SARS-CoV-2 in inflammatory bowel disease population:Antibodies,disease and correlation with therapy

lNTRODUCTlON

A new β-coronavirus (SARS-CoV-2) spread in November 2019 in China and then worldwide,becoming a pandemic.The related disease,known as coronavirus disease 2019 (COVID-19),mainly involves the respiratory system.The elderly and patients affected by chronic diseases seem to be at a higher risk to develop severe pneumonia and acute distress syndrome[1].In this scenario,the patients affected by inflammatory bowel diseases (IBD) appeared to be an at-risk population for severe COVID-19,considering the possible gastrointestinal system involvement[2-6].Indeed,it seems that the high expression of angiotensin-converting enzyme 2 in the intestinal tract,above all in the absorptive enterocytes of the ileum and colon and in the epithelial cells of the esophagus,makes these tissues highly susceptible to SARS-CoV-2 infection.Mucosal damage was observed in the esophagus,stomach,duodenum and rectum by histological examinations as plasma cells and lymphocytes infiltrated the lamina propria.Approximately 3% of COVID-19 cases have only digestive symptoms.Moreover,the detection of SARS-CoV-2 in the stool suggested that the virus could replicate in the digestive tract[6].

In his young days Simon had been one of the gayest and most active youths of the neighbourhood, but as he grew old and stiff he found it very difficult to walk, and his faithful servant urged him to get a horse so as to save his poor old bones

Initial indications from an IBD center in Wuhan,China was to discontinue all biological and immunosuppressive treatments.They reported that among 318 registered IBD patients,none developed COVID-19[7].Nevertheless,scientific societies suggested that IBD patients should continue the ongoing treatment to avoid relapse,including the biological therapies[1].However,regarding IBD patients affected by COVID-19,guidelines suggest handling the treatments with more caution.In particular,the American Gastroenterological Association guidelines divided them into three different categories:IBD patients without SARS-CoV-2 infection;IBD patients with SARS-CoV-2 infection but no symptoms of COVID-19;and IBD patients with COVID-19 symptoms.The first category should continue all treatments.The second category should discontinue thiopurines,methotrexate and tofacitinib and delay biological therapies for 2 wk while monitoring symptoms of COVID-19.The third category should discontinue thiopurines,methotrexate,tofacitinib and biological therapy during the illness[1].

Since the scientific community had to develop new guidelines in a short time with a new and unknown disease,the recommendations carry a low grade of evidence.In an Italian cohort of 522 IBD patients,none were hospitalized for SARS-CoV-2 infection,and 16% of the patients were under biologic treatment.However,11% of the patients were children,a population with an unclear susceptibility to the virus[8].Moreover,some interesting observational studies report COVID-19 prevalence and symptoms/outcomes in IBD cohorts[9,10].However,little is known about the possible role of IBD treatments in the development of severe COVID-19 disease.Importantly,it remains unclear whether IBD patients are at a higher or lower risk of severe COVID-19.

Systemic inflammation is a crucial target for the treatment of COVID-19 pneumonia,as the severity of the respiratory disease seems to be linked to the upregulation of inflammatory cytokines by creating a “cytokine storm,” producing interleukin (IL)-6,IL-1,tumor necrosis factor (TNF) and interferon-γ.The exaggerated synthesis of IL-6 can lead to an acute severe systemic inflammatory response.It should be noted that cytokine blockers and Jak inhibitors were considered for clinical therapy of COVID-19 acute respiratory distress syndrome[11-13].Interestingly,TNF inhibition has also been suggested in selected patients with high IL-6 levels.Indeed,when TNF is blocked,there is a serial decrease of IL-6 and IL-1 within 12 h in patients with active rheumatoid arthritis.A reduction of adhesion molecules and vascular endothelial growth factor was observed as well[14].Nevertheless,no definitive treatment has been approved.Therefore,many hypotheses but few certainties are present.In particular,COVID-19 outcomes in patients with IBD immunomodulant/immunosuppressive treatments remains under debate.

The present study aimed to investigate the prevalence of SARS-CoV-2 antibody positivity and COVID-19 disease severity in an IBD cohort,in both symptomatic and asymptomatic patients and to evaluate the correlation with clinical/therapeutic variables.

MATERlALS AND METHODS

Study design

We conducted a prospective cohort study.The informed consent for the study was obtained from all the patients in accordance with the World Medical Association’s 2008 Declaration of Helsinki:Ethical Principles for Medical Research Involving Human Subjects.The privacy rights of patients were always observed.All authors had access to the study data and reviewed and approved the final manuscript.

Patients

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Data collection

Each IBD patient was asked about his/her recent clinical history (respiratory and gastrointestinal symptoms) from the beginning of the COVID-19 pandemic in Europe (February 21,2020) by completing a questionnaire,and all the information was validated with the doctor who conducted the interview.Data collected in the questionnaire were summarized in the Supplementary Material.

I was trying to arrange to take a bus tomorrow, she said. But my friend said, ‘No way! She s going to drive me there right now. We ll get in about 2 am.

Age,sex,body mass index (BMI),IBD type,treatments and clinical activity and other comorbidities were anonymously collected in a database.Charlson Comorbidity Index was calculated for each patient.

Antibody testing

A single blood test was performed for each patient to search for anti-SARS-CoV-2 IgG.The LIAISON

SARS-CoV-2 S1/S2 IgG test [Diasorin S.p.A,Saluggia (VC) - Italy] was used according to manufacturer’s instructions.S1 and S2 are subunits of the spike protein and are responsible for binding (S1) and fusion (S2) of the virus to cells.The spike protein is the target of neutralizing antibodies.They are defined as antibodies that protect cells from pathogens or infectious particles by neutralizing their biological effects.The manufacturer reports a positive agreement of 94.4% [95% confidence interval (CI):88.8%-97.2%] with the plaque reduction neutralization test.The IgG test has diagnostic specificity of 98.5% (95%CI:97.5%-99.2%) in blood donors and 98.9% in presumably SARS-Cov-2 negative diagnostic routine samples.The IgG values are considered negative when＜12.0 kAU/L,equivocal from 12 kAU/L to 15.0 kAU/L and positive when ≥ 15.0 kAU/L.When applying a cutoff of＞15 kAU/L,the reported test’s sensitivity is time-dependent:25% (14.6%-39.4%) ≤ 5 d after reverse transcriptase-PCRconfirmed diagnosis;90.4% (79.4%-95.8%) from day 5 to day 15;and 97.4% (86.8%-99.5%) after＞15 d from PCR diagnosis[15].However,Plebani

[16] found that 6.2 kAU/L was the appropriate cutoff for the DiaSorin method to reach a sensitivity of 97.1% and a specificity of 88.9%.Moreover,in our hospital,all health care workers (HCW) were tested for serology immediately after the first 2 mo of pandemic (between April and May 2020).Among the HCW who were previously confirmed ill,only the 85% of them resulted having IgG value＞15,whereas 14% of them had values between 7 and 15 (data from National Institute of Heath,2020).

Thus,in the present study we decided to perform the analysis using both 15 and 7 as cutoffs,considering 7 as the most reliable value.

Swab throat test

All patients who resulted positive for SARS-CoV-2 IgG were tested with a SARS-CoV-2 swab throat test during the same week using the Allplex 2019-nCoV assay (Arrow Diagnostics S.r.l.,Genova,Italy),which is a single-tube assay able to detect the three target genes (

gene,

gene and

gene) as recommended by the World Health Organization.

All the 103 patients of the study had been clinically followed up for 10 mo after the beginning of the study.None of them hold the IBD treatments or developed new symptoms of COVID-19 until April 2021.After this period of time all our IBD patients had been received the vaccine against COVID-19.

Statistical analysis

Categorical variables were described as count and percentage and compared between groups with the

test.Continuous variables were described as mean and standard deviation or median and interquartile range if not normally distributed (Shapiro-Wilks test) and compared with independent

-test or Mann-Whitney.

The patients treated with biologic therapy don’t seem to carry a high risk of developing severe COVID-19.

The analysis was performed using SARS-CoV-2 IgG value cutoff of＞7 kAU/L (15-16).

To investigate the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)antibodies positivity and COVID-19 disease severity in IBD patients.Evaluate the correlation with clinical/therapeutic variables.

RESULTS

In total,103 IBD patients were consecutively enrolled;54 had Crohn’s disease and 49 ulcerative colitis.Among these,36 patients (35.0%) were treated with biologic treatment,14 (13.6%) with azathioprine (AZA) and 53 (51.4%) with mesalazine.Demographic,clinical and therapeutic characteristics of the cohort were summarized in Table 1.The survey’s results were summarized in Table 2.

Prevalence of SARS-CoV-2 IgG positivity in IBD cohort

SARS-CoV-2 IgG positivity with value＞7 was found in 19 out of 103 patients (18.4%).Among them:10 were under biological treatment;5 under AZA;and 4 under mesalazine.Symptoms related to COVID-19 disease were reported in 12 out of 19 patients (63%).Among them,2 were treated with mesalazine,4 with AZA and 6 with biologic treatment.Among the 7 out of 19 patients without a history of COVID-19-related symptoms but positive for antibodies,2 were treated with mesalazine,1 with AZA and 4 with biologic therapy.All but one patient,who had pneumonia and was under AZA treatment,did not require hospitalization.Data regarding the patients with IgG＞7 were summarized in Table 3.

Cohort of patients affected by IBD (Crohn’s disease or ulcerative colitis).From April 22,2020 to May 31,2020,each IBD patient followed-up at ASST Cremona was offered to participate in the study.The patients were consecutively enrolled.

Swab throat test

All the patients with IgG＞7 were tested with a swab throat test.All of them were negative.The patient with a history of COVID-19 pneumonia had tested positive before the enrollment and tested negative after enrollment.

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Correlation between SARS-CoV-2 IgG positivity and clinical/therapeutic variables in the IBD cohort

SARS-CoV-2 IgG value ≥ 7 correlated at multivariate analysis only with IBD treatment.In detail,stratifying the population for treatment,the relative risk of having SARS-COV-2 IgG ≥ 7 was higher for patients treated with AZA and lower with mesalazine.The odds ratios for AZA was 1.44 (95%CI:0.27-7.56) and 0.16 (95%CI:0.03-0.71) for mesalazine

biologic drug (

=0.0157 between them).The relative risk for patients under mesalazine was lower than for those under biologic therapy (

=0.016).

Correlation between the presence of COVID-19-related symptoms and clinical/therapeutic variables in IBD cohort

The presence of COVID-19-related symptoms were correlated after multivariate analysis with BMI (

=0.05) and with IBD therapy.The relative risk of having symptoms was higher for patients treated with AZA and lower with mesalazine

biologic drug:odds ratios 7.47 (95%CI:1.22-45.73) and 0.52 (95%CI:0.17-1.72,

=0.03) for AZA and mesalazine,respectively (

=0.004 between them).

103 IBD consecutive patients were enrolled:54 with Crohn’s disease and 49 ulcerative colitis.36 patients (35%) were treated with biologic treatment,14 (13.6%) with azathioprine (AZA) and 53 (51.4%) with mesalazine.19 out of 103 patients (18.4%) had SARS-CoV-2 IgG positivity,with value＞7.Among them:10 were under biological treatment,5 under AZA and 4 under mesalazine.12 out of 19 (63%) reported symptoms related to COVID-19 disease.Among them,2 were treated with mesalazine,4 with AZA and 6 with biologic treatment.Among the 7 out 19 patients without history of COVID-19 related symptoms,but positive for antibodies,2 were treated with mesalazine,one with AZA and 4 with biologic therapy.All but one patient,who had pneumonia and was under AZA treatment,did not require hospitalization.All the patients with IgG＞7 were tested for swab throat test.All of them resulted negative at the enrollment.SARS-CoV-2 IgG value ≥ 7 correlated at multivariate analysis only with IBD treatment.The relative risk of having SARS-COV-2 IgG ≥ 7 was higher for patients treated with AZA and lower with mesalazine:odds ratio (OR) 1.44 (95%CI:0.27-7.56) and 0.16 (95%CI:0.03-0.71),for AZA and mesalazine,respectively,

biologic drug (

=0.0157 between them).The relative risk for patients under mesalazine was lower than for those under biologic therapy,

=0.016.The presence of COVID-19 related symptoms resulted correlated at multivariate analysis with Body Mass Index (BMI),

=0.05 and with IBD therapy.The relative risk of having symptoms was strongly higher for patients treated with AZA and lower with mesalazine

biologic drug:odds ratio (OR) 7.47 (95%CI:1.22-45.73) and 0.52 (95%CI:0.17-1.72,

=0.03),for AZA and mesalazine,respectively (

=0.004 between them).

DlSCUSSlON

The use of SARS-Cov-2 antibodies to monitor the immunity against COVID-19 remains a matter of debate in the general population.However,the presence of SARS-CoV-2 IgG antibodies certify the previous or recent infection[17].In our hospital,all health care workers (HCW) were tested for serology immediately after the first 2 mo of pandemic,in the same week of the start of our study on IBD cohort.364 out of 1600 operators were diagnosed as affected by COVID-19 between February 21 and April 22 and all of them tested positive for SARS-CoV-2 swab throat test.Among the HCWs who were previously confirmed ill,the 99% resulted having IgG3 value＞7.Interestingly,20% of operators who did not report symptoms suggestive for COVID-19 resulted having SARS-CoV-2 antibodies ≥ 7.(data from National Institute of Health,2020).This observation confirms the presence of an unknown number of asymptomatic infected people[18].The available studies on the serum concentration of IgG after COVID-19 infection revealed conflicting results and the duration of antibodies rises is currently unknown,but is estimated around 9 mo (data from National Institute of Health,2021).There is a possible decrease of IgG title after the first two wk of infection and it is unclear whether the test is able to detect lower antibody levels in milder and asymptomatic COVID-19 disease[17-20].Plebani group tried to harmonize the thresholds to allow a larger agreement on IgG anti Sars-Cov-2 antibodies determination.They found 6.2 KAU/L as the cut off for Diasorin method to reach a sensitivity of 97.1% and a specificity of 88.9% for the diagnosis of SARS-CoV-2 infection[16].Our data are thus in line with this latter observation.The COVID-19 symptoms occurred in IBD patients at least 1 mo before the interview.During the time between the symptoms and the enrollment,they lived the complete lock down,established in Italy from March 9 to May 18.They tested all negative at the swab test performed at the enrollment.This is in line with the overall sensitivity of the test,ranging from 56 to 83%:66.7% in the first week of the infection and lower in the following wk observation that the SARS-CoV-2 positivity in the swab[21].

Prevalence of patients with SARS-CoV-2 IgG positivity in our cohort was 18.4%.This means that those patients got infected with SARS-CoV-2 virus in the previous period,but only 63% of them developed the disease,reporting symptoms.Moreover,only one patient required hospitalization for pneumonia.The patients with history of COVID-19 related symptoms mainly had mild respiratory symptoms or minor manifestations.None but one patient (5%) required hospitalization,but without the need of intensive care unit.Conversely,in the general population,during both the first and the second wave of the pandemic,10% of people required hospitalization in intensive care unit (data from the National Institute of Health,2021).Half of the IBD patients that resulted positive to antibody test remained asymptomatic and in 48% of cases they developed only mild symptoms.We can thus conclude that the IBD population does not seem at higher risk to develop severe COVID-19 disease in comparison with the general population,confirming the observation of Bezzio

[9].Only the patient with pneumonia hold the IBD treatment.This happened because,due to the mildness of the disease,the patients informed the general practitioner but not the IBD center about the symptoms.These data,even if do not confirm the American Gastroenterological Association guidelines strategy,gave us the opportunity to evaluate the cohort[1].The results obtained are encouraging,as it seems that IBD patients with COVID-19 ongoing disease with symptoms could continue any treatments both avoiding IBD relapse and without a significant higher risk of developing severe COVID-19 requiring hospitalization.Differently from Bezzio

[9],nobody died in our cohort;moreover,nor age neither active IBD were significantly associated with a COVID-19 worse prognosis.

SARS-coV-2 serology resulted associated only with the ongoing IBD treatment.Among the patients having a positive serology there was a prevalence of biologic therapy.The presence of COVID-19 disease was associated with both IBD therapy and BMI.The patients who reported previous symptoms were treated with mesalazine in 2 cases,with AZA in 4 and with biological treatment in 6;the only patient with pneumonia was treated with AZA.The calculated relative risk of being infected was higher for patients treated with AZA,then for patients treated with biologic drugs and the lowest risk was found for patients treated with mesalazine.We decided to separate the different treatments in the analysis,as the AZA and the biologic therapy have a different mechanism of action:AZA is an immunosuppressive agent,whereas the biologic therapies are known as immunomodulating agents.None of the patients treated with biologic therapy developed a severe COVID-19 disease.Our results show that the use of biologic therapy does not seem to expose the patients to higher risk of severe COVID-19 disease,even when the infection is present.We did not perform a sub-analysis of the different type of biologic treatment for the small sample size.However,we report that the 80% of patients was treated with anti-TNF agents.More studies are needed to confirm whether it is appropriate to continue biological drugs for IBD patients who are affected with Sars-cov-2.The other variable associated with the presence of COVID-19 related symptoms was the BMI.This data is supported by the literature,as obesity is a factor associated with bad prognosis in the patients with COVID-19 pneumonia[22].Interestingly,nor the old age neither the comorbidities or the type of IBD were associated with the antibody positivity or the development of COVID-19 symptoms in our study.This could be explained by the fact that these variables were associated in literature to death or very bad outcome,and none of our patients reported such complication[23].

You know, every morning for the past week, a fine looking gentleman in a military uniform has been standing10 across the corner watching you when you get off the bus. He makes sure you cross the street safely and he watches you until you enter your office building. Then he blows you a kiss, gives you a salute11 and walks away. You are one lucky lady, the bus driver said.

The main limitation of the study is the small sample.Therefore,further studies with larger populations are needed to confirm our observations.

CONCLUSlON

We investigated both the SARS-CoV-2 IgG positivity in symptomatic and asymptomatic IBD patients and the relationship between IBD therapy and COVID-19 disease severity.The results are interesting and seem encouraging for the patients treated with biologic therapy,since they don’t seem to carry a high risk of developing severe COVID-19.However,further and larger studies are needed to confirm these observations.

ARTlCLE HlGHLlGHTS

Research background

Guidelines recommend to hold inflammatory bowel diseases (IBD) biologic therapy during coronavirus disease 2019 (COVID-19).It is still not clear if the IBD patients carry a high risk of developing severe COVID-19.

Research motivation

IBD patients could carry a high risk of relapse or worsening of the intestinal disease in holding the therapy.

Research objectives

He eagerly inquired whose it was, and the Queen, with many tears, told him it was all that was left her of her beloved daughter, who had suddenly been carried off, she knew neither where nor how

The service was over, and thecongregation passed out into the churchyard, where not a tree orbush was to be seen; no flowers were planted there, and they had notplaced a single wreath upon any of the graves

Research methods

Prospective cohort study.Patients with IBD were consecutively enrolled from April 22nd to May 31st 2020.Age,sex,BMI,IBD type,treatments and clinical activity and other comorbidities were anonymously collected in a Database.Charlson Comorbidity Index was calculated for each patient.A single blood test was performed to each patient to search for Immunoglobulin IgG anti SARS-Cov-2.The LIAISON? SARS-CoV-2 S1/S2 IgG test [DiasorinS.p.A,Saluggia (VC) - Italy] was used according to manufacturers’ instructions.The analysis was performed using SARS-CoV-2 IgG value cut off of＞7 kAU/L.All patients who resulted positive to SARS-CoV-2 IgG were tested with SARS-CoV-2 swab throat test during the same week,using the Allplex 2019-nCoV assay (Arrow Diagnostics S.r.l.,Genova,Italy) a single-tube assay able to detect the three target genes (E gene,RdRP gene and N gene) as in the WHO recommended protocols.Categorical variables were described as count and percentage and compared between groups with chi square test;continuous variables were described as mean and standard deviation or median and inter-quartile range if not normally distributed (Shapiro-Wilks test) and compared with independent t- test or Mann-Whitney.Through univariate and multivariate logistic regression models were assessed:association between age,sex,BMI,IBD type,IBD treatments,IBD clinical activity,Charlson Comorbidity Index and SARS-CoV-2 IgG positivity or the presence of COVID-19 symptoms.

Research results

Near the end of the evening, a flower vendor10 made her way through the cafe. We watched as the old gentleman purchased a bouquet11. Artfully, he plucked two flowers from the bunch, presented the bouquet to his lady, and gave her a kiss. Then, bowing smartly in our direction, he held out a rose, one for each of us.

Research conclusions

Univariate and multivariate logistic regression models were used to assess:(1) Association between age,sex,BMI,IBD type,IBD treatments,IBD clinical activity,Charlson Comorbidity Index and SARSCoV-2 IgG positivity;and (2) Association between age,sex,BMI,IBD type,IBD treatments,IBD clinical activity,Charlson Comorbidity Index and presence of COVID-19 symptoms.

Research perspectives

The patients treated with biologic therapy don’t seem to carry a high risk of developing severe COVID-19.Therefore,further and larger studies are needed to confirm these observations and to understand if the strategy to hold the IBD treatment during COVID-19 disease could be modified.

With these words he continued his way, and very soon met the third knave13, who said to him, God bless you, sir; are you by any chance coming from the market? Yes, I am, replied Simon

Conti CB and Grassia R conceived and planned the study;Mainardi E,Grassia R,Drago A,Cereatti F,Soro S and Testa S carried out the tests and collected the data;Testa S and Mainardi E contributed to the interpretation of the results;De Silvestri A performed the statistical analysis;Conti CB wrote the manuscript;all authors provided critical feedback and helped the research and analysis of the manuscript.

The study was reviewed and approved by the Institutional Review Board Comitato EticoVal Padana.

9. Black and blue: The idea that the princess has actually been bruised14 by the pea(s) is a haunting image of her sensitivity. Some modern writers have explored the sado-masochist possibilities of this story element, wondering why the prince would want to marry a woman with such physical sensitivity.Return to place in story.

All study participants,or their legal guardian,provided informed written consent prior to study enrollment.

No conflict of interest.

Technical appendix,statistical code,and data set available from the corresponding author at benedetta.conti1@gmail.com.Participants gave informed consent for data collection and data are recorded anonymized.Risk of identification is very low.

The authors have read the STROBE Statement-checklist of items,and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.

This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BYNC 4.0) license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is noncommercial.See:https://creativecommons.org/Licenses/by-nc/4.0/

She didn t wait. Making her way home, she chose the longer route that twisted along the foot of the moor5. They had a long way to go, but, like the weather, maybe the outlook was promising13.

At early dawn the giants went off to the wood, and quite forgot about the little tailor, till all of a sudden they met him trudging17 along in the most cheerful manner

Italy

:Clara Benedetta Conti 0000-0001-9774-2374;Elsa Mainardi 0000-0003-3522-105X;Sara Soro 0000-0002-4802-8403;Sophie Testa 0000-0002-3512-0243;Annalisa De Silvestri 0000-0003-3128-8441;Andrea Drago 0000-0002-9777-8665;Fabrizio Cereatti 0000-0003-0628-4473;Roberto Grassia 0000-0003-4491-4050.

:Wang LL

Filipodia

:Wang LL

1 Rubin DT,Feuerstein JD,Wang AY,Cohen RD.AGA Clinical Practice Update on Management of Inflammatory Bowel Disease During the COVID-19 Pandemic:Expert Commentary.

2020;159:350-357 [PMID:32283100 DOI:10.1053/j.gastro.2020.04.012]

2 Cheung KS,Hung IFN,Chan PPY,Lung KC,Tso E,Liu R,Ng YY,Chu MY,Chung TWH,Tam AR,Yip CCY,Leung KH,Fung AY,Zhang RR,Lin Y,Cheng HM,Zhang AJX,To KKW,Chan KH,Yuen KY,Leung WK.Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort:Systematic Review and Meta-analysis.

2020;159:81-95 [PMID:32251668 DOI:10.1053/j.gastro.2020.03.065]

3 Ling Y,Xu SB,Lin YX,Tian D,Zhu ZQ,Dai FH,Wu F,Song ZG,Huang W,Chen J,Hu BJ,Wang S,Mao EQ,Zhu L,Zhang WH,Lu HZ.Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients.

2020;133:1039-1043 [PMID:32118639 DOI:10.1097/CM9.0000000000000774]

4 Zhang J,Wang S,Xue Y.Fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia.

2020;92:680-682 [PMID:32124995 DOI:10.1002/jmv.25742]

5 Wu Y,Guo C,Tang L,Hong Z,Zhou J,Dong X,Yin H,Xiao Q,Tang Y,Qu X,Kuang L,Fang X,Mishra N,Lu J,Shan H,Jiang G,Huang X.Prolonged presence of SARS-CoV-2 viral RNA in faecal samples.

2020;5:434-435 [PMID:32199469 DOI:10.1016/S2468-1253(20)30083-2]

6 Ma C,Cong Y,Zhang H.COVID-19 and the Digestive System.

2020;115:1003-1006 [PMID:32618648 DOI:10.14309/ajg.0000000000000691]

7 An P,Ji M,Ren H,Su J,Ding NS,Kang J,Yin A,Zhou Q,Shen L,Zhao L,Jiang X,Xiao Y,Tan W,Lv X,Li J,Liu S,Zhou J,Chen H,Xu Y,Liu J,Chen M,Cao J,Zhou Z,Tan S,Yu H,Dong W,Ding Y.Prevention of COVID-19 in patients with inflammatory bowel disease in Wuhan,China.

2020;5:525-527 [PMID:32311321 DOI:10.1016/S2468-1253(20)30121-7]

8 Norsa L,Indriolo A,Sansotta N,Cosimo P,Greco S,D'Antiga L.Uneventful Course in Patients With Inflammatory Bowel Disease During the Severe Acute Respiratory Syndrome Coronavirus 2 Outbreak in Northern Italy.

2020;159:371-372 [PMID:32247695 DOI:10.1053/j.gastro.2020.03.062]

9 Bezzio C,Saibeni S,Variola A,Allocca M,Massari A,Gerardi V,Casini V,Ricci C,Zingone F,Amato A,Caprioli F,Lenti MV,Viganò C,Ascolani M,Bossa F,Castiglione F,Cortelezzi C,Grossi L,Milla M,Morganti D,Pastorelli L,Ribaldone DG,Sartini A,Soriano A,Manes G,Danese S,Fantini MC,Armuzzi A,Daperno M,Fiorino G;Italian Group for the Study of Inflammatory Bowel Disease (IG-IBD).Outcomes of COVID-19 in 79 patients with IBD in Italy:an IGIBD study.

2020;69:1213-1217 [PMID:32354990 DOI:10.1136/gutjnl-2020-321411]

10 D'Amico F,Danese S,Peyrin-Biroulet L.Systematic Review on Inflammatory Bowel Disease Patients With Coronavirus Disease 2019:It Is Time to Take Stock.

2020;18:2689-2700 [PMID:32777550 DOI:10.1016/j.cgh.2020.08.003]

11 Neurath MF.COVID-19 and immunomodulation in IBD.

2020;69:1335-1342 [PMID:32303609 DOI:10.1136/gutjnl-2020-321269]

12 Buonaguro FM,Puzanov I,Ascierto PA.Anti-IL6R role in treatment of COVID-19-related ARDS.

2020;18:165 [PMID:32290847 DOI:10.1186/s12967-020-02333-9]

13 Cortegiani A,Ippolito M,Greco M,Granone V,Protti A,Gregoretti C,Giarratano A,Einav S,Cecconi M.Rationale and evidence on the use of tocilizumab in COVID-19:a systematic review.

2021;27:52-66 [PMID:32713784 DOI:10.1016/j.pulmoe.2020.07.003]

14 Monteleone G,Ardizzone S.Are Patients with Inflammatory Bowel Disease at Increased Risk for Covid-19 Infection?

2020;14:1334-1336 [PMID:32215548 DOI:10.1093/ecco-jcc/jjaa061]

15 Perkmann T,Perkmann-Nagele N,Breyer MK,Breyer-Kohansal R,Burghuber OC,Hartl S,Aletaha D,Sieghart D,Quehenberger P,Marculescu R,Mucher P,Strassl R,Wagner OF,Binder CJ,Haslacher H.Side-by-Side Comparison of Three Fully Automated SARS-CoV-2 Antibody Assays with a Focus on Specificity.

2020;66:1405-1413[PMID:32777031 DOI:10.1093/clinchem/hvaa198]

16 Plebani M,Padoan A,Negrini D,Carpinteri B,Sciacovelli L.Diagnostic performances and thresholds:The key to harmonization in serological SARS-CoV-2 assays?

2020;509:1-7 [PMID:32485157 DOI:10.1016/j.cca.2020.05.050]

17 Gaebler C,Wang Z,Lorenzi JCC,Muecksch F,Finkin S,Tokuyama M,Cho A,Jankovic M,Schaefer-Babajew D,Oliveira TY,Cipolla M,Viant C,Barnes CO,Bram Y,Breton G,H?ggl?f T,Mendoza P,Hurley A,Turroja M,Gordon K,Millard KG,Ramos V,Schmidt F,Weisblum Y,Jha D,Tankelevich M,Martinez-Delgado G,Yee J,Patel R,Dizon J,Unson-O'Brien C,Shimeliovich I,Robbiani DF,Zhao Z,Gazumyan A,Schwartz RE,Hatziioannou T,Bjorkman PJ,Mehandru S,Bieniasz PD,Caskey M,Nussenzweig MC.Evolution of antibody immunity to SARS-CoV-2.

2021;591:639-644 [PMID:33461210 DOI:10.1038/s41586-021-03207-w]

18 Grassia R,Testa S,De Silvestri A,Drago A,Cereatti F,Conti CB.Lights and shadows of SARS-CoV-2 infection risk assessment in endoscopy.

2020;52:816-818 [PMID:32601027 DOI:10.1016/j.dld.2020.06.013]

19 Melga?o JG,Azamor T,Ano Bom APD.Protective immunity after COVID-19 has been questioned:What can we do without SARS-CoV-2-IgG detection?

2020;353:104114 [PMID:32361409 DOI:10.1016/j.cellimm.2020.104114]

20 Long QX,Liu BZ,Deng HJ,Wu GC,Deng K,Chen YK,Liao P,Qiu JF,Lin Y,Cai XF,Wang DQ,Hu Y,Ren JH,Tang N,Xu YY,Yu LH,Mo Z,Gong F,Zhang XL,Tian WG,Hu L,Zhang XX,Xiang JL,Du HX,Liu HW,Lang CH,Luo XH,Wu SB,Cui XP,Zhou Z,Zhu MM,Wang J,Xue CJ,Li XF,Wang L,Li ZJ,Wang K,Niu CC,Yang QJ,Tang XJ,Zhang Y,Liu XM,Li JJ,Zhang DC,Zhang F,Liu P,Yuan J,Li Q,Hu JL,Chen J,Huang AL.Antibody responses to SARS-CoV-2 in patients with COVID-19.

2020;26:845-848 [PMID:32350462 DOI:10.1038/s41591-020-0897-1]

21 Zou L,Ruan F,Huang M,Liang L,Huang H,Hong Z,Yu J,Kang M,Song Y,Xia J,Guo Q,Song T,He J,Yen HL,Peiris M,Wu J.SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients.

2020;382:1177-1179 [PMID:32074444 DOI:10.1056/NEJMc2001737]

22 Kassir R.Risk of COVID-19 for patients with obesity.

2020;21:e13034 [PMID:32281287 DOI:10.1111/obr.13034]

23 Yu C,Lei Q,Li W,Wang X,Liu W.Epidemiological and clinical characteristics of 1663 hospitalized patients infected with COVID-19 in Wuhan,China:a single-center experience.

2020;13:1202-1209 [PMID:32718894 DOI:10.1016/j.jiph.2020.07.002]