key: cord-262796-syu4wbpi authors: Wei, Xiao-Shan; Wang, Xu; Niu, Yi-Ran; Ye, Lin-Lin; Peng, Wen-Bei; Wang, Zi-Hao; Yang, Wei-Bing; Yang, Bo-Han; Zhang, Jian-Chu; Ma, Wan-Li; Wang, Xiao-Rong; Zhou, Qiong title: Diarrhea is associated with prolonged symptoms and viral carriage in COVID-19 date: 2020-04-18 journal: Clin Gastroenterol Hepatol DOI: 10.1016/j.cgh.2020.04.030 sha: doc_id: 262796 cord_uid: syu4wbpi Abstract Background & Aims We compared clinical, laboratory, radiological, and outcome features of patients with SARS-CoV-2 infection (COVID-19) with pneumonia, with vs without diarrhea. Methods We performed a retrospective, single-center analysis of 84 patients with SARS-CoV-2 pneumonia in Wuhan Union Hospital, China, from January 19 through February 7, 2020. Cases were confirmed by real-time reverse-transcriptase PCR of nasal and pharyngeal swab specimens for SARS-CoV-2 RNA. Blood samples were analyzed for white blood cell count, lymphocyte count, alanine aminotransferase, creatine kinase, lactate dehydrogenase, D-dimer, C-reactive protein, and in some cases, immunoglobulins, complement, lymphocyte subsets, and cytokines. Virus RNA was detected in stool samples by real-time PCR. Results Of the 84 patients with SARS-CoV-2 pneumonia, 26 (31%) had diarrhea. The duration of fever and dyspnea in patients with diarrhea was significantly longer than those without diarrhea (all P<.05). Stool samples from a higher proportion of patients with diarrhea tested positive for virus RNA (69%) than from patients without diarrhea (17%) (P<.001). As of February 19, a lower proportion of patients with diarrhea had a negative result from the latest throat swab for SARS-CoV-2 (77%) than patients without diarrhea (97%) (P=.010), during these patients’ hospitalization. Of 76 patients with a negative result from their latest throat swab test during hospitalization, a significantly higher proportion of patients with diarrhea had a positive result from the retest for SARS-CoV-2 in stool (45%) than patients without diarrhea (20%) (P=.039). Conclusions At a single center in Wuhan, China, 31% of patients with SARS-CoV-2 pneumonia had diarrhea. A significantly higher proportion of patients with diarrhea have virus RNA in stool than patients without diarrhea. Elimination of SARS-CoV-2 from stool takes longer than elimination from the nose and throat. Since December 2019, pneumonia caused by novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) has broken out in Wuhan, Hubei province, China 1 . The novel coronavirus has spread to other cities in China, and even around the world [1] [2] [3] [4] . As of April 10, 2020, 1,521,252 confirmed cases have been reported globally. Among them, 83,305 confirmed cases were from China, furthermore, 3345 cases had died 5 . The clinical manifestations of novel coronavirusinfected pneumonia (Corona Virus Disease 2019, have been reported in several recent studies 1,6-9 . Some COVID-19 patients had gastrointestinal symptoms, especially diarrhea. It has been reported that the stool from confirmed cases were tested positive by RT-PCR for SARS-CoV-2, suggesting the possibility of "fomite transmission" 1, 3 . However, the difference in clinical characteristics between diarrhea and non-diarrhea cases has not been reported. In this study, we analyzed the differences of the clinical characteristics, laboratory examinations, imaging manifestations and outcomes between COVID-19 patients with diarrhea and those without diarrhea. Consecutive patients with confirmed COVID-19 who were admitted to Wuhan Union Hospital from January 19 to February 7, 2020, were enrolled retrospectively. All patients were diagnosed according to World Health Organization interim guidance. A confirmed case was defined as a positive result to real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR) assay of nasal and pharyngeal swab specimens for SARS-CoV-2 RNA. Only the confirmed cases were enrolled in this study. Stool samples were also tested for SARS-CoV-2 RNA by RT-PCR. The definition of diarrhea by the WHO is having three or more loose or liquid stools per day or having more stools than a person's health condition. All medical records of the enrolled patients were collected, including clinical symptoms, laboratory findings, imaging manifestations and outcomes. The clinical outcomes (ie, discharges, mortality) were monitored up to February 19, 2020, the final date of follow-up. Data collection and analysis of cases were determined by the National Health Commission of the People's Republic of China to be part of a continuing public health outbreak investigation and were thus considered exempt from institutional review board approval. The SARS-CoV-2 laboratory test assays were performed for throat-swab specimens and stool samples based on the previous WHO recommendation 10 . RNA in patients' specimens were extracted and tested for SARS-CoV-2 by real-time RT-PCR using the same protocol as previously described in the studies from Wuhan Jinyintan Hospital 6,7 . Continuous variables were expressed as the means and standard deviations. Categorical variables were summarized as the counts and percentages in each category. Comparisons were determined by unpaired t test or chi-square tests as appropriate. All statistical analyses were performed with SPSS22.0 (SPSS Inc., Chicago, IL). All authors had access to the study data, reviewed and approved the final manuscript. A total of 84 hospitalized health-care workers with confirmed COVID-19 were enrolled in this study population, including 17 doctors, 66 nurses and 1 allied health worker. All the doctors worked in the wards and went to outpatient clinic intermittently, for two half-days per week. Most of the nurses worked in the wards, except 3 nurses who worked in the fever clinic. The only allied health worker worked in the Network Center of Union Hospital. All of these patients were admitted to isolation wards. The median age of the patients was 37 years (range:24-74 years), and 28(33%) of the 84 patients were male. Among them, 26(31%) patients had diarrhea, the rest 58 patients had no diarrhea. The comparison of clinical characteristics between these two groups was shown as Table 1 . Several clinical symptoms were more common in diarrhea group, as compared with non-diarrhea group, including headache (58% vs 22%, P= .003), myalgia or fatigue (65% vs 34%，P = .010), cough(85% vs 45%, P < .001), sputum production(54% vs 21%, P = .004), nausea(38% vs 10%, P = .005) and vomiting(19% vs 2%, P = .010). Table 2 . shows the comparison of laboratory findings between the diarrhea group and non-diarrhea group, at the time of hospitalization. Most of the laboratory findings had no difference between these two groups, including white blood cell count, lymphocyte count, alanine aminotransferase, creatine kinase, lactate dehydrogenase, D-Dimer and C-reactive protein. Some of the 84 patients were tested for immunoglobulins, complement, lymphocyte subsets and cytokines. As shown in Appendix Table 1 , several cytokine including interleukin-2, interleukin-4 and interferon-γ decreased significantly in diarrhea group, as compared with non-diarrhea group (2.26 ± 0.27 vs 2.59 ± 0.24 pg/ml, P = .001; 1.54 ± 0.37 vs 2.11 ± 0.44 pg/ml, P = .001; 1.90 ± 0.49 vs 2.85 ± 1.27 pg/ml, P = .012; respectively). However, there were no differences in immunoglobulins, complement and lymphocyte subsets between these two groups, as shown in Appendix Table 2 and 3. The most common finding in CT images were ground-glass opacifications (96%), as shown in Appendix Table 4 . There were no differences in the location or number of ground-glass opacifications between these two groups. On admission to hospital, all confirmed COVID patients were tested for SARS-CoV-2 RNA from stool samples. Stool samples from a higher proportion of patients with diarrhea tested positive for virus RNA (69%) than from patients without diarrhea (17%) (P<.001) ( Table 3) . As of Feb 19, none of these 84 patients had died or admitted to ICU; 63 patients had been discharged; 21 patients were still in hospital. All patients received antibiotics and antiviral agents during hospitalization, 39(46%) patients received two kinds of antibiotics. After a period of time for treatment, throat swab specimens from patients were regathered and tested for SARS-CoV-2. If the result of real-time RT-PCR turned to be negative two times consecutively, body temperature returned to normal for more than 3 days, respiratory symptoms and lung lesions on CT scan improved significantly, patients were permitted to discharge. The duration of fever and dyspnea in patients with diarrhea was significantly longer than those without diarrhea (10.5±4.7 vs 7.6±3.4day, P = .005; 8.1±3.2 vs 4.7±2.3day, P = .002; respectively) ( Figure 1A&B ). By the end of Feb. 19, a lower proportion of patients with diarrhea had a negative result from the latest throat swab for SARS-CoV-2 (77%) than patients without diarrhea (97%) (P=.010), during these patients' hospitalization (Table 4 ). The mean time of SARS-CoV-2 in throat swab turning to be negative was longer in diarrhea group, as compared to non-diarrhea group (12.5±4.0 vs 9.2±3.9day, P = .002) ( Figure 1C ). Patients' stool specimens were also retested for SARS-CoV-2. Of 76 COVID-19 patients who had a negative result from their latest throat swab test during hospitalization, a significantly higher proportion of patients with diarrhea had a positive result from the retest for SARS-CoV-2 in stool (45%) than patients without diarrhea (20%) (P=.039) ( Table 5) . All patients reexamined a CT scan during hospitalization. By the end of Feb. 19, the improvement rate and deterioration rate show no differences between diarrhea group and non-diarrhea group, as shown in Appendix Table 5 . Discharged patients were more common in non-diarrhea group, as compared to diarrhea group (83% vs 58%, P = .028) (Appendix Table 6 ). Meanwhile, the hospital stays were longer in diarrhea group than non-diarrhea group (16.5±5.2 vs 11.8±5.6day, P < .001) ( Figure 1D ). respectively, all P < .0001), as shown in Appendix Table 7 . This study has shown that diarrhea occurred in 31% of SARS-CoV-2 infectious pneumonia patients and is the first report which focus on the difference between COVID-19 patients with diarrhea and those without. Despite most of the laboratory and radiologic findings show no difference between these two groups, we do find some characteristics differs between them. The COVID-19 patients with diarrhea suffered headache, myalgia or fatigue, cough, sputum production, nausea, vomiting more frequently than those patients without diarrhea, but seldomly suffered abdominal pain, abdominal distension and tenesmus. The characteristics of diarrhea in SARS-CoV-2 pneumonia patients includes increased defecation frequency (3~14 times per day), pastry stool with no mucous or purulent blood. The diarrhea in some patients gradually alleviates and disappears during hospitalization; but in other patients, the frequency of diarrhea increased, and smear for stool fungus and stool test for occulted blood showed positive. The intestinal epithelial injury caused by the infection of novel coronavirus might be an important cause of the diarrhea in COVID-19 patients. Full-genome sequencing and phylogenic analysis showed that SARS-CoV-2 and SARS-CoV belong to the same genus of coronaviruses (betacoronaviruses), with about 80% sequence identity 11, 12 . SARS viral particles and genomic sequence were detected in the mucosa of the intestine 13 . Recently, researchers from Guangzhou Institute of Respiratory Health successfully isolate SARS-CoV-2 from a COVID-19 patient's stool (not published data). Studies indicated that SARS-CoV-2 and SARS-CoV utilize the same receptor, angiotensin-converting enzyme 2 (ACE2), to get access into host cells 11, 14 . Through single-cell RNA sequencing technology, Yu Zhao and his colleagues found that the expression of ACE2 was concentrated in a small population of type Ⅱ alveolar cells (AT2 cells) in the normal human lungs 15 . However, the lung AT2 cells was not the only highly expressing ACE2 cells, but also in esophagus upper and stratified epithelial cells and absorptive enterocytes from ileum and colon 16 . Digestive system is also a potential pathway for SARS-CoV-2 infection. Diarrhea, as a common symptom in COVID-19 patients, also indicates the involvement of the digestive system. The absorptive enterocytes were the most vulnerable intestinal epithelial cells and can be invaded by coronavirus and norovirus, leading to malabsorption, unbalanced intestinal secretion and activated enteric nervous system, resulting in diarrhea finally 17, 18 . The diarrhea symptom may be caused by the invaded ACE2-expressing enterocytes. The underlying molecular pathogenesis needs to be further investigated. Another reason for the diarrhea in COVID-19 patients might be antibiotic-associated diarrhea. Broad-spectrum antibiotic use can disrupt the gastrointestinal microbiota resulting in diarrhea 19 . All patients in this study had received oral or intravenous antibiotics (100%), and some had received two antibiotics (46%). Because this is a small size study, we failed to analyze the correlation between antibiotic and diarrhea. The diarrhea in these patients significantly relieved after taking intestinal probiotics, indicating that the use of antimicrobial drugs might be an important cause of diarrhea in COVID-19 patients. We also find that the duration of symptoms in patients with diarrhea was significantly longer than those without diarrhea, including fever and dyspnea. Patients with diarrhea took much more time to eliminate SARS-CoV-2 from respiratory system, leading to longer hospital stay time. The frequency of positive rate for testing SARS-CoV-2 from stool was higher in patients with diarrhea, as compared with patients without diarrhea at admission. It indicated that SARS-CoV-2 infection in digestive system maybe more common and severer in patients with diarrhea. Moreover, stool specimens retesting to SARS-CoV-2 in patients with persistent diarrhea persist positive, even after the throat swab testing to SARS-CoV-2 has turn to be negative. The elimination of SARS-CoV-2 from digestive system may be much later and harder than that from respiratory system. Patients with negative throat swab test for SARS-CoV-2, may still be able to spread infection to other people through fomite transmission. We supposed the delay elimination of SARS-CoV-2 in digestive system might partly related with the use of antibiotic. Studies indicated that antibiotics had profound effect on gut microbiota, leading to altered immune system, including antibody production and T cell differentiation [20] [21] [22] . Further studies are needed to investigate the relationship between gut microbiota and antibiotics in COVID-19 patients. The proportion of patients with diarrhea (31%) in this study is much higher than what was reported in other series 1, 7, 8 . The possible reasons are the precise and timely descriptions of symptoms of the 84 patients, which are all infected medical staff. According to their descriptions, we noticed 26 patients complained of diarrhea among the total 84 patients. However, the sample size was relatively small and it couldn't represent the overall situation. The collection and analysis of such data are still ongoing in our group, and expected to give a more comprehensive explanation in the near future. None of these 84 infected health care workers died or required ICU admission. One explanation might be that almost all the medical staff with COVID-19 pneumonia in our cohort were younger people and had mild disease. Another one explanation might be that our patients did not had significant underlying chronic diseases and receive timely treatment. This study has several limitations. First, the sample size of COVID-19 patients with diarrhea was relatively small than those without diarrhea (26 vs 58). Second, all of the 84 patients were confirmed with throat swab specimens, and no paired lower respiratory tract specimens were obtained to see the difference of the viral RNA detection rate between them. In this single-center case series of 84 confirmed COVID-19 patients in Wuhan, China, 26 (31%) patients suffered diarrhea. COVID-19 patients with diarrhea suffered discomfort longer, as compared with COVID-19 patients without diarrhea. In patients with diarrhea, stool specimens testing to SARS-CoV-2 may persist positive, even after the throat swab testing to SARS-CoV-2 has turn to be negative. (20) no. (%) Clinical characteristics of 2019 novel coronavirus infection in China Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany First Case of 2019 Novel Coronavirus in the United States 2019-nCoV acute respiratory disease Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China Epidemiological and clinical features of the 2019 novel coronavirus outbreak in China COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans A pneumonia outbreak associated with a new coronavirus of probable bat origin A new coronavirus associated with human respiratory disease in China Multiple organ infection and the pathogenesis of SARS The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes Replication of human noroviruses in stem cell-derived human enteroids Role of sialic acids in feline enteric coronavirus infections Effectiveness of Lactobacillus helveticus and Lactobacillus rhamnosus for the management of antibiotic-associated diarrhoea in healthy adults: a randomised, double-blind, placebo-controlled trial Protection of the Human Gut Microbiome From Antibiotics Maternal Antibiotic Treatment Impacts Development of the Neonatal Intestinal Microbiome and Antiviral Immunity Contributions of the intestinal microbiome in lung immunity We thank all the patients involved in the study. Special thank Dr.Hui Zhang for his dedication to data analysis and encouragement. Appendix Table 1 . Cytokine Detection Assays in patients infected with SARS-CoV-2