key: cord-0932606-9b6fxy33
authors: Díaz, Luis Antonio; García-Salum, Tamara; Fuentes-López, Eduardo; Reyes, Diego; Ortiz, Javier; Chahuan, Javier; Levican, Jorge; Almonacid, Leonardo I.; Valenzuela, Gonzalo H.; Serrano, Eileen; Budnik, Sigall; Gandara, Vicente; Gallardo, Andrea; Seydewitz, María Francisca; Ferrés, Marcela; Cofré, Colomba; Álvarez, Manuel; Pavez, Carolina; Candia, Roberto; Monrroy, Hugo; Espino, Alberto; Rada, Gabriel; Ortiz, Luis; Valderrama, Sebastián; Salinas, Erick; Toro, Adriana; Ortega, Marcos; Pizarro, Margarita; Medina, Rafael A.; Riquelme, Arnoldo
title: High prevalence of SARS-CoV-2 detection and prolonged viral shedding in stools: A Systematic Review and Cohort Study
date: 2022-01-22
journal: Gastroenterol Hepatol
DOI: 10.1016/j.gastrohep.2021.12.009
sha: 2bc375654a37f0c2c1abf7a6185a3da3838edd52
doc_id: 932606
cord_uid: 9b6fxy33

Objectives: To: 1. Describe the frequency of viral RNA detection in stools in a cohort of patients infected with SARS-CoV-2, and 2. Perform a systematic review to assess the clearance time in stools of SARS-CoV-2. Methods: We conducted a prospective cohort study in two centers between March-May 2020. We included SARS-CoV-2 infected patients of any age and severity. We collected seriated nasopharyngeal swabs and stool samples to detect SARS-CoV-2. After, we performed a systematic review of the prevalence and clearance of SARS-CoV-2 in stools (PROSPERO-ID: CRD42020192490). We estimated prevalence using a random-effects model. We assessed clearance time by using Kaplan-Meier curves. Results: We included 32 patients; mean age was 43.7;17.7 years, 43.8% were female, and 40.6% reported gastrointestinal symptoms. Twenty-five percent (8/32) of patients had detectable viral RNA in stools. The median clearance time in stools of the cohort was 11[10-15] days. Systematic review included 30 studies (1,392 patients) with stool samples. Six studies were performed in children and 55% were male. The pooled prevalence of viral detection in stools was 34.6% (twenty-four studies, 1,393 patients; 95%CI:25.4-45.1); heterogeneity was high (I2:91.2%, Q:208.6; p=<0.001). A meta-regression demonstrates an association between female-gender and lower presence in stools (p=0.004). The median clearance time in stools was 22 days (nineteen studies, 140 patients; 95%CI:19–25). After 34 days, 19.9% (95%CI:11.3-29.7) of patients have a persistent detection in stools. Conclusions: Detection of SARS-CoV-2 in stools is a frequent finding. The clearance of SARS-CoV-2 in stools is prolonged and it takes longer than nasopharyngeal secretions.

The current pandemic of a novel Severe Acute Respiratory Syndrome coronavirus, named SARS-CoV-2, has constituted a new threat worldwide 1 . More than 13,165,000 cumulative cases have been confirmed in 188 countries across five continents, reaching a mortality rate of 4.9% 2 .

The novel coronavirus was first described in patients with pneumonia, and the most common symptoms included fever, dry cough, and dyspnea 3 . Gastrointestinal symptoms are less common and include diarrhea, nausea, vomiting, and abdominal discomfort. The prevalence of these symptoms varies significantly among different studied populations, which can be mild at the early onset, but is frequently followed by typical respiratory manifestations 4 . A recent systematic review showed that 17.6% of individuals infected with SARS-CoV-2 presented with gastrointestinal symptoms; the most frequent being anorexia 26.8%, diarrhea 12.5%, nausea and vomiting 10.2%, and abdominal pain 9.2% 5 . The presence of gastrointestinal symptoms is frequent in hospitalized patients, and these became more pronounced as the severity of the disease increased 6, 7 .

Several reports have shown the detection of SARS-CoV-2 RNA in stools for prolonged periods, suggesting that the virus could be transmitted through the digestive tract 8 . The viral detection in stools is variable among several studies, with frequencies ranging between 15 .3% and 70.3% 5, [8] [9] [10] . Interestingly, recent publications have described enterocyte viral Page 9 of 40 J o u r n a l P r e -p r o o f 9 invasion and detection of viable virus in stools, suggesting that the fecal-oral route could provide a source of transmission 4, 10 .

To date, the relevance of viral presence in stools and length of shedding has not been established adequately. In this study, we aimed to assess the viral presence and clearance of SARS-CoV-2 in stools of infected Chilean patients. We also performed a systematic review to establish the frequency of SARS-CoV-2 detection in stools, and median clearance time in nasopharyngeal secretions and stools.

Patient clinical-epidemiological data and clinical samples were collected after informed written consent was obtained. This study was reviewed and approved by the Scientific For clinical information, we performed an interview and a survey to characterize the presence and timing of symptoms. We recorded the physical examination and primary laboratory data obtained during medical evaluation. All the patients were characterized by sociodemographic data and comorbidities. We collected NPS and stool or rectal swabs between days 2 to 35 after the onset of symptoms and tested for the presence of SARS-Page 11 of 40 J o u r n a l P r e -p r o o f 11 CoV-2 by qRT-PCR. All the information was registered in an anonymous database where sensitive information was excluded. The stages of this process, collecting, transporting and processing samples are illustrated in Figure 1 . Additional data of RNA extraction and viral genome detection are described in Supplementary Methods.

We assessed the normality assumption by means of the Shapiro-Wilk test. Descriptive statistics were obtained using the mean and standard deviation (SD) for continuous variables with normal distribution and non-normal distributions were described with the median and interquartile range. In the case of categorical variables, the proportions and percentage were estimated.

We performed a systematic review to establish the prevalence of viral detection in stools and median clearance time among previous published data and our cohort. This systematic review with meta-analysis was registered on PROSPERO (CRD42020192490) and followed a prespecified analysis plan. This study is reported in accordance with the Preferred Reporting Items for a Review and Meta-analysis (PRISMA) guidelines 11 .

Eligible studies had to include patients diagnosed with COVID-19, regardless of age and gender. The diagnosis of COVID-19 had to be based on a compatible clinical history and molecular evidence with a qRT-PCR for SARS-CoV-2. We included studies reporting the frequency of positive viral RNA testing in stool. We considered stool samples at any time of the course of the disease.

We included studies that reported the main outcome, regardless of the design (casereports, case-series, descriptive cases, case-control studies, cross-sectional studies, cohort studies and randomized controlled trials). We excluded studies performed in vitro, animal models, or lacking evidence of SARS-CoV-2 infection from this systematic review.

We conducted an electronic search from December 1, 2019, to June 3, 2020 in MEDLINE (via PubMed) database. We used included terms related to SARS-CoV-2 and stools in the search strategy. References of studies searched were also included. We hand searched (up to June 3, 2020) preprint servers (bioRxiv, medRxiv, and SSRN) and coronavirus resource centers of The Lancet, JAMA, and New England Journal of Medicine. We did not limit our search by language. Two investigators (LAD and DR) independently screened the titles and abstracts to ascertain whether each study met the eligibility criteria. The full texts of the identified eligible articles were then evaluated to determine whether they should be included in the analysis. Disagreements between the two reviewers were resolved by consensus. In case of persistent disagreement, arbitration by a third reviewer (JO) settled the discrepancy. 

The main outcome was the prevalence of detection of viral RNA in stools of COVID-19 patients. This data was registered as an absolute number and percentage of the total cases.

The secondary outcome was the clearance time (defined as the days between onset of symptoms and a negative test result for viral RNA in stools).

We estimated prevalence or event rates in the form of a proportion (with confidence interval 95%). Proportions were pooled using random-effects models. Only those studies with a sample size of at least 10 patients were included in the meta-analysis to decrease J o u r n a l P r e -p r o o f 14 bias caused by sampling error 12 . We used Q statistic and I 2 to quantify heterogeneity. We planned a subgroup analysis according to age, gender, ethnicity, presence of gastrointestinal symptoms and hospitalization. We stratified age in three subgroups: pediatrics (patients with less than 18 years old), adults (patients over 18 years old), and general (patients of all ages). Ethnicity was defined per region of origin (Asian versus not-Asian studies). We stratified gender by predominant gender (the gender with a prevalence higher than 50%). Presence of gastrointestinal symptoms was arbitrarily grouped in high (50% or more) and low (lesser than 50%) prevalence of gastrointestinal symptoms. In the case of studies with very low or very high proportion, we used a double arcsine transformation 13 . Small study effect was evaluated with a funnel plot. Random effects meta-regression was used to examine whether baseline characteristics covariates explained heterogeneity of proportions between studies 14 . As such, subgroup analysis should be considered as exploratory. Clearance of viral RNA in stools was defined as the time between onset of symptoms and the last detection of SARS-CoV-2 in stools. We registered the disaggregated data from studies that reported clearance in NPS and stools for each participant. Based on this data, we performed Kaplan-Meier survival curves to estimate viral clearance in NPS and stools. We compared clearance in NPS and stools curves with log-rank test. The analyses were done with the use of STATA (version 16, StataCorp, College Station, Texas) and R software for statistical computing version 3.6.1 15 . We included the STATA metaprop command to perform meta-analyses 16 .

The quality of evidence for the outcomes was graded with the GRADE framework.

The funding source only provided support for the analysis of the nasopharyngeal and stool samples. The researchers did not receive a fee or other incentives for the systematic review process.

We included 32 patients recruited between the 3 rd March 2020 and 28 th May 2020. Median age was 43.717.7 years and 14 (43.8%) were females. Nineteen (59.4%) patients were hospitalized and 6 (31.6%) required invasive mechanical ventilation. One of our patients died at the end of this study (mortality rate of 3.1%).

In our cohort, the most common extraintestinal manifestations were fever (90.6%), cough 

We identified 1,683 records and 671 were duplicated. After a screening process against title and abstract, we obtained 59 full-text articles that were assessed for eligibility. We finally selected 30 studies for our systematic review from 6 countries (China, France, Germany, Hong Kong, Singapore, and the United States). We included 23 articles in the meta-analysis of prevalence of SARS-CoV-2 detection in stools and 18 articles were used to estimate clearance in stools. We also included data of the current study in both analyses. The selection process is described in Figure 2 . All studies were observational in design; no randomized trials were identified. Among the 30 studies, 14 were case series and 16 cohort studies. The study with the largest number of stool samples included a total of 273 cases 17 .

The risk of bias assessment results is presented in supplementary material.

The pooled studies included a total of 1,392 patients that were tested by SARS-CoV-2 qRT-PCR in stools. Fifty-five percent were male and 6 studies were performed exclusively in children. Twenty-two out of 30 (73.3%) studies included only hospitalized patients. The grade of evidence was considered very low and low, since all the information emerged from case series and cohort studies, respectively. We performed subgroup analyses per age, gender, ethnicity, presence of gastrointestinal symptoms, and hospitalization. Table 2 summarizes the main baseline characteristics summary of each study.

We identified 1,393 patients from 23 studies and our current study, which tested the presence of viral RNA in stools (including our cohort). The pooled prevalence of viral detection in stools was 34 

In this study, we analyzed general clinical data from 32 SARS-CoV-2 infected patients. In the first stage, we found that 40.6% of the studied population presented with gastrointestinal symptoms: 37.5% reported diarrhea, 12.5% nausea, 6.3% abdominal pain, 3.1% vomiting, and 3.1% constipation. A significant percentage of our cohort required hospitalization 59.4%, reaching a mortality of 3.1%. We determined that 25% of patients had detectable Remarkably, the Kaplan-Meier clearance curves showed a median clearance time in NPS of 13 days (95%CI: [12] [13] [14] , which was significantly lower than clearance in stools (p<0,001). In fact, viral RNA detection in stools could occur for at least another nine (for 22 days or more) in the half proportion of individuals tested (Figure 4) . These findings suggest a longer shedding period in the gastrointestinal tract, which is in agreement with reports from cohort studies in Hong Kong, Macau, Beijing and Germany 27-30 , and a case study in Italy 31 .

The identification of prolonged SARS-CoV-2 RNA shedding in rectal swabs was reported as early as mid-February 2020 by Zhang et al. 32 Therefore, the potential of viral transmission through the oral-fecal route has been cautioned since then, and recent additional evidence supports this idea. First, it was reported that viral RNA can be detected in fecal samples of infected individuals for up to 11.2 days after respiratory samples became negative and up to 47 days after symptom onset 33 ; secondly, virus RNA was found at high concentrations in stool, and occasionally viral sub genomic messenger RNA (only present in replicating cells)

can also be detected in these specimens; third, endoscopic studies of SARS-CoV-2 infected The limitations of the cohort study were the limited number of patients followed-up and the difficult in obtaining daily stool samples. In addition, there is not a gold standard of SARS-CoV-2 infection and SARS-CoV-2 qRT-PCR has sensitivity near to 70% in NPS 44 . This sensitivity could be even lower according to a recent study, reaching 47.3% in real life and underestimating detection of SARS-CoV-2 in organs, fluids, and stools 45 . Also, we identified a high heterogeneity among the studies to estimate proportion of SARS-CoV-2 RNA detection in stools. We performed several analyses to elucidate the cause of this heterogeneity including a meta-regression. We identified that gender can importantly contribute to this high heterogeneity. This interesting observation could be explained by the higher expression of ACE2 and a less robust T cell-mediated immunity in male patients 46, 47 . However, this heterogeneity could also be explained by important differences and quality among prior published studies. Future research questions include the establishment of additional data to support the fecal-oral transmission and the assessment of the longterm impact of SARS-CoV-2 infection in the gastrointestinal tract. Finally, although anal swabs to detect SARS-CoV-2 have been performed as a screening tool in countries such as Indonesia, the sensitivity of this technique is low (36.7%) 48 . Therefore, we require more sensitive anal-swabs-based tests to recommend SARS-CoV-2 detection in anal swabs as a screening tool.

In conclusion, this cohort study and systematic review demonstrated that detection of SARS-CoV-2 in stools is a frequent finding. The clearance of SARS-CoV-2 in stools is significantly prolonged as compared to clearance in nasopharyngeal secretions. The 

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