Gastrointestinal Endoscopy in the Era of COVID-19

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, 1 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. 2 ACE2 messenger RNA is highly expressed and stabilized by B0AT1 in gastrointestinal system, 3 , 4 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. Methods 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 China Disease Control and Prevention–standardized quantitative polymerase chain reaction assay. 5 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. Figure 1 Images of histologic and immunofluorescent staining of gastrointestinal tissues. Shown are images of histologic and immunofluorescent staining of esophagus, stomach, duodenum, and rectum. The scale bar in the histologic image represents 100 μm. The scale bar in the immunofluorescent image represents 20 μm. Results 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). Discussion 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, 6 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, 7 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), 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. 8 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.

Due to the rapid spread and increasing number of coronavirus disease 19 (COVID-19) cases caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapid and accurate detection of virus and/or disease is increasingly vital to control the sources of infection and help patients to prevent the illness progression. Since December 2019, there has been considerable challenge regarding the use of nucleic acid test or clinical characteristics of infected patients as the reference standard to make a definitive diagnose of COVID-19 patients. As the early diagnosis of COVID-19 is critical for prevention and control of this pandemic, clinical characteristics cannot alone define the diagnosis of COVID-19, especially for patients presenting early-onset of symptoms. Along with the advancement in medical diagnosis, nucleic acid detection-based approaches have become a rapid and reliable technology for viral detection. Among nucleic acid tests, the polymerase chain reaction (PCR) method is considered as the ‘gold standard’ for the detection of some viruses and is characterized by rapid detection, high sensitivity, and specificity. As such, real-time reverse transcriptase-PCR (RT-PCR) is of great interest today for the detection of SARS-CoV-2 due to its benefits as a specific and simple qualitative assay [1–3]. Moreover, real-time RT-PCR has adequate sensitivity to help us much for diagnosing early infection. Therefore, the ‘criterion-referenced’ real-time RT-PCR assay can be considered as a main method to be applied to detect the causative agent of COVID-19, SARS-CoV-2. An important issue with the real-time RT-PCR test is the risk of eliciting false-negative and false-positive results. It is reported that many ‘suspected’ cases with typical clinical characteristics of COVID-19 and identical specific computed tomography (CT) images were not diagnosed [4]. Thus, a negative result does not exclude the possibility of COVID-19 infection and should not be used as the only criterion for treatment or patient management decisions. It seems that combination of real-time RT-PCR and clinical features facilitates management of SARS-CoV-2 outbreak. Several factors have been proposed to be associated with the inconsistency of real-time RT-PCR [5]. In the following, we attempt to discuss various challenges regarding the detection of SARS-CoV-2 by real-time RT-PCR. It is expected that this could provide beneficial information for the comprehension of the limitations of the obtained results and to improve diagnosis approaches and control of the disease. It is well known that results from real-time RT-PCR using primers in different genes can be affected by the variation of viral RNA sequences. Genetic diversity and rapid evolution of this novel coronavirus have been observed in different studies [6,7]. False-negative results may occur by mutations in the primer and probe target regions in the SARS-CoV-2 genome. Although it was attempted to design the real-time RT-PCR assay as precisely as possible based on the conserved regions of the viral genomes, variability causing mismatches between the primers and probes and the target sequences can lead to decrease in assay performance and potential false-negative results. In this regard, multiple target gene amplification could be used to avoid invalid results. Several types of SARS-CoV-2 real-time RT-PCR kit have been developed and approved rapidly, but with different quality. Importantly, the sensitivity and specificity of the real-time RT-PCR test is not 100%. All of them behind the laboratory practice standard and personnel skill in the relevant technical and safety procedures explain some of the false-negative results. According to the natural history of the COVID-19 and viral load kinetics in different anatomic sites of the patients, sampling procedures largely contribute to the false-negative results. Optimum sample types and timing for peak viral load during infections caused by SARS-CoV-2 remain to be fully determined. A study has reported sputum as the most accurate sample for laboratory diagnosis of COVID-19, followed by nasal swabs, while throat swabs were not recommended for the diagnosis [8]. They also suggested the detection of viral RNAs in bronchoalveolar lavage fluid (BALF) for the diagnosis and monitoring of viruses in severe cases. However, gathering of BALF needs both a suction tool and an expert operator, in addition to being painful to the patients. While BALF samples are not practical for the routine laboratory diagnosis and monitoring of the disease, collection of other samples such as sputum, nasal swab, and throat swab is rapid, simple, and safe. To avoid inconsistent results, it would be better to use different specimen types (stool and blood) besides respiratory specimen during different stages. It is worth noting that samples should be obtained by dacron or polyester flocked swabs and should reach the laboratory as soon as possible after collection. False-negative results may occur due to the presence of amplification inhibitors in the sample or insufficient organisms in the sample rising from inappropriate collection, transportation, or handling. Viral load kinetics of SARS-CoV-2 infection have been described in two patients in Korea, suggesting a different viral load kinetics from that of previously reported other coronavirus infections [9]. In the first patient, the virus was detected from upper respiratory tract (URT) and lower respiratory tract (LRT) specimens on days 2 and 3 of symptom onset, respectively. On day 5, the viral load was increased from day 3 in the LRT specimen. However, the viral loads decreased from around day 7 in both URT and LRT specimens. Real-time RT-PCR continued to be positive at a low level until day 13 (LRT specimens) and day 14 (URT specimens). Finally, the assay became undetectable for two consecutive days from day 14 (LRT specimen) and day 15 (URT specimen), respectively. In the second patient, SARS-CoV-2 was detected in both URT and LRT specimens on day 14 of symptom onset. However, the initial viral loads were relatively lower than those of patient 1 in whom the test was performed on day 2 of symptom onset. From day 18 (URT specimen) and day 20 (LRT specimen), real-time RT-PCR became undetectable for two consecutive days, respectively. URT sample of day 25 was again positive for RdRp and E genes. However, it was interpreted as negative due to high Ct value of the RdRp gene (Ct value of 36.69). These findings indicate the different viral load kinetics of SARS-coV-2 in different patients, suggesting that sampling timing and period of the disease development play an important role in real-time RT-PCR results. Finally, the Centers for Disease Control and Prevention (CDC) has designed a SARS-CoV-2 Real-Time RT-PCR Diagnostic Panel to minimize the chance of false-positive results [10]. In accordance, the negative template control (NTC) sample should be negative, showing no fluorescence growth curves that cross the threshold line. The occurrence of false positive with one or more of the primer and probe NTC reactions is indicative of sample contamination. Importantly, the internal control should be included to help identify the specimens containing substances that may interfere with the extraction of nucleic acid and PCR amplification. Because of the several risks to patients in the event of a false-positive result, all clinical laboratories using this test must follow the standard confirmatory testing and reporting guidelines based on their proper public health authorities. 1. Expert opinion In conclusion, according to the mentioned reasons, the results of real-time RT-PCR tests must be cautiously interpreted. In the case of real-time RT-PCR negative result with clinical features suspicion for COVID-19, especially when only upper respiratory tract samples were tested, multiple sample types in different time points, including from the lower respiratory tract if possible, should be tested. Importantly, combination of real-time RT-PCR and clinical features especially CT image could facilitate disease management. Proper sampling procedures, good laboratory practice standard, and using high-quality extraction and real-time RT-PCR kit could improve the approach and reduce inaccurate results.

Summary Background There is little published evidence on the gastrointestinal features of COVID‐19. Aims To report on the gastrointestinal manifestations and pathological findings of patients with COVID‐19, and to discuss the possibility of faecal transmission. Methods We have reviewed gastrointestinal features of, and faecal test results in, COVID‐19 from case reports and retrospective clinical studies relating to the digestive system published since the outbreak. Results With an incidence of 3% (1/41)‐79% (159/201), gastrointestinal symptoms of COVID‐19 included anorexia 39.9% (55/138)‐50.2% (101/201), diarrhoea 2% (2/99)‐49.5% (146/295), vomiting 3.6% (5/138)‐66.7% (4/6), nausea 1% (1/99)‐29.4% (59/201), abdominal pain 2.2% (3/138)‐6.0% (12/201) and gastrointestinal bleeding 4% (2/52)‐13.7% (10/73). Diarrhoea was the most common gastrointestinal symptom in children and adults, with a mean duration of 4.1 ± 2.5 days, and was observed before and after diagnosis. Vomiting was more prominent in children. About 3.6% (5/138)‐15.9% (32/201) of adult and 6.5% (2/31)‐66.7% (4/6) of children patients presented vomiting. Adult and children patients can present with digestive symptoms in the absence of respiratory symptoms. The incidence of digestive manifestations was higher in the later than in the early stage of the epidemic, but no differences in digestive symptoms among different regions were found. Among the group of patients with a higher proportion of severe cases, the proportion of gastrointestinal symptoms in severe patients was higher than that in nonsevere patients (anorexia 66.7% vs 30.4%; abdominal pain 8.3% vs 0%); while in the group of patients with a lower severe rate, the proportion with gastrointestinal symptoms was similar in severe and nonsevere cases (nausea and vomiting 6.9% vs 4.6%; diarrhoea 5.8% vs 3.5%). Angiotensin converting enzyme 2 and virus nucleocapsid protein were detected in gastrointestinal epithelial cells, and infectious virus particles were isolated from faeces. Faecal PCR testing was as accurate as respiratory specimen PCR detection. In 36% (5/14)‐53% (39/73) faecal PCR became positive, 2‐5 days later than sputum PCR positive. Faecal excretion persisted after sputum excretion in 23% (17/73)‐82% (54/66) patients for 1‐11 days. Conclusions Gastrointestinal symptoms are common in patients with COVID‐19, and had an increased prevalence in the later stage of the recent epidemic in China. SARS‐CoV‐2 enters gastrointestinal epithelial cells, and the faeces of COVID‐19 patients are potentially infectious.