive immune response causing viral clearance. In patients with predominant GI symptoms, viral factors override innate immune mechanisms and cause delayed and weak innate antiviral response. The subsequential adaptive response is a mishit response with mucosal injury and GI symptoms. In the later part of this infection, a resurgent adaptive immunity induction can occur, causing persistent fecal shedding followed by viral clearance. In severe disease, there is heightened dysregulation of the adaptive immune response leading to cytokine storm and severe complications like pyroptosis, endothelitis, and hemophagocytic lymphohistiocytosis.

1. Introduction

Humankind should be aware that many of its greatest enemies are paradoxically microscopic. Twentieth-century saw deadly pandemic of Spanish flu in 1918. The twenty-first century saw the emergence of human coronaviruses, severe acute respiratory syndrome causing coronavirus (SARS-CoV) in 2002, Middle East respiratory syndrome causing coronavirus (MERS-CoV) in 2012 and now in December 2019, a novel β-coronavirus infection, SARS CoV-2 emerged in Wuhan, China. As of July 10, COVID-19 has emerged into a pandemic of 21st century affecting 215 countries with over 12 million confirmed cases, more than 5 hundred thousand deaths ¹. COVID-19 virus is a single positive-stranded RNA virus belonging to the corona viridae family in the nidovirales order. SARS CoV-2 genome research showed more than 95% sequence identity to the genome of Bat CoV, suggesting a common ancestry. The route of human transmission remains elusive, but it is suspected to be zoonotic due to direct contact with intermediate hosts or the consumption of infected animals.

2. Methods

The review was based on literature search in PubMed and Google internet information sources including Google Scholar, reports, publications, reviews and data collected by WHO from December 2019 to July 2020. Manual hand searching and reading of unpublished relevant articles were also done. We used search terms ‘SARS-CoV-2’ or ‘COVID-19’ combined with ‘gastrointestinal’, ‘GI symptoms’, ‘clinical feature’ or ‘immune system’.

3. Pathogenesis

COVID-19 virus genome encodes nonstructural proteins (NSP), four essential structural proteins, including spike (S) glycoprotein, small envelope (E) protein, matrix (M) protein, nucleocapsid (N) protein and also several accessory proteins,that interfere with the host innate immune response. Like SARS CoV, the COVID-19 virus requires the presence of the ACE2 receptor and the cellular protease TMPRSS2 enzyme in the cell membrane of host cells, which cleaves the S spike protein on the virus envelope to be able to enter the cell. S glycoprotein includes two subunits, S1 and S2. S1 glycoprotein includes a receptor-binding domain (RBD) and determines the virus-host range and cellular tropism. The S2 mediates virus-cell membrane fusion. After membrane fusion, the viral genome RNA gets released into the cytoplasm. The viral RNA further undergoes transcription and translation using host cell organelles to produce virion-containing vesicles that fuse with the plasma membrane to release the virus ². The genomic differences aid COVID-19 virus to readily transmit while causing less severe human infection than human SARS-CoV. COVID-19 infection has an incubation period of 1-14days, contagious during this latency period ³. COVID-19 infection can be roughly divided into stage I, an asymptomatic incubation period with or without detectable virus; stage II, nonsevere symptomatic period with the presence of virus; stage III, a severe respiratory symptomatic stage with high viral load ⁴.

4. GI Presentation in COVID-19

GI mucosal involvement can have three presentations. Firstly, patients with predominantly respiratory symptoms and mild self-limited GI mucosal involvement. A cohort of over a thousand patients with COVID-19 from 552 hospitals in China, 5.0 percentage of cases presented with nausea or vomiting, and 3.8 percentage presented with diarrhea ⁵. Several other cohorts have reported frequencies of diarrhea ranging from 2.0-10.1%, and nausea and vomiting ranging from 1.0-10.1% ⁶ [Table 1].

Coronaviruses are known to cause respiratory and intestinal tract infections but with respiratory tract being the readiest transmission approach for the virus. ACE2 is the most critical mechanism for viral infection of cells. Lungs had a moderate expression of ACE2 among all tissues and found concentrated in a small population of type II alveolar cells. ACE2 and TMPRSS2 expression levels were among the highest in the small intestine, stomach, colon, liver, and bile ducts, among other tissues ⁷ [Figure 1 and Figure 2].

Following a respiratory infection, viral dissemination into GI cells can be via the bloodstream or mucosal route. The postulated mechanism for early resolution of symptoms can be a crucial early viral control by an effective innate immune response. This initial non-specific immune response in which monocytes, macrophages, innate lymphoid cells, NK cells, neutrophils, and dendritic cells dampen the progress of the viral infection and may even prevent it from developing symptoms. The dynamics of interferon response is crucial in this innate immunity. If SARS-CoV-2 viral structural and nonstructural protein modulation of type I interferon fails, then viral replication ceases. ACE2 GI expression also gets regulated by interferon molecules ⁸. ACE2 expression is also affected by host factors like age, gender, presence of diabetes, hypertension, and other cardiovascular diseases ⁹. NK cells play a crucial role in the innate immune response. SARS-CoV-2 virus attempts to override NK cell innate activity of the host by inducing over-expression of NK group 2 member A (NKG2A) receptor on NK and CD 8 T cells. NKG2A receptor transduces inhibitory signalling, suppressing NK cytokine secretion and cytotoxicity. This can culminate in functional exhaustion of the immune response against the viral pathogen ¹⁰. The GI mucosal secretory IgA antibodies are another factor that can have a significant role in limiting GI infection. Secretory IgA can entrap viruses and reduce docking and entry of the virus into cells. Studies suggest that Secretory IgA reduces the coronavirus titer significantly in cell lines ¹¹. IgA is present in the serum of COVID-19 patients, although a small number of patients were involved, and a study on IgA detection was so far limited ^{12, 13}.

Mucosal memory IgA cells and anamnestic response from previous exposure to coronaviruses are other factors that need to be studied. There is only scarce data available on the effect of COVID-19 virus on intestinal microbiota. A small case series from China revealed that some patients with COVID-19 showed microbial dysbiosis with decreased Lactobacillus and Bifidobacterium. Lactobacilli and Bifidobacteria are only two strains from the gut microbiota, and we must consider whether they can tip the balance of a diverse gut ecosystem in combating COVID-19 virus ¹⁴. In contrast, another study from China found that probiotics such as Segmented Filamentous Bacteria may elevate the expression of coronaviruses receptors in the murine small intestine ¹⁵. So the role of gut microbiota in combating COVID-19 virus is presently unclear.

The culmination of the above responses can be an early Specific Th1/Th17 adaptive immune response that may effectively enhance inflammatory responses. B cells/plasma cells produce SARS-CoV-2 specific antibodies that may help neutralize viruses. The COVID-19 virus can stimulate the co-expression of CD38 and HLA-DR, causing early specific activation of CD8+ T cells. This strong perforin and granulysin-positive CD8+ T cells or inflammatory Th17 cells play a vital role in viral clearance ¹⁶ [Figure 3].

5. Predominant GI manifestations and Feco-oral Transmission

A delayed innate immune response can be harmful to the patient as prolonged viral persistence can exacerbate adaptive inflammatory responses deleteriously, leading to immune enervation and later immune suppression as a regulatory feedback mechanism. The delayed innate immune response is a characteristic of the second group of patients who present with predominant gastrointestinal manifestations [Figure 4].

In their descriptive multicenter study, Lei Pan et al. found that 204 patients with COVID-19 (mean age 52.9 years) presented to three hospitals in Hubei, China, 103(50.5%) patients presented with digestive symptoms as their chief complaint. Six patients had only digestive symptoms. The group with digestive symptoms had a greater delay in admission from the onset of symptoms and also higher liver dysfunction ¹⁷. Xi Jin et al. investigated 74 COVID-19 patients with GI symptoms in the Zhejiang province of China. Up to 28% of those with GI symptoms did not have respiratory symptoms. They noted that patient groups with digestive symptoms compared with patients without GI symptoms (n=577) had overall more severe/critical disease, more patients with fever >38.5°C, family clustering, and higher rates of liver dysfunction ¹⁸. A feco oral route of direct GI infection with significant viral load overwhelming and delaying the immune response could be another explanation. Prominent GI expression of ACE 2 and significant tropism of COVID-19 virus can result in docking, entry, and mucosal cell replication. Viral nucleocapsid staining detected in the cytoplasm of gastric, duodenal, and rectal epithelial cells ¹⁹. Subsequently, it can result in portal viremia and cause liver dysfunction and systemic viral dissemination. A neuroenteric route of infection is also possible ²⁰. The analogy is during the SARS epidemic; the fecal source leads to a major outbreak caused by toilet fume in Amoy Garden in Hong Kong. Amoy gardens with faulty sewage management in Hong Kong showed high GI involvement (50-70%) compared to China (10-20% diarrhea). The inferences drawn were the possible difference in the mode of viral transmission and variability in the virus's specificity to GI versus the respiratory system. Genomic mutations in the virus isolate, contributing to the observed difference in presentation ²¹. In COVID-19 patients, following GI infection, persistent mucosal replication can cause prolonged fecal shedding. In the first case of COVID-19 infections reported from the USA, the patient initially had GI symptoms. The virus detected in the stool on day 7 of illness ²². Xiao et al. investigated 73 COVID-19 hospitalized patients in China, and 53.4% of patients were tested positive for the virus in the stool ranging from day 1 to 12 of infection. Importantly, in this study, >20% of infected patients had a positive virus in feces even after clearance of the virus in the respiratory tract ²³. Wang et al. studied the detection of COVID-19 in different types of clinical specimens (1070 specimens from 205 patients of mean age 44 years) using RT-PCR. They found that 126 (32%) of 398 pharyngeal swabs (126 of 398), 44 (29%) of 153 fecal specimens (44 of 153), and 3(1%) of blood samples were positive for COVID-19 virus.The authors cultured four COVID-19 virus positive fecal specimens with high copy numbers and detected live virus using electron microscopy detection. Live COVID-19 virus detected in the stool sample from two patients who did not have diarrhea ²⁴. The hypothesized mechanism could be the interplay of mucosal immunity and the degree of viral replication. The continuous positive detection of viral RNA from feces suggests release of infectious virions from gastrointestinal cells ²⁵. A delayed innate immune response is likely followed by the initial adaptive immune response, which is protracted and inefficient in eradicating the epithelial viral replication. The immune signature in GI results in cytokine-mediated mucosal injury, resulting in symptoms and finally apoptosis of infected epithelial cells. The mucosal lamina propria of the stomach, duodenum, and rectum, shows numerous infiltrating plasma cells and lymphocytes with interstitial edema. The GI cells relatively unaffected due to lack of ACE2 expression are goblet cells, Paneth cells, tuft, or enteroendocrine cells ²⁶. The hypothesis that a line of defense beyond the epithelium may result in viral replication limited to epithelial cells and persistent viral shedding with no GI symptoms [Figure 5]. Towards later part of infection circulating bone marrow-derived monocytes and immature dendritic cells take up COVID-19 viral particles by micropinocytosis and recognize viral pathogen associated molecular patterns (PAMPS) ²⁷. These cells home into GI mucosa and subsequently process and present viral peptides to immune cells translating as a resurgent adaptive immune response. T cell receptor (TCR) repertoire is highly dynamic and exposure to antigen triggers massive expansion of T cells capable of recognizing the particular antigen (antigen-specific T cells), leading to skewing of the TCR repertoire to favor antigen-specific T cells ²⁸. This resurgent adaptive immune response capable of viral clearance can be detected more than one week after onset of symptoms. The innovative COVID-19 viral mRNA based vaccine research stands on this principle of enhanced immunogenicity and translation of adaptive immunity ²⁹

6. Severe GI Disease in COVID-19

The third group of patients has a severe disease with GI symptoms. These patients have severely dysregulated mucosal immune mechanisms [Figure 6]. ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. COVID-19 viral interaction with ACE2 can alter enzyme activity and induce severe colitis susceptibility. ACE 2 deficiency in a mouse model causes intestinal epithelial neutral amino acid deficiency especially critical impairment of tryptophan homeostasis. The effects are severe colitis, blunted intestinal epithelial regenerative and repair mechanisms, down regulated gut immunity mediated by antimicrobial peptides ^{30, 31}. In COVID-19 severe disease, there is low interferon type I and III molecules and a robust response of cytokine storm due to cytokines and chemokines by numerous cells, including epithelial cells, neutrophils, monocytes, and T cells, mirrors the aberrant overwhelming hyper-responsiveness of the immune system. Severe COVID-19 patients exhibit lower lymphocyte counts, higher neutrophil counts, and an elevated neutrophil-lymphocyte ratio. Direct virus killing of lymphocytes could contribute to lymphopenia ³². There is the suppression of regulatory T cells in severe cases, which indicates insufficient counter-regulation of pro-inflammatory immune responses. Significant release of CXCL9 and CXCL16, chemoattractant molecules of T or NK cells, CCL8 and CCL2, which recruit monocytes/macrophages, and CXCL8, a classic neutrophil chemoattractant molecule, suggest that the recruitment of these inflammatory cells may be a primary driver of the signature pathology of severe COVID-19. High levels of inflammatory cytokines like IL-1beta, IL-6, IL-7, IL-8, IL-9, IL-10, MCP-1, TNF-alpha, IFN-gamma in plasma causes pyroptosis. Pyroptosis is a novel inflammatory form of programmed cell death ^{33, 34, 35}. Histological analysis in severe disease and post mortem has revealed intestinal endothelial viral inclusion bodies, prominent submucosal endothelitis, hepatic lymphocytic endothelitis, pyroptosis, and formation of apoptotic bodies ³⁶. Cytokine storm in COVID-19 may lead to secondary hemophagocytic lymphohistiocytosis, a hyperinflammatory disorder triggered by viral infections associated with multiorgan failure and high lethality ³⁷.

7. Conclusion

COVID-19 infection is associated with varied gastrointestinal manifestations ranging from asymptomatic to severe GI disease. The interaction of the host immune system with COVID-19 virus is responsible for this variability. Viral factors like viral genomics, infective viral load, and mode of transmission play a key role in establishing host infection. Host factors like ACE-2 and TMPRSS2 expression, early innate immune response, and adaptive immune response appear to play a vital role in COVID-19 infection. The cat and mouse theory illustration attempts to highlight this critical host viral interaction producing variable clinical manifestations. The scientific data regarding gut microbiota, gut immunity, and COVID-19 virus interaction needs further research.

Acknowledgements

We would like to thank Mr. Sivi S Nair for helping us with the figure of cat and mouse concept in the manuscript.

References