COVID-19 Original research Depicting SARS- CoV-2 faecal viral activity in Gut: first published as 10.1136/gutjnl-2020-322294 on 20 July 2020. Downloaded from association with gut microbiota composition in patients with COVID-19 Tao Zuo ,1,2,3 Qin Liu,1,2,3 Fen Zhang,1,2,3 Grace Chung- Yan Lui,3,4 Eugene YK Tso,5 Yun Kit Yeoh ,1,6 Zigui Chen ,1,6 Siaw Shi Boon,6 Francis KL Chan ,1,3 Paul KS Chan,1,6 Siew C Ng 1,2,3 ► Additional material is ABSTRACT Significance of this study published online only. To view, Objective Although severe acute respiratory syndrome please visit the journal online coronavirus 2 (SARS- CoV-2) RNA was detected in faeces (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2020- 322294). of patients with COVID-19, the activity and infectivity of the virus in the GI tract during disease course is largely ► GI symptoms are present in a substantial For numbered affiliations see proportion of patients with COVID-19. end of article. unknown. We investigated temporal transcriptional activity of SARS- CoV-2 and its association with ► Severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2) RNA has been detected and Correspondence to longitudinal faecal microbiome alterations in patients remained positive in the faecal samples of Professor Siew C Ng, with COVID-19. some patients with COVID-19 even after Department of Medicine and Design We performed RNA shotgun metagenomics Therapeutics, The Chinese respiratory specimens were negative for viral sequencing on serial faecal viral extractions from 15 University of Hong Kong, Hong RNA. Kong, Hong Kong; hospitalised patients with COVID-19. Sequencing In vitro transcriptional analysis on SARS- CoV- siewchienng@ cuhk. edu. hk ► coverage of the SARS- CoV-2 genome was quantified. We 2-infected cell model showed that the 3’ end of assessed faecal microbiome composition and microbiome TZ, QL and FZ contributed SARS- CoV-2 genome was substantially highly equally. functionality in association with signatures of faecal covered than the 5’ end indicating a signature SARS- CoV-2 infectivity. of active viral replication and infection. Received 21 June 2020 Results Seven (46.7%) of 15 patients with COVID-19 Revised 6 July 2020 What are the new findings? http://gut.bmj.com/ Accepted 12 July 2020 had stool positivity for SARS-CoV -2 by viral RNA Published Online First metagenomic sequencing. Even in the absence of GI ► We found for the first time a signature of active 20 July 2020 manifestations, all seven patients showed strikingly gut viral infection in a subset (47%) of patients higher coverage (p=0.0261) and density (p=0.0094) of with COVID-19 even in the absence of GI the 3’ vs 5’ end of SARS- CoV-2 genome in their faecal symptoms, suggesting ‘quiescent’ GI infection viral metagenome profile. Faecal viral metagenome of SARS- CoV-2. of three patients continued to display active viral ► The transcriptional activity of viral infection on September 28, 2021 by guest. Protected copyright. infection signature (higher 3’ vs 5’ end coverage) up to and replication persisted in the gut even after respiratory clearance of SARS- CoV-2. 6 days after clearance of SARS- CoV-2 from respiratory Faecal samples with a signature of high SARS- samples. Faecal samples with signature of high SARS- ► CoV-2 infectivity harboured a higher abundance CoV-2 infectivity had higher abundances of bacterial of opportunistic pathogens, Collinsella species Collinsella aerofaciens, Collinsella tanakaei, aerofaciens, Collinsella tanakaei, Streptococcus Streptococcus infantis, Morganella morganii, and higher infantis, Morganella morganii and an enhanced functional capacity for nucleotide de novo biosynthesis, capacity for biosynthesis of nucleotide and amino acid biosynthesis and glycolysis, whereas faecal amino acid and carbohydrate metabolism samples with signature of low- to- none SARS- CoV-2 (glycolysis), whereas faecal samples with a infectivity had higher abundances of short-chain fatty signature of low-to- none SARS-CoV -2 infectivity acid producing bacteria, Parabacteroides merdae, had a higher abundance of short- chain fatty Bacteroides stercoris, Alistipes onderdonkii and acid producing bacteria, Parabacteroides Lachnospiraceae bacterium 1_1_57FAA. merdae, Bacteroides stercoris, Alistipes Conclusion This pilot study provides evidence for onderdonkii and Lachnospiraceae bacterium active and prolonged ’quiescent’ GI infection even in the 1_1_57FAA. © Author(s) (or their absence of GI manifestations and after recovery from employer(s)) 2021. Re- use respiratory infection of SARS-CoV -2. Gut microbiota permitted under CC BY- NC. No of patients with active SARS-CoV -2 GI infection was INTRODUCTION commercial re- use. See rights characterised by enrichment of opportunistic pathogens, COVID-19, an acute respiratory illness caused and permissions. Published by novel coronavirus (severe acute respiratory by BMJ. loss of salutary bacteria and increased functional capacity for nucleotide and amino acid biosynthesis and syndrome coronavirus 2 (SARS-CoV -2)), is charac- To cite: Zuo T, Liu Q, Zhang F, carbohydrate metabolism. terised by active virus replication in the upper respi- et al. Gut 2021;70:276–284. ratory tract.1 Early reports from Wuhan showed 276 Zuo T, et al. Gut 2021;70:276–284. doi:10.1136/gutjnl-2020-322294 COVID-19 extrapolated from indirect observations of findings based on Significance of this study intestinal organoid and mammalian cell models: (1) SARS-CoV -2 receptor, ACE2, is highly expressed in intestinal enterocytes and Gut: first published as 10.1136/gutjnl-2020-322294 on 20 July 2020. Downloaded from How might it impact on clinical practice in the foreseeable colonocytes10 11 and (2) SARS-CoV -2 infects enterocyte lineage future? cells in human and bat intestinal organoids.12 13 To date, there is ► Active and prolonged SARS- CoV-2 activity in the gut a lack of data of replication-competent and infection- competent of patients with COVID-19, even in the absence of GI SARS- CoV-2 virus in the human gut. Deeper understanding of manifestations and after recovery highlights the importance the life cycle and pathogenicity of SARS-CoV -2 in the human gut of long- term coronavirus and health surveillance and the is an urgent unmet need. threat of potential faecal- oral viral transmission. In this pilot observational study, we postulate that SARS- CoV-2 ► Therapeutic approaches including nullifying gut SARS-CoV -2 is active in the gut of patients with COVID-19, and therefore activity and modulating gut microbiome composition and depicted the temporal transcriptional activity and infectivity of functionality should be explored. SARS- CoV-2 in the gut of hospitalised patients with COVID-19 both during disease course and after disease clearance. We prospectively included 15 hospitalised patients with COVID-19 that 2%–10% of patients with COVID-19 had GI symptoms admitted between 16 February 2020 and 2 March 2020 in Hong including diarrhoea, but a recent meta-analysis reported that up Kong, China, followed from hospital admission until discharge. 2–5 to 20% had GI symptoms. Moreover, faecal calprotectin, an Faecal viral RNA was extracted, followed by shotgun metage- indicator of inflammatory responses in the gut, was found to nomics sequencing and profiling to investigate SARS- CoV-2 6 be elevated in patients with COVID-19 with diarrhoea. These transcriptional activity. evidence suggest that the digestive tract might be an extrapulmo- nary site for SARS- CoV-2 infection in patients with COVID-19 with GI manifestations. However, viral activity and infectivity of METHODS SARS- CoV-2 in the GI tract of patients with COVID-19 during Study subject and design disease course and after disease resolution is largely unknown. This prospective study involved 15 patients with COVID-19 SARS- CoV-2 RNA has been detected in anal swabs and stool hospitalised with laboratory-confirmed SARS- CoV-2 infection samples based on RT- PCR in a substantial proportion of patients (table 1). SARS-CoV -2 infection was confirmed by two consec- with COVID-19.1 7 In addition, viral particles of SARS-CoV -2 utive RT-PCR tests targeting different regions of the RdRp gene were observed from faeces of patients with COVID-19 through performed by the local hospitals and Public Health Laboratory electron microscopy.8 The presence of SARS- CoV-2 RNA in Service (Hong Kong, China). All patients with COVID-19 were faeces can last longer after respiratory specimens became viral admitted to the Prince of Wales Hospital or the United Chris- negative in a subset of patients with COVID-197 9. However, tian Hospital, Hong Kong, between 5 February 2020 and 17 it is unknown about the activity of SARS- CoV-2 virus in the March 2020. They were followed until discharged from hospital gut of those patients with COVID-19 with yet faecal positivity or until 4 April 2020. All patients provided informed consent http://gut.bmj.com/ for SARS-CoV -2 after respiratory clearance of SARS-CoV -2. to participate in this study and agreed for publication of the Currently, the viral activity of SARS-CoV -2 in the gut was mostly research results. Data including demographic, epidemiological, Table 1 Clinical characteristics of subjects with COVID-19 Patients with Disease COVID-19 severity Age Sex Comorbidities Respiratory symptom GI symptom Chest X- ray findings on September 28, 2021 by guest. Protected copyright. 1 Critical 65 F Hypertension, chronic hepatitis Fever, cough, sputum Nil Bilateral lung infiltrates B carrier 2 Moderate