Sonographic Assessment in Severe Ulcerative Colitis Patients Admitted for Intravenous Corticosteroids and Eligible for Infliximab Rescue Therapy; a Prospective Clinician-blinded Observational Study Protocol.
Sonographic Assessment in Severe Ulcerative Colitis Patients Admitted for Intravenous Corticosteroids and Eligible for Infliximab Rescue Therapy; a Prospective Clinician-blinded Observational Study Protocol.
Introduction Acute severe ulcerative colitis (ASUC) occurs in 15-25 % of all ulcerative
 colitis (UC) patients. Initial treatment with intravenous corticosteroids fails in 30-50 % of
 patients, for whom the next line of treatment is biological therapy or colectomy. Acute
 colectomy has a higher risk of morbidity and mortality than a scheduled colectomy. Data
 suggest that an accelerated administration of biological treatment in corticosteroid
 non-responders compared to clinical practice, 5-7 days with intravenous corticosteroids, may
 be superior in inducing disease remission, thus potentially avoiding acute colectomy.
 However, there are currently no patient friendly and objective diagnostic tool to preselect
 patients for such a treatment. The aim of this study is to examine if gastrointestinal
 ultrasound (GIUS) could preselect corticosteroid non-responders to biological treatment after
 48 hours to increase effectiveness of the second line therapy and thereby reduce the
 morbidity and mortality of ASUC.

 Methods and analysis The study is a clinician blinded observational multi-center study
 derived from the Department of Gastroenterology, Herlev Hospital, Denmark. Fifty ASUC
 patients will be included at the time of hospitalization and followed for 12 months. Baseline
 clinical activity scores, endoscopic scores, blood samples, fecal-calprotectin, vital
 parameters and GIUS measurements will be obtained prior to administration of intravenous
 corticosteroids. All examinations except fecal-calprotectin and endoscopy will be repeated at
 48 ± 24 hours, 5-7 days and 3 months after treatment start. Endoscopic scores and
 fecal-calprotectin will be obtained after 3 months and an additional fecal-calprotectin after
 6 ± 1 days. Treatment outcome will be registered at each event and after 12 months. Patients
 will be divided into corticosteroid responders and non-responders and compared to GIUS
 measurements at each event using non-parametric statistics (Mann-Whitney and Wilcoxon test)
 and time to endpoints by survival statistics (Kaplan Meier). ROC statistics will determine
 the best cutoff values for GIUS parameters for optimal sensitivity, specificity and accuracy.

 Ethics and dissemination The study is approved by the National committee on health research
 ethics (H-18031264). Results will be published in relevant scientific journals and presented
 at international conferences. Fully anonymized data will be accessible from authors upon
 request.
Introduction

 Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD), typically diagnosed
 between the age of 15 and 40 and characterized by colonic mucosal inflammation and
 ulcerations. These mucosal changes lead to bloody diarrhea and in severe cases, fever,
 anemia, weight loss and death if not treated appropriately. The condition requires prompt
 immunosuppressive treatment and monitoring. To characterize disease severity and guide
 treatment decisions, clinicians typically use a combination of clinical scores based on
 patients symptoms, endoscopic scoring systems based on mucosal appearance endoscopic
 assessments of disease extent and blood and stool markers of inflammation.

 Most patients experience a mild to moderate disease course with varying periods of activity
 and remission. However, 15-25 % will experience a flare of acute severe UC (ASUC), a
 condition characterized by extensive and deep ulcerations on endoscopy combined with a high
 clinical score of severity. Due to the risk of bowel wall perforation, these patients are
 usually admitted to the hospital ward for intensive monitoring and medical therapy. The
 medical therapy ultimately aims at avoiding acute colectomy, a procedure that still carries a
 30-day mortality rate of 5 %, which is higher compared to elective surgery (OR 1.82; CI 95 %
 1.19 - 2.62). Initial therapy is well established and consists of intravenous
 corticosteroids, which in early clinical trials dramatically improved the disease outcome by
 reducing the mortality rate from 24 to 7 %. Unfortunately, even today 30 - 50 % of patients
 with ASUC fails to respond to initial corticosteroid treatment and are therefore eligible for
 medical rescue treatment if surgery is not imminent.

 Accordingly, primary non-responders to high dose intravenous corticosteroids are usually
 switched to infliximab (or at certain centers ciclosporine) after 5-7 days, if an acute
 colectomy is not needed. Infliximab is an anti-tumor necrosis factor (TNF) alpha antibody,
 which blocks TNFα, a key inflammatory mediator in UC,and has been proven effective in
 corticosteroid resistant UC reducing the risk of acute colectomy from 58 % to 29 %.

 Despite advances in medical treatment ASUC still carries a 10 % risk of acute colectomy.
 Further improvement in medical therapy and strategy is therefore highly warranted. A decrease
 in serum albumin is associated with an increased clearance of infliximab, and it has been
 suggested that postponement of infliximab treatment in ASUC could reduce the bioavailability
 of the drug as a result of severe hypoalbuminemia. In addition, infliximab is lost in feces
 in the setting of ASUC, suggesting that an accelerated infliximab induction regimen or an
 increased starting dose compared to standard procedures could be beneficial. However, a
 recent review of retrospective studies showed no difference in colectomy rates when comparing
 an accelerated infliximab induction regimen with standard induction regimen (both induced
 after 3-5 days of corticosteroid treatment), although confounding by disease severity cannot
 be excluded. Hence it is currently unresolved if optimized timing and dosing of infliximab
 rescue treatment could be of major importance to reduce the need of acute colectomies and
 thereby the morbidity and mortality among ASUC patients. Therefore, data to support
 clinician's recognition of corticosteroid non-responders early in the process are warranted.

 Unfortunately, there are currently no patient friendly, effective or objective point-of-care
 tool to identify corticosteroid non-responders early in the process. A high clinical score,
 C-reactive protein (CRP) and a low albumin is correlated with an inferior response, but due
 to poor positive and negative predictive values they are not routinely used to pre-select
 patients for specific medical therapies. Endoscopic severity is predictive for the outcome of
 ASUC, but the use of close monitoring with endoscopy is limited in this setting due to the
 risk of perforation and the need for repeated bowel cleansing.

 Transabdominal gastrointestinal ultrasound (GIUS) is potentially a relevant objective
 point-of-care tool, which could be used for treatment outcome assessment in ASUC. A recent
 study in ulcerative colitis patients using GIUS to monitor anti-TNFα treatment demonstrated a
 high correlation between change in GIUS parameters, bowel wall thickness (BWT) and color
 Doppler Signal (DS), compared to baseline with treatment response after 6 and 12 weeks. The
 results are in accordance with other studies showing a correlation between endoscopic
 activity, clinical symptom scores, CRP and GIUS parameters after 2-3 months of treatment. In
 this setting, GIUS can also predict treatment outcome after 12-15 months. Ultrasound has
 several advantages compared to other modalities as it is non-invasive and does not inflict
 pain, is easily repeatable and available and does not expose the patient to radiation.
 Although some studies suggest 4 hours of fasting to diminish peristalsis and intraluminal
 air, fasting before examination is generally not necessary, making it preparation free.

 Not all bowel segments can be easily visualized, which can be an issue when assessing disease
 extent. Especially the rectum and the left colonic flexure can be difficult to visualize and
 examine with the transabdominal approach. The combined reported sensitivity and specificity
 for detection of colonic inflammation is 74 % and 93 % per segment, whereas the sigmoid colon
 and the terminal ileum are easy to identify and measure, showing a higher diagnostic
 performance (sensitivity 92 % and specificity 87 %) compared to endoscopy. There is no
 significant difference in diagnostic accuracy per bowel segment compared to other radiologic
 modalities and in the latest ECCO guidelines GIUS is recommended as a valid alternative to CT
 or MR.

 There are different signs of inflammation to look for when examining the bowel using GIUS;
 the most reported and prominent one being BWT. BWT in healthy subjects ranges between 0.5-3.0
 mm depending on anatomical location. Studies on UC patients use different cutoff values,
 usually ranging between 3.0-4.0 mm. There is an ongoing discussion within the scientific
 community about cutoff values. In general, sensitivity decreases and specificity increases
 with a higher value. Vascularization is another important measure and can be estimated with
 DS and the Limberg score to define the presence of a high blood flow; a high blood flow
 correlates well with inflammation. Further, the bowel wall layers are easily identified due
 to echo-stratification on ultrasound. These layers are still present in the mild and moderate
 UC cases, whereas the stratification can be lost in severe UC. Severe inflammation can also
 cause colonic de-haustration, although this parameter might also correlate to the chronicity
 of the condition. Inflammatory mesenteric fat wrapped around the affected intestine is rarely
 seen in UC patients but frequently in Crohn's disease.

 No study examining the ability of GIUS to predict treatment response in ASUC patients has yet
 been conducted on a shorter timeframe than 6 weeks. The investigators have unpublished
 preliminary data suggesting a rapid detectable change in GIUS parameters in patients
 responding to high-dose corticosteroids. If GIUS can be used as an objective point-of-care
 tool for treatment response, it may be useful to identify corticosteroid non-responders at an
 early stage, thus providing an opportunity for optimized individual treatment, i.e. rescue
 infliximab treatment, and hopefully a decreased morbidity and mortality rate.

 Methods and analysis

 Study aims The primary aim is to determine if early changes in GIUS parameters (BWT,
 vascularization, echostratification, inflammatory mesenteric fat and haustration) from
 baseline to 48 ± 24 hours after intravenous corticosteroids initiation in hospitalized
 patients with ASUC, can predict clinical corticosteroid non-response and need for biological
 treatment and/or colectomy.

 The secondary aims are to:

 1. Determine if changes in GIUS parameters from baseline to day 6 ± 1 and 3 months after
 intravenous corticosteroids initiation can predict treatment non-response and need for
 colectomy.

 2. Identify the potential interrater variability between investigators and central reading
 for GIUS parameters.

 3. Correlate GIUS parameters with changes in fecal calprotectin and endoscopic disease
 activity (Mayo endoscopic sub-score and Ulcerative Colitis Endoscopic Index of Severity
 (UCEIS)) at baseline and 3 months after hospitalization.

 4. Correlate GIUS parameters with changes in clinical scores (Mayo score and Simple
 Clinical Colitis Activity Index (SCCAI)), blood samples (hemoglobin, CRP, albumin and
 alpha-1-antitrypsin), pulse, blood pressure, temperature and time since last meal at 48
 ± 24 hours, 6 ± 1 days and 3 months after hospitalization.

 5. Examine GIUS´s ability to assess disease extent compared to endoscopy and CT (if a
 CT-scan is performed after an ordination by a treating physician).

 6. Determine the ideal cutoff values for GIUS parameters for optimal sensitivity,
 specificity and accuracy.

 Study design The study design is a prospective clinician blinded multi-center observational
 study. Fifty patients will be included and followed for 12 months. The study is approved by
 the Danish National committee on health research ethics H-18031264 and the Danish data
 protection agency VD-2018-319.

 Procedures and scheduled follow-up visits

 Included patients will prior to intravenous corticosteroid treatment have the following
 information registered as baseline characteristics:

 -  Date of birth, age at disease diagnoses, previous and present IBD medication, previous
 intestinal surgery, previous extent of disease, other known chronic diseases, smoking
 status, extra-intestinal manifestations, weight and height, symptoms (blood and
 frequency of stools, urgency of defecation, general well-being, physician's general
 assessment), family history of IBD and previously known gastrointestinal infections
 (Clostridium difficile, Salmonella, Campylobacter jejuni, CMV, Yersinia enterocolitica
 or other).

 -  Mayo endoscopic sub-score, UCEIS and current endoscopic extent of inflammation.

 -  Blood samples (hemoglobin, CRP, albumin, alpha - 1 - antitrypsin), fecal calprotectin
 results, pulse, blood pressure and temperature.

 -  GIUS assessment of the BWT, vascularization, echostratification, inflammatory mesenteric
 fat and haustration.

 -  Time since last meal prior to GIUS examination. The intestinal ultrasound together with
 blood samples and recordings of pulse, blood pressure, temperature, time since last
 meal, time since first intravenous corticosteroid administration as well as clinical
 scores will be repeated 48 ± 24 hours, 6 ± 1 days and 3 months after the first dose of
 intravenous corticosteroids. Endoscopic examination and fecal calprotectin will be
 repeated after 3 months. Treatment outcome (response to corticosteroid, infliximab or
 colectomy) will be registered at 48 ± 24 hours, 6 ± 1 days, 3 months and 12 months after
 the first dose of intravenous corticosteroids. Results of eventual CT-scans or extra
 endoscopies will be stored.

 Clinical assessment Patient disease status is monitored during hospitalization using the Mayo
 clinical score and SCCAI. Mayo clinical score is based on blood and stool frequency, baseline
 endoscopy results and the physician's global assessment. SCCAI is based on bowel frequency
 divided by night and day, urgency of defecation, blood in stool, patient general well-being
 and extra-intestinal features. Endoscopic severity is graded using the Mayo endoscopic
 subscore and the UCEIS, which is based on the severity of the vascular pattern, presence of
 blood, erosions and ulcers. The clinical assessment and endoscopies will be performed by a
 physician blinded to the GIUS measurements. Endoscopic assessment is based on the worst
 segment and documented with still images on a secure drive.

 GIUS assessment and measurements Each examination will be performed by a GIUS trained
 gastroenterologist using the same high-end ultrasound scanner and 9L probe for the same
 patient. Probe frequency will be between 5-8 MHz. Scans are performed by localizing the
 sigmoid colon at the site of the iliac vessels and then followed in both anal and oral
 direction. The most severe part of the bowel segment will be assessed and documented by still
 images and cineloop files and stored in DICOM format on a secure drive. Sigmoid colon is
 always assessed as the primary point of interest. However, if topical treatment has been
 applied or a more oral located bowel segment has a higher BWT this segment will also be
 documented as above and stored. GIUS examination recordings will be subjected to central
 reading by an independent GIUS specialist, blinded to the original scans. Fasting will not be
 required, but the time passed since patients last meal will be registered. The GIUS examiner
 will have no knowledge about the patient disease status (blood and stool sample results,
 clinical scores or endoscopic activity).

 • BWT is measured in the most severe segment (largest BWT) using the average value of four
 measurements with one decimal (two longitudinal scans, more than 1 cm apart and two
 cross-sectional scans, which can be in the same image, but in different quadrants). A BWT
 value < 3.0 mm is regarded as no inflammation and ≥ 3.0 mm is regarded as active disease.

 In segments with a BWT ≥ 3.0 mm, vascularization will be classified using a color Doppler and
 a modified Limberg score, as well as echostratification and the eventual presence of
 inflammatory mesenteric fat and absence of haustration.

 -  Vascularization is measured in both the cross-sectional and longitudinal plane.
 Pathological vascularization is defined as a modified Limberg score > 1 and normal
 vascularization is defined as ≤ 1. No signal, 0 points; minimal pixels, 1 point;
 increased doppler signal limited to the wall, 2 points; and if the signal is significant
 in the wall as well as the mesentery, 3 points.

 -  Normal echostratification is measured in a longitudinal plane and defined as 3-5 echo
 layers in each bowel wall (mucosa, submucosa, lamina muscularis) and will be
 characterized as 1. normal/preserved, 2. uncertain, 3. focal disruption < 3 cm or 4.
 extensive disruption ≥ 3 cm.

 -  Inflammatory mesenteric fat will be defined as a bright looking mass surrounding or
 wrapping the bowel, usually brighter and more homogenous than normal intraabdominal fat
 in obese; characterized as either 1. absent, 2. uncertain or 3. present.

 -  Haustration will be defined as either 1. present (normal), 2. uncertain or 3. absent.

 Centre selection The study is devised by leading researchers in IBD and trained bowel
 ultrasound experts at the Department of Gastroenterology, Herlev Hospital, Denmark. The IBD
 clinic at Herlev Hospital is one of the largest clinical and academic IBD centers in Denmark
 and an international training center for GIUS. Recruitment and examination of the patients
 will be conducted at the gastroenterological departments of the university hospitals in
 Herlev, Aarhus, Vejle and Hvidovre. All GIUS gastroenterologists engaged in the study meet
 and train together to make the procedures identical and standardized before patient
 inclusion.

 Sample size calculation The sample size was calculated to be able to detect a 0.7-fold
 decrease in thickness of the intestinal wall with a common standard deviation (sigma) of 30 %
 and a desired power of 80 % to determine a statistically significant difference (α = 0.05,
 two-sided test). The estimated sample size is 17 subjects in each group
 (responders/non-responders). The investigators aim to include 20 UC patients in each group,
 and since 40 % are expected to be non-responders to steroids, the overall aim is to include
 50 patients. Our department currently admits 3-5 patients/month with ASUC. Together with the
 other departments, the estimated inclusion period is 15 months.

 Statistical analysis Clinical data will be entered into a validated data capture system
 provided by the Danish Capital Region. The data system will include password protection and
 internal quality checks, such as automatic range checks, to identify data that appear
 inconsistent, incomplete or inaccurate. Descriptive data will be provided for all outcomes
 (table 2) according to data type; number of patients, mean, SD (for interval data), median 25
 % and 75 % quartiles (for ordinal data). Frequency (absolute and relative) distributions will
 be provided for categorical data. Two-sided p values will be presented throughout. Data
 management and analysis will be performed using REDCap and SPSS.

 Primary endpoint analysis Patients will be divided into steroid responders and steroid
 non-responders and compared to GIUS parameters (BWT, modified Limberg score,
 echostratification, inflammatory mesenteric fat and haustration at baseline to 48 ± 24 hours)
 using non-parametric statistics (Mann-Whitney and Wilcoxon test) and time to endpoints by
 survival statistics (Kaplan Meier).

 Secondary endpoints analysis The same statistical tests as used for primary endpoint analysis
 will be applied to predict responders versus non-responders throughout the observational
 period. GIUS measurements at baseline compared to 6 ± 1 days and 3 months, with a follow up
 at 12 months. GIUS interrater-variability between GIUS examiners and proof reader will be
 calculated as kappa values, interclass correlation co-efficiency and Limits of Agreement for
 continues variables. Correlation between the GIUS measurements and endoscopic variables will
 be assessed using Spearman's rank correlation coefficient, multiple logistic regression and
 receiver operating characteristics (ROC) analyses. An additional ROC analyses will be
 performed to determine the best cutoff values for GIUS parameters for optimal sensitivity,
 specificity and accuracy.

 Variables associated with non-responders and responders GIUS measurements (CRP, Hemoglobin,
 Albumin, alpha-1-antitrypsin, Mayo clinical score, SCCAI, pulse, blood pressure, temperature,
 time since last meal and fecal calprotectin) will be assessed with univariate analyses. Those
 who achieve a p-value of < 0.2 will be included into a multivariable logistic regression
 analysis.

 Discussion There is an unfulfilled need for a reliable, easy, non-invasive and safe modality
 to repeatedly assess treatment response in patients admitted with ASUC. GIUS presents itself
 as such a modality and the investigators hypothesize that within 48 hours after
 corticosteroid treatment GIUS has the potential to identify steroid non-responders. This has
 not previously been investigated. An early identification allows for an accelerated treatment
 decision in steroid resistant ASUC patients and thus a potential decrease in the need of
 acute colectomies. To achieve this goal, the investigators first need to validate GIUS as a
 point-of-care tool to predict treatment outcome in patients with ASUC.

 Ethics and dissemination In accordance with the Helsinki V declaration and the Ethics
 committee of the Capital Region of Denmark enrollment in the study is based on voluntary
 informed written consent. All patients can at any time withdraw their participation without
 any change in standard care or treatment. Written information about the project will ensure
 patient knowledge of their rights. Ultrasound is a safe and patient friendly examination
 without any side effects, damage or radiation for the patient. The trial will contribute to a
 new partially unexplored field with promises of improving disease activity monitoring and
 consequently more rational and timely treatment intervention and decreased morbidity and
 mortality. Results will be published in international journals and disseminated at
 international conferences.
Inclusion Criteria:

 -  Definitive diagnoses of Ulcerative colitis.

 -  Mayo clinical score ≥8.

 -  Need for Hospitalization and intravenous corticosteroid treatment.

 -  Age between 18-70.

 -  Ability and willingness to give written consent and comply with study protocol.

 Exclusion Criteria:

 -  Contraindicators for infliximab.

 -  Bowel infection.

 -  Crohn's disease.

 -  Ultrasonographic inflammation in terminal ileum other than backwash ileitis.

 -  Bowel wall thickness <3 in Sigmoid colon.

 -  Minors.

 -  Known malignant disease.

 -  Pregnancy.

 -  Immune modulating therapy at admission apart from corticosteroids, mesalazine or
 azathioprine.

 Contraindicators = Patients suffering from moderate to severe heart failure,
 hypersensitivity to murine proteins, severe bacterial infection.