Evidence for Gastrointestinal Infection of SARS-CoV-2

Abstract

See Covering the Cover synopsis on page 1515. See Covering the Cover synopsis on page 1515. Since the novel coronavirus (SARS-CoV-2) was identified in Wuhan, China, at the end of 2019, the virus has spread to 32 countries, infecting more than 80,000 people and causing more than 2600 deaths globally. The viral infection causes a series of respiratory illnesses, including severe respiratory syndrome, indicating that the virus most likely infects respiratory epithelial cells and spreads mainly via respiratory tract from human to human. However, viral target cells and organs have not been fully determined, impeding our understanding of the pathogenesis of the viral infection and viral transmission routes. According to a recent case report, SARS-CoV-2 RNA was detected in a stool specimen,1Holshue M.L. et al.N Engl J Med. 2020; 382: 929-936Crossref PubMed Scopus (3818) Google Scholar raising the question of viral gastrointestinal infection and a fecal-oral transmission route. It has been proven that SARS-CoV-2 uses angiotensin-converting enzyme (ACE) 2 as a viral receptor for entry process.2Zhou P. et al.Nature. 2020; 579: 270-273Crossref PubMed Scopus (12486) Google Scholar ACE2 messenger RNA is highly expressed and stabilized by the neutral amino acid transporter B0AT1 (SLC6A19) in gastrointestinal system,3Harmer D. et al.FEBS Lett. 2002; 532: 107-110Crossref PubMed Scopus (600) Google Scholar,4Yan R. et al.Science. 2020; 367: 1444-1448Crossref PubMed Scopus (3014) Google Scholar providing a prerequisite for SARS-CoV-2 infection. To further investigate the clinical significance of SARS-CoV-2 RNA in feces, we examined the viral RNA in feces from 71 patients with SARS-CoV-2 infection during their hospitalizations. The viral RNA and viral nucleocapsid protein were examined in gastrointestinal tissues from 1 of the patients. From February 1 to 14, 2020, clinical specimens, including serum, nasopharyngeal, and oropharyngeal swabs; urine; stool; and tissues from 73 hospitalized patients infected with SARS-CoV-2 were obtained in accordance with China Disease Control and Prevention guidelines and tested for SARS-CoV-2 RNA by using the Chinese Center for Disease Control and Prevention–standardized quantitative polymerase chain reaction assay.5Li Q. et al.N Engl J Med. 2020; 382: 1199-1207Crossref PubMed Scopus (9428) Google Scholar Clinical characteristics of the 73 patients are shown in Supplementary Table 1. The esophageal, gastric, duodenal, and rectal tissues were obtained from 1 of the patients by using endoscopy. The patient’s clinical information is described in the Supplementary Case Clinical Information and Supplementary Table 2. Histologic staining (H&E) as well as viral receptor ACE2 and viral nucleocapsid staining were performed as described in the Supplementary Methods. The images of fluorescent staining were obtained by using laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging, Oberkochen, Germany) and are shown in Figure 1. This study was approved by the Ethics Committee of The Fifth Affiliated Hospital, Sun Yat-sen University, and all patients signed informed consent forms. From February 1 to 14, 2020, among all of the 73 hospitalized patients infected with SARS-CoV-2, 39 (53.42%), including 25 male and 14 female patients, tested positive for SARS-CoV-2 RNA in stool, as shown in Supplementary Table 1. The age of patients with positive results for SARS-CoV-2 RNA in stool ranged from 10 months to 78 years old. The duration time of positive stool results ranged from 1 to 12 days. Furthermore, 17 (23.29%) patients continued to have positive results in stool after showing negative results in respiratory samples. Gastrointestinal endoscopy was performed on a patient as described in the Supplementary Case Clinical Information. As shown in Figure 1, the mucous epithelium of esophagus, stomach, duodenum, and rectum showed no significant damage with H&E staining. Infiltrate of occasional lymphocytes was observed in esophageal squamous epithelium. In lamina propria of the stomach, duodenum, and rectum, numerous infiltrating plasma cells and lymphocytes with interstitial edema were seen. Importantly, viral host receptor ACE2 stained positive mainly in the cytoplasm of gastrointestinal epithelial cells (Figure 1). We observed that ACE2 is rarely expressed in esophageal epithelium but is abundantly distributed in the cilia of the glandular epithelia. Staining of viral nucleocapsid protein was visualized in the cytoplasm of gastric, duodenal, and rectum glandular epithelial cell, but not in esophageal epithelium. The positive staining of ACE2 and SARS-CoV-2 was also observed in gastrointestinal epithelium from other patients who tested positive for SARS-CoV-2 RNA in feces (data not shown). In this article, we provide evidence for gastrointestinal infection of SARS-CoV-2 and its possible fecal-oral transmission route. Because viruses spread from infected to uninfected cells,6Xiao F. et al.PLoS Pathog. 2014; 10e1004128Crossref PubMed Scopus (79) Google Scholar viral-specific target cells or organs are determinants of viral transmission routes. Receptor-mediated viral entry into a host cell is the first step of viral infection. Our immunofluorescent data showed that ACE2 protein, which has been proven to be a cell receptor for SARS-CoV-2, is abundantly expressed in the glandular cells of gastric, duodenal, and rectal epithelia, supporting the entry of SARS-CoV-2 into the host cells. ACE2 staining is rarely seen in esophageal mucosa, probably because the esophageal epithelium is mainly composed of squamous epithelial cells, which express less ACE2 than glandular epithelial cells. Our results of SARS-CoV-2 RNA detection and intracellular staining of viral nucleocapsid protein in gastric, duodenal, and rectal epithelia demonstrate that SARS-CoV-2 infects these gastrointestinal glandular epithelial cells. Although viral RNA was also detected in esophageal mucous tissue, absence of viral nucleocapsid protein staining in esophageal mucosa indicates low viral infection in esophageal mucosa. After viral entry, virus-specific RNA and proteins are synthesized in the cytoplasm to assemble new virions,7Weiss S.R. et al.Microbiol Mol Biol Rev. 2005; 69: 635-664Crossref PubMed Scopus (769) Google Scholar which can be released to the gastrointestinal tract. The continuous positive detection of viral RNA from feces suggests that the infectious virions are secreted from the virus-infected gastrointestinal cells. Recently, we and others have isolated infectious SARS-CoV-2 from stool (unpublished data, 2020), confirming the release of the infectious virions to the gastrointestinal tract. Therefore, fecal-oral transmission could be an additional route for viral spread. Prevention of fecal-oral transmission should be taken into consideration to control the spread of the virus. Our results highlight the clinical significance of testing viral RNA in feces by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) because infectious virions released from the gastrointestinal tract can be monitored by the test. According to the current Centers for Disease Control and Prevention guidance for the disposition of patients with SARS-CoV-2, the decision to discontinue transmission-based precautions for hospitalized patients with SARS-CoV-2 is based on negative results rRT-PCR testing for SARS-CoV-2 from at least 2 sequential respiratory tract specimens collected ≥24 hours apart.8Centers for Disease Control and Preventionhttps://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-hospitalized-patients.htmlGoogle Scholar However, in more than 20% of patients with SARS-CoV-2, we observed that the test result for viral RNA remained positive in feces, even after test results for viral RNA in the respiratory tract converted to negative, indicating that the viral gastrointestinal infection and potential fecal-oral transmission can last even after viral clearance in the respiratory tract. Therefore, we strongly recommend that rRT-PCR testing for SARS-CoV-2 from feces should be performed routinely in patients with SARS-CoV-2 and that transmission-based precautions for hospitalized patients with SARS-CoV-2 should continue if feces test results are positive by rRT-PCR testing. On January 17, 2020, a 78-year-old man, who, along with his wife, had come from Wuhan 6 days earlier to visit his daughter, presented to the outpatient clinic at our hospital in Zhuhai, Guangdong Province, China, with a 7-day cough and fever. He was admitted to the negative-pressure isolation room in the Department of Infectious Diseases at our hospital as a suspected case of SARS-CoV-2 infection. On admission, the physical examination showed a body temperature of 37.5°C, blood pressure of 105/56 mm Hg, pulse of 67 beats per minute, and respiratory rate of 22 breaths per minute with oxygen saturation of 97%. On physical examination, auscultation indicated rhonchi and cracks on bilateral lungs. Initial arterial blood gas analysis showed the arterial partial pressure of oxygen (Pao2)/fraction of inspiration oxygen (Fio2) was 176 mmHg. Nasopharyngeal and oropharyngeal swab specimens tested positive by rRT-PCR for SARS-CoV-2. Chest computed tomography presented with multiple ground-glass opacities, coinciding with previous reports,1Kanne J.P. Chest CT Findings in 2019 Novel Coronavirus (2019-nCoV) Infections from Wuhan, China: Key Points for the Radiologist.Radiology. 2020; : 200241Google Scholar,2Huang C. Wang Y. Li X. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; Abstract Full Text Full Text PDF Scopus (28324) Google Scholar showing evidence of pneumonia in both the left and right lungs. The patient’s wife and daughter tested positive for SARS-CoV-2 RNA and were admitted to the hospital on January 18, 2020. On hospital days 1 through 3, the patient remained febrile, with stable vital signs. The oxygen saturation remained above 95% with high-flow oxygen therapy. Empiric antimicrobials with oseltamivir and moxifloxacin was given during this period of time. On hospital day 4, the patient developed severe respiratory distress, with the Pao2/Fio2 decreasing to 130 mmHg and was immediately transferred to the intensive care unit, receiving an intubation and mechanical ventilation. Along with sedation, prone-position mechanical ventilation was applied for 12 hours per day, and low tidal volume was set. The Pao2/Fio2 increased to 350 mmHg immediately after intubation but decreased gradually again in the following several days to the lowest level of 70 at 10 days after admission. Meanwhile, the chest radiograph showed extensive bilateral consolidation; emergent veno-venous extracorporeal membrane oxygenation was applied at the same day. On day 10, coffee ground gastric contents were observed from the gastric drainage tube and fecal occult blood tested positive, indicating upper gastrointestinal bleed. Gastrointestinal endoscopy was performed to determine the exact location of bleeding. Mucosa damage in the esophagus was observed under endoscopy. Biopsy samples were taken from esophagus, gastric, duodenum, and colon for histopathologic and immunofluorescent staining. One day after treatment with octreotide, esomeprazole, etc, gastrointestinal bleeding stopped. As of February 12, 2020, the patient remained hospitalized. The vital signs were stable with mechanical ventilation, veno-venous extracorporeal membrane oxygenation, and low-dose vasopressors. There is no obvious evidence of other organ dysfunction. Esophageal, gastric, duodenal, and rectal tissues were obtained using endoscopy on day 10. Samples were embedded with paraffin and then stained with H&E. For immunofluorescent staining, 3-μm-thick sections were dewaxed in xylene, rehydrated in alcohol, and washed in distilled water 3 times before microwave repair. After washing 3 times in phosphate-buffered saline with Tween (PBST), sections were incubated with 10% goat serum in PBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (anti-ACE2, Sino Biological, Beijing, China, 10108-T56, 1:500; anti-nucleoprotein, Sino Biological, 40143-T62, 1:500). The slides were incubated with secondary antibodies (Alexa Fluor 647–conjugated goat anti-rabbit IgG, bs-0296G-AF647, 1:100; Bioss, London, UK) for 1 hour at room temperature followed by washing 3 times with PBST. Nuclei were then counterstained with 4′,6-diamidino-2-phenylindole after washing 3 times with PBST. Slides were imaged by using a laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging). Supplementary Table 1Clinical Characteristics of the 73 Hospitalized Patients Infected With SARS-CoV-2S+R+S+(R+S+/S+)%∼R+S+(∼R+S+/R+S+)%∼R-S+(∼R-S+/S+)%∼R-S-(∼R-S-/R+S+)%No. or % of patients733953.42%615.38%1743.59%1641.03%Female321443.75%214.29%535.71%750.00%Male412569.98%416.00%1248.00%936.00%Age (years)43 (0.83-7)49 (0.83-78)/52.5 (3-78)/44 (0.83-69)/47 (19-75)/Tumors7342.86%133.00%133.00%133.00%Surgical history17847.06%112.50%450.00%337.50%Ulcer00/0/0/0/Smoking9444%00250.00%250.00%Respiratory symptoms533056.60%413.33%1343.33%1343.33%Typical chest CT663654.55%513.89%1644.44%1541.67%Diarrhea261765.38%211.76%635.29%952.94%Gastrointestinal bleeding10440%125.00%125.00%250.00%Use of corticosteroid211257.14%216.67%325.00%758.33%Antibiotic therapy603552.05%617.14%1440.00%1542.86%Antiviral therapy733849.32%615.79%1642.11%1642.11%PPIs therapy512447.06%416.67%625.00%1458.33%NSAID12650.00%116.67%233.33%350.00%ICU44100%125.00%125.00%250.00%CT, computerized tomography; ICU, intensive care unit; NSAID, nonsteroidal anti-inflammatory drugs; PPI, proton pump inhibitor; R, respiratory specimens; R+, SARS-CoV-2 RNA tested positive in R on hospital admission; R+S+, SARS-CoV-2 RNA tested positive in both R and S during hospitalization; ∼R+S+, SARS-CoV-2 RNA remained positive in both R and S until the date of writing the manuscript on February 14th, 2020; ∼R-S+, SARS-CoV-2 RNA converted to negative in R during hospitalization but remained positive in S until the date of writing the manuscript on February 14th, 2020; ∼R-S-, SARS-CoV-2 RNA converted to negative in both R and S during hospitalization; S, stool specimens; S+, SARS-CoV-2 RNA tested positive in stool during hospitalization; /, not applicable. Open table in a new tab Supplementary Table 2Timeline of Detection of Viral RNA in Different Specimens of the Patient Infected With SARS-CoV-2SpecimenDay 1Day 2Day 3Day 5Day 7Day 9Day 10Day 11Day 13Day 14Day 16Day 18Day 20Day 21Day 22Day 24Day 26RespiratoryNTPositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositiveStoolNTNTNegativeNegativeNegativePositivePositivePositivePositivePositivePositiveNTPositivePositivePositivePositivePositiveSerumNTNTNegativeNegativeNegativeNegativeNegativePositiveNegativeNegativeNegativeNTNTNTNegativeNTNegativeUrineNTNTNegativeNegativeNegativeNegativeNegativeNTNegativeNTPositiveNTNTNTNTNTNTEsophagusNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTStomachNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTDuodenumNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTRectumNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTNT, denotes not tested. Open table in a new tab CT, computerized tomography; ICU, intensive care unit; NSAID, nonsteroidal anti-inflammatory drugs; PPI, proton pump inhibitor; R, respiratory specimens; R+, SARS-CoV-2 RNA tested positive in R on hospital admission; R+S+, SARS-CoV-2 RNA tested positive in both R and S during hospitalization; ∼R+S+, SARS-CoV-2 RNA remained positive in both R and S until the date of writing the manuscript on February 14th, 2020; ∼R-S+, SARS-CoV-2 RNA converted to negative in R during hospitalization but remained positive in S until the date of writing the manuscript on February 14th, 2020; ∼R-S-, SARS-CoV-2 RNA converted to negative in both R and S during hospitalization; S, stool specimens; S+, SARS-CoV-2 RNA tested positive in stool during hospitalization; /, not applicable. NT, denotes not tested. Covering the CoverGastroenterologyVol. 158Issue 6PreviewA Swedish nationwide study showed an increased risk of inflammatory bowel disease among individuals with microscopic colitis. Full-Text PDF Presence of SARS-Coronavirus-2 in the Ileal Mucosa: Another Evidence for Infection of GI Tract by This VirusGastroenterologyVol. 159Issue 4PreviewWe have read with great interest the recent article published entitled “Evidence for Gastrointestinal Infection of SARS-CoV-2.” In this article, Xiao et al1 report how ≤20% of patients with negative viral RNA in their respiratory tract present positive RNA in feces. They present a case of a patient with severe pneumonia owing to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and gastrointestinal bleeding, with viral RNA in the gastrointestinal mucosa, as well as the presence by immunofluorescence of abundant angiotensin-converting enzyme (ACE)2 receptors in the gastrointestinal tract. Full-Text PDF Fecal–Oral Transmission of SARS-COV-2: Practical ImplicationsGastroenterologyVol. 159Issue 4PreviewWe read with great interest the study by Xiao et al1 on evidence for gastrointestinal infection of coronavirus disease-19 (COVID-19). Testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA in stool specimens of 73 hospitalized patients resulted in virus detection in 53.4% of patients, both with and without gastrointestinal manifestations (ie, diarrhea, nausea, vomiting, gastrointestinal bleeding). In addition, COVID-19 nucleic acid was positive in feces of 23.3% of patients in which respiratory samples had already turned negative. Full-Text PDF Gastrointestinal ACE2, COVID-19 and IBD: Opportunity in the Face of Tragedy?GastroenterologyVol. 159Issue 4PreviewWe read with interest the articles by Xiao et al1 and Du et al2 regarding severe acute respiratory syndrome coronavirus-2 (SARS Co-V 2) shedding in feces, staining of viral nucleocapsid protein in the cytoplasm of gastrointestinal epithelial cells, and the characterization of angiotensin-converting enzyme 2 (ACE2) receptors across tissues in the human body. The relationship between coronavirus disease 2019 (COVID-19), intestinal ACE2 expression, and gastrointestinal symptoms is worth exploring further, and may offer unique clues to the pathogenesis of intestinal inflammation. Full-Text PDF