Gastrointestinal SARS-CoV-2 Infection and the Dynamic of its Detection in Stool

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Introduction
The novel coronavirus (SARS-CoV-2), Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) were reported to have significant intestinal tropism and these three virus strains were detected in stool of patients. [1,2] Corman et al. examined adult patients infected with MERS-CoV and detected MERS-CoV RNA in 14.6% of stool samples. [3] Furthermore, in-vitro studies using human primary intestinal epithelial cells of prior coronavirus outbreaks revealed sustained primary intestinal epithelial inflammation and massive viral replication with sequential development of lung infection via lymphatics and/or bloodstream. [4] As the emerging novel coronavirus has been identified, gastrointestinal symptoms became major findings. It has been reported that up to 79% of patients in Wuhan, China presented with gastrointestinal symptoms such as diarrhea, decreased appetite, nausea, vomiting, abdominal pain and gas-trointestinal bleeding. [5] This generated the hypothesis that the GI tract could be a major source of viral shedding and fecal-oral transmission. The first COVID-19 case reported in the WA, USA on January 20, 2020 experienced two loose bowel movements on day 6 of the illness. [6] Real-time reverse transcriptase PCR (rRT-PCR) for stool specimen came back positive for the SARS-CoV-2, before initiating any antiviral or antimicrobial therapy. Interestingly, higher loads of SARS-CoV-2 were detected in nasopharyngeal/ oropharyngeal swabs compared to the stool specimen. [6] It has been suggested that SARS-CoV-2 is at least as well adapted to the angiotensin converting enzyme 2 (ACE2) as the SARS-CoV, or even 10 to 20 fold higher binding affinity. [7][8][9] An immunofluorescent study by Xiao et al. showed abundance of the ACE2 receptor and viral nucleocapsid proteins in the glandular cells of the stomach, duodenum and the rectum, however it is rarely expressed in the esophageal epithelium. [2] De-

Study design
This was a secondary analysis of studies published in English language peer-reviewed journals on COVID-19 that performed stool SARS-CoV-2 RT-PCR tests.

Database search strategies
We did a literature review for studies of patients with COVID-19 that reported positive stool SARS-CoV-2 in PubMed and Google Scholar published from April to June 30, 2020. Search queries included: "COVID-19", "SARS-CoV-2", and "stool SARS-CoV-2 RT-PCR".

Inclusion criteria
Studies that examined patients with proven COVID-19 that reported stool SARS-CoV-2 RT-PCR test.

Exclusion criteria
Studies with less than eight patients with positive stool SARS-CoV-2 RT-PCR. Institutional review board approval was not required given this study did not involve direct human participant research.

COVID-19 patients tested for SARS-CoV-2 in stool samples
Eighteen studies examined stool and respiratory samples for SARS-CoV-2 in patients with COVID-19 ( Table  1)  Abbreviations: RT-PCR, reverse transcription-polymerase chain reaction. Patients with positive stool SARS-CoV-2 RT-PCR after negative respiratory samples SARS-CoV-2 RT-PCR A total of six studies reported positive SARS-CoV-2 RT-PCR in stool after the respiratory SARS-CoV-2 RT-PCR became negative ( Table 2). Out of 198 patients with positive stool RT-PCR, 101 patients (51%) had prolonged fecal viral shedding after the respiratory samples RT-PCR. The mean and range of the positive stool SARS-CoV-2 RT-PCR were 51% and from 20.3% to 78% respectively.

Gastrointestinal symptoms and positive stool SARS-CoV-2 RT-PCR
Nine studies reported the presence of gastrointestinal symptoms in COVID-19 patients, either as sole presentation or in combination with respiratory symptoms, see Table 3. Among a total of 200 patients who tested positive for SARS-CoV-2 through stool samples, 95 (47.5%) patients had at least one gastrointestinal symptom. The GI symptoms were diarrhea, anorexia, abdominal pain, nausea, vomiting or GI bleeding. Anorexia and diarrhea were the most prevalent GI symptoms. [11,18] Patients with positive SARS-CoV-2 in stool had no higher occurrence of GI symptoms compared to those tested negative as per Chen et al. [12] Meanwhile, patients presenting with gastrointestinal symptoms were more likely to test positive for fecal virus (73.3% vs. 14.3%, P=0.033) where 11 out of 12 patients with positive fecal samples had digestive symptoms. [13] The proportion of patients with detectable stool viral RNA was higher among those with diarrhea than those without diarrhea, and 38% of patients experiencing diarrhea tested positive for fecal RT-PCR. [ trointestinal symptoms was not associated with fecal sample viral RNA positivity (P=0·45). [14] Larger scale studies are required to come to a more reasonable answer addressing the relation of gastrointestinal symptoms to the possibility of positive fecal testing.

Discussion
Our review included a total of 18 studies of patients with COVID-19 and positive SARS-CoV-2 RT-PCR in stool. These studies were predominantly from China and included a relatively small number of patients. Our study revealed that about 50% of patients with COVID-19 have positive stool SARS-CoV-2 RT-PCR. Factors affecting fecal viral shedding is a topic still under research, but may be affected by antiviral regimens, gastrointestinal genetic susceptibility, microbiota or corticosteroid management. Ling et al. have tested five patients who received corticosteroids during hospitalization. The duration of viral RNA detection for throat swabs and feces in the corticosteroid treatment group was longer than that in the non-corticosteroid treatment group, which were 15 days compared to 8.0 days (P=0.013) and 20 days compared to 11 days (P<0.001), respectively. [17] The duration of viral shedding was significantly higher in patients treated with glucocorticoids for more than 10 days compared to those received treatment for less than 10 days. [20] This could be a recommendation against steroid management if no other comorbid conditions require such treatment.
Our study revealed the range duration of 4.5 to 48 days of positive stool SARS-CoV-2 RT-PCR in patients with COVID-19 and 51% of the cases had positive tests after the respiratory tests turned negative. Chen et al. identified that patients who tested positive in stool samples can continue fecal viral shedding after negative conversion in respiratory samples for 6 to 10 days. [12] Wu et al. reported that the stool of 4 out of 41 patients turned negative for SARS CoV-2 before the respiratory samples. [14] In the same series, 32 patients continued with fecal shedding of the virus after the respiratory samples tested negative, while the respiratory and stool samples of 5 patients turned negative on the same day. In addition, one patient continued testing positive in stool for 33 days after negative respiratory samples. Xiao et al. reported that 17 out of 39 patients (44%) who tested positive for SARS-CoV-2 by RT-PCR continued testing positive for SARS-CoV-2 after their respiratory samples turned negative. [2] It has been evident that viral detection in stool usually extends beyond that of the respiratory samples, however an exact pathophysiology is not yet clear. The longest duration of SARS CoV-2 RT-PCR positive in stool was 48 days and the longest viral positive stool after clearance of the upper respiratory samples was 33 days. [14] This adds more concern about when to announce to a patient that is free of the COVID-19 disease.
Our study revealed that a mean of 47.5% of patients with positive stool SARS-CoV-2 RT-PCR had at least one GI symptom either as a sole presentation or in combination with the respiratory symptoms. Interestingly, only 47% of patients with positive fecal RT-PCR for SARS-CoV-2 had gastrointestinal symptoms. Current protocols encourage discharging infected patients after relief of symptoms and double negative RT-PCR testing of nasopharyngeal swabs. However, this can be not a favorably safe outcome in controlling further viral transmission, especially after proving positive anal swabs in the setting of negative oral test. Our study has some strengths and limitations. One of the strengths of our study is that to our knowledge this is the most updated review of GI COVID-19 and positive stool SARS-CoV-2 RT-PCR that included studies published until June 30, 2020. Also, in our review we reported the largest pool of 707 COVID-19 patients tested in stool samples for SARS-CoV-2, among other published reviews. Some of the weaknesses are the fact that most of the studies are from China and only one study from the US therefore this is a limitation for the data generalizability. Secondly, all the studies were observational with a small sample size. Regardless of these limitations, our study is very timely for enhancing the knowledge of GI COVID-19 in relation to the presence of positive stool SARS-CoV-2 RT-PCR.
In conclusion, our study suggests that about a half of COVID-19 patients have positive stool SARS-CoV-2 RT-PCR and 51% of patients have positive stool SARS-CoV-2 RT-PCR after the respiratory samples became negative for SARS-CoV-2 RT-PCR. In addition, at least one GI symptom was reported in 47.5% of patients with positive stool SARS-CoV-2 RT-PCR.