key: cord-329193-xuxbqbsf authors: Park, Soo-kyung; Lee, Chil-Woo; Park, Dong-Il; Woo, Hee-Yeon; Cheong, Hae Suk; Shin, Ho Cheol; Ahn, Kwangsung; Kwon, Min-Jung; Joo, Eun-Jeong title: Detection of SARS-CoV-2 in Fecal Samples from Patients with Asymptomatic and Mild COVID-19 in Korea date: 2020-06-10 journal: Clin Gastroenterol Hepatol DOI: 10.1016/j.cgh.2020.06.005 sha: doc_id: 329193 cord_uid: xuxbqbsf Abstract: Background & Aims Although COVID-19 is characterized by fever and respiratory symptoms, some patients have no or mild symptoms. SARS-CoV-2 has been detected in feces of patients. We investigated gastrointestinal symptoms and shedding of virus into feces of patients with asymptomatic or mild COVID-19. Methods We collected data from 46 patients (median age, 26 years; 46% men) with asymptomatic or mild COVID-19 (without fever and pneumonia) and prolonged respiratory shedding of SARS-CoV-2, quarantined from April 4, 2020 through April 24, 2020 in Korea. Respiratory specimens included upper respiratory specimens (nasopharyngeal and oropharyngeal swabs) and lower respiratory specimens (sputum) and were collected twice per week. The median interval between COVID-19 diagnosis to the start of fecal sample collection was 37 days (range, 29–41); 213 stool specimens were collected from 46 patients. We used real-time reverse transcription PCR to detect SARS-CoV-2 in the respiratory and fecal specimens. Results Gastrointestinal manifestations were observed in 16 of the 46 patients (35%); diarrhea was the most common (15%), followed by abdominal pain (11%), dyspepsia (11%), and nausea (2%). Virus RNA was detected in feces from 2 patients without gastrointestinal symptoms (4%). Mean cycle threshold values from the time of quarantine to the time of fecal collection tended to be lower in patients with virus detected in fecal samples than patients without virus in fecal samples (29.91 vs 33.67 in the first week; 29.47 vs 35.71 in the fifth week, respectively). Shedding of virus into feces persisted until day 50 after diagnosis; fecal samples began to test negative before or at approximately the time that respiratory specimens also began to test negative. Conclusions In an analysis of fecal and respiratory specimens from patients with COVID-19 in quarantine in Korea, we found that the gastrointestinal tract could be a route of transmission of SARS-CoV-2 even in patients with asymptomatic or mild disease, with no gastrointestinal symptoms. The viral load of the respiratory specimens appears be related to shedding of the virus into feces in this group of patients. Only a few weeks after the first case of coronavirus disease 2019 was reported in Wuhan, China, the first case of COVID-19 was confirmed in the Republic of Korea on January 20, 2020. 1 On February 18, when a suspicious index case was identified in a religious group, a sharp increase in the number of COVID-19 cases was observed, with most infections being reported in specific clusters and geographical regions; the highest number of COVID-19 cases was reported in Daegu, followed by Gyeongbuk. 1 It is well established that most patients with COVID-19 have fever along with respiratory symptoms, and human-to-human transmission occurs among close contacts, mainly through respiratory droplets and direct contact. 4, 5 However, the diseases caused by novel coronaviruses are commonly found to be accompanied with gastrointestinal (GI) manifestations, 6, 7 and these viruses may be viable in environments/conditions that facilitate fecal-oral transmission. 8 In the SARS outbreak of 2002-2003, viral RNA in the feces was detectable for more than a month in some patients. 9 In experimental conditions, the stability of SARS-CoV-2 is found to be similar to that of SARS-CoV-1. 10 However, SARS-CoV-2 and SARS-CoV-1 have different viral kinetics at high viral loads, with prolonged shedding in the upper respiratory tract even in patients with asymptomatic and mildly symptomatic disease (not published, in process); hence, the GI symptoms and fecal viral shedding in prolonged respiratory shedders with asymptomatic or mild COVID-19, who were in quarantine for over a month, must be evaluated. We conducted a prospective cohort study involving the patients diagnosed with asymptomatic or mild COVID- 19 LTC was a human resource training center of Samsung, but on March 2, 2020, "out-ofhospital quarantine and care" for patients with asymptomatic or mild COVID-19 was mandated by the KCDC. Therefore, the accommodation facilities including Youngdeok Samsung human resource training centers were transformed to "LTC", and COVID-19 patients in Daegu and Gyeongbuk were quarantined there on March 4. 11 This center has 300 individual rooms in seven buildings, and each patient was quarantined in a single-bed room. COVID-19 was confirmed by real-time reverse-transcriptase polymerase-chain reaction (rRT-PCR) assay for SARS-CoV-2 in upper respiratory tract specimens using nasopharyngeal (NP) and oropharyngeal (OP) swabs and/or lower respiratory tract specimens, i.e., sputum. 12 Although none of the patients showed fever or respiratory symptoms with suspected pneumonia, rRT-PCR assays were performed as they were close-contacts. This study was approved by the Institutional Review Board of Kang Buk Samsung Medical Center (KBSMC-2020-03-065). Written informed consent was obtained from all the patients. Starting from March 4, 2020, all quarantined patients' respiratory specimens were collected at Youngdeok Samsung LTC every Monday by medical teams tested for SARS-CoV-2 nucleic acid by rRT-PCR assay. Respiratory specimens included upper respiratory specimens (NP swab and OP swab) and lower respiratory specimens (sputum). Those who showed cyclic threshold (Ct) values were reported the following day. Then, the specimens showing SARS-CoV-2-positive results were re-tested the following Monday. The patients whose specimens showed negative or inconclusive results on Monday, were re-tested on Wednesday, and in cases of inconclusive and negative results on Monday and Wednesday, an additional test was performed on Thursday. Since April 13, respiratory specimens were collected on Mondays and Thursdays regularly. Only patients with two consecutive negative results on RT-PCR assay at least 24 to 48 hours apart were discharged from the LTC. Official reports including past medical history and symptoms at the time of diagnosis were obtained from the district public health centers. Since the beginning of the operation of Youngdeok Samsung LTC, 11 medical staff (four doctors and seven nurses) had been carefully monitoring the patients' symptoms twice a day (9 am and 5 pm) by a mobile application. Thermometers were given to each patient, and the patients were monitored for the following symptoms every day: fever (over 37.5°C), headache, myalgia, cough, sputum, rhinorrhea, nasal obstruction, chest pain, anosmia, fatigue, nausea, vomiting, diarrhea, and loss of appetite. For this study, we collected additional data on underlying digestive diseases including reflux esophagitis, gastritis, gastric ulcer, irritable bowel disease, inflammatory bowel disease, fatty liver, viral hepatitis, cirrhosis, pancreatitis, cholecystitis, and cholangitis. Since the day we started collecting fecal specimens, we also collected additional data on GI manifestations, such as abdominal pain and dyspepsia, once a day by phone call, as they were not included in the list of symptoms that were investigated using the mobile application. Data regarding abdominal pain and dyspepsia that occurred at the time of diagnosis and before the day we started collecting fecal specimens were collected by patient recall. To ensure proper collection and testing of fecal specimens for SARS-CoV-2, fecal specimens of patients who were admitted to KangBuk Samsung hospital were tested in various conditions (See supplementary Method and Table for further details). Considering the above results, from April 4 and 5, 2020 for groups 1 and 2, respectively, stool collection kits and refrigerators were provided in each room of the LTC and stool collection was conducted by patients until April 24 (See supplementary Method for further details). Among them, seven patients agreed to provide fecal specimens. Finally, a total of 46 patients were enrolled in this study ( Figure 1 ). The clinical characteristics of study participants with asymptomatic and mildly symptomatic COVID-19 are described in Table 1 . The median age was 26, and 46% were men. The median number of days from diagnosis to quarantine was 6.5 days (range, 4.0-18.0 days). The median number of days from diagnosis to fecal sample examination was 37.0 (range, 29.0-41.0); this indicates that patients had prolonged respiratory shedding of SARS-CoV-2 for a median of 37 days at the start of fecal examination. We followed the patients up to 61 days (median, 52; range, 39-61). Medical history of digestive diseases was reported in 24% of the study population: reflux esophagitis (13%), gastritis (7%), irritable bowel disease (7%), and fatty liver (2%). A total of 38 patients (83%) complained of symptoms during the monitoring period after diagnosis, mostly sputum (57%) and coughing (37%). Detailed digestive symptoms are shown in Table 2 . A total of 16 patients (35%) presented with digestive symptoms, and diarrhea was the most common (15%), followed by abdominal pain (11%), dyspepsia (11%), and nausea (2%). Only one patient (2%) presented with abdominal pain, but no respiratory symptoms at diagnosis. Most of the patients (94%, 15/16) complained of digestive symptoms within one month from diagnosis. Five patients presented with digestive symptoms after1 month from diagnosis (Supplement Figure) . Eight (17%) patients did not have any symptoms (general or digestive) from the time of diagnosis to the last follow-up. Fecal specimens were collected from April 4 to April 24 (21 days), and the median interval between COVID-19 diagnosis to the start of fecal sample collection was 37 days (range, 29-41) (Table 1, Figure 2 ). A total of 213 fecal specimens were collected from 46 patients ( Figure 2 ). During the study period, 30 (65%) patients were discharged from the LTC, and one patient was transferred to a hospital for persistent fever. After follow-up, she was discharged at day 62 since two consecutive negative results were obtained from her respiratory specimens on day 57 and day 61 (Figure 2 ). We investigated the chronological changes in RT-PCR test results in both fecal and respiratory specimens of the two patients with positive fecal samples ( Figure 4 ). In patient-40, RT-PCR results of both respiratory and fecal specimens were positive on day 50, but fecal specimens converted to negative on day 52, and the negative results were sustained at day 54, whereas the respiratory specimens still yielded positive results. Respiratory specimens were converted to negative at day 57 (Figures 2 & 4A) . In patient-7, the RT-PCR results of the fecal specimens converted to negative at day 47, and the respiratory specimens converted to negative at day 50 (Figures 2 & 4B ). In this prospective study of 46 patients with asymptomatic or mild COVID-19 in an out-ofhospital LTC, GI manifestations were found in 35% of the study participants during 2 months of evaluation, in 2% at the time of diagnosis, in 33% within 1 month from diagnosis, and in 11% after 1 month from diagnosis. Viral RNA was detected in the feces of two (4%) patients although they did not have any GI manifestations since diagnosis to discharge. Of note, the In a meta-analysis of 60 studies, comprising 4,243 patients, the pooled prevalence rate of all GI symptoms was 17.6%, and when compared to the prevalence of GI symptoms according to the severity of COVID-19, pooled prevalence rates of GI symptoms were 11.8% and 17.1% in patients with non-severe and severe COVID-19, respectively. 13 In others studies, which were not included in previous meta-analysis, the prevalence rates of GI symptoms were 11.4% (75/651), 14 50.5% (101/204), 15 and 61.1% (58/95), respectively. 16 In our study, although the enrolled patients had non-severe, mild-to-asymptomatic disease, the prevalence of GI symptoms was 34.7%. Considering the possibility of selection bias, we also investigate digestive symptoms in 22 patients who did not agree with the stool test. There were no difference in digestive symptoms and diarrhea between those who participated in the study and those who were not. (digestive symptoms, 34.8% vs.31.8%, p=0.81, diarrhea, 15.2% vs.18.2%, p=0.75) (data not shown). The reason for this high prevalence compared with that in the meta-analysis might be explained by the following reasons. First, we enrolled patients who had prolonged respiratory viral shedding of SARS-CoV-2 for at least 39 days (days since diagnosis to last follow-up; median, 52; range, 39-61), and 15 patients could not be discharged during the study period because of persistent respiratory shedding on days 49-61 ( Figure 2) . Second, we prospectively collected data from Youngdeok Samsung LTC, where medical teams carefully monitored symptoms two times a day and automatically recorded them by a mobile application, whereas due to their retrospective design, most previous studies in the meta-analysis might have underestimated the prevalence. 13 After starting fecal collection, we checked the Bristol stool scale for all fecal samples, and when we defined diarrhea as Bristol scale score of more than 6 (Supplement Figure) , the prevalence of diarrhea after 1 month of diagnosis increased up to 26%, and prevalence of all GI symptoms increased up to 57% (data not shown). The pooled prevalence rate of fecal samples that were positive for SARS-CoV-2 was 48.1% in the meta-analysis 13 , and one study reported a viral RNA positive rate of 29% (44/153) according to the number of stool specimens. 17 In our study, the viral RNA positive rates were low at 4% of patients and 6% of specimens. However, we collected fecal specimens after a median of 37.0 days from diagnosis and all our patients were non-hospitalized with asymptomatic or mild conditions, as compared with the patients in the meta-analysis who accounted for 1.3-62.3% of severe disease cases in the studies, and feces were collected at 1-33 days. 13 In another study, the viral RNA positive rate was 47.7%, with 20% of the patients being severe cases. 16 Although our patients showed prolonged respiratory shedding over 1 month, the viral loads in the respiratory specimen (mean Ct) of group 1 (n = 39) were low at 33.46 and 35.39, respectively, at the time of quarantine in LTC and at the time of fecal collection. A remarkable finding in our study is that the respiratory specimens of two patients with positive fecal samples showed low Ct values from the time of quarantine to the time of fecal collection compared to those with negative fecal samples. Thus, the Ct values of respiratory specimens may predict positive findings on fecal testing. Regarding the relation between digestive symptoms and rate of positive fecal samples, one study with 65 patients reported positive rates of 52.4% and 39.1% for patients with and without GI symptoms, respectively, without statistical significance (p = 0.31). 16 However, Cheung et el., who studied the cases of 59 patients, reported that viral RNA was more prevalent in the stool of patients with diarrhea than in the stool of those without diarrhea (38.5% vs. 8.7%; p = 0.019) 13 . In our study, two patients with positive fecal samples had no digestive symptoms from diagnosis to discharge; therefore, further large-scale studies are needed to confirm this correlation. In our study, in the two patients with positive fecal samples, the positive fecal samples converted to negative earlier than the respiratory specimens in one patient, and fecal and respiratory specimens converted to negative at a similar time in another patient. These results are not in agreement with those of previous studies, e.g., 70.3% of the fecal specimens collected after loss of virus from respiratory specimens tested positive for the virus. 13 The persistence of viral RNA in the stool samples was longer than that in respiratory specimens in 34% (13/38) of the patients. 13 However, there were 7 (53.8%, 7/13) pediatric patients and several studies demonstrated that SARS-CoV-2 may exist for a longer time in children's GI tracts than in their respiratory system. [18] [19] [20] Thus, whether viral shedding is sustained for a longer time in the respiratory system of adults than in the respiratory system of children needs to be further evaluated. One feature of respiratory viral shedding in our study was that there were dynamic changes in respiratory specimens, as even after reporting negative results, positive or inconclusive results were observed repeatedly. On the other hand, when fecal specimens were tested negative or converted to negative, the finding persisted ( Figure 2 ). Thus, it remains to be determined whether the viral RNA in the respiratory system or feces is the source of the recurrent infection. The strength of our study is that it is the first prospective cohort study involving asymptomatic and mild COVID-19 patients who were quarantined in the LTC, not hospitalized. We carefully monitored them for not only diarrhea but also other GI symptoms up to 61 days of quarantine. In addition, with systematic specimen collection protocols, we reported prolonged fecal viral shedding up to 50 days in the patients with prolonged respiratory viral shedding, indicating a correlation between the viral loads of respiratory and fecal specimens. However, there are several limitations. First, as we started collecting fecal specimens 1 month after diagnosis, the full extent of the positive findings on fecal testing, including the peak timing and extent of fecal shedding, was limited. Second, as there were only two patients with positive fecal samples, the comparison of Ct values and digestive symptoms between patients with positive and negative findings on fecal testing was also limited. Third, as we did not perform endoscopy in these patients, the understanding of viral replication in the intestinal mucosa was limited, and whether these were live virus particles or just RNA fragments released from the intestinal cells was unclear. In conclusion, we found that GI symptoms were present in 35% of the patients with asymptomatic and mild COVID-19, who had prolonged respiratory shedding of SARS-CoV-2. Viral shedding in the feces was detected in 4% of the patients who did not show any GI manifestations and could persist for up to ≥50 days. Relatively high viral load in respiratory specimens might be related to fecal viral shedding. Future research needs to focus on this correlation in order to predict the fecal shedding of SARS-CoV-2 and its clinical significance. In addition, it is also important to understand whether the GI tract is a potential transmission route even in asymptomatic or mild COVID-19 cases with no GI symptoms so that appropriate pandemic prevention and control measures can be developed. What you need to know: Background and Context: This study investigated gastrointestinal symptoms and shedding of virus into feces of patients with asymptomatic or mild COVID-19. New Findings: An analysis of fecal and respiratory specimens from patients with COVID-19 in quarantine in Korea found that the gastrointestinal tract could be a route of transmission of SARS-CoV-2 even in patients with asymptomatic or mild disease, with no gastrointestinal symptoms. The viral load of the respiratory specimens appears be related to shedding of the virus into feces in this group of patients. Impact: Even patients with mild or no symptoms of COVID-19, and no gastrointestinal symptoms, still shed virus into feces. What you need to know: Background and Context: This study investigated gastrointestinal symptoms and shedding of virus into feces of patients with asymptomatic or mild COVID-19. New Findings: An analysis of fecal and respiratory specimens from patients with COVID-19 in quarantine in Korea found that the gastrointestinal tract could be a route of transmission of SARS-CoV-2 even in patients with asymptomatic or mild disease, with no gastrointestinal symptoms. 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