BMJ Open · Coventry CV2 2DX [email protected] Tel: 02476 966098 Fax: 02476 966090:...

For peer review only

Clinical outcomes at 12 months and risk of IBD in patients

with an intermediate raised faecal calprotectin (FC): A “real

world” view

Journal: BMJ Open

Manuscript ID bmjopen-2016-011041

Article Type: Research

Date Submitted by the Author: 07-Jan-2016

Complete List of Authors: McFarlane, Michael; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Chambers, Samantha; University Hospitals Coventry and Warwickshire

NHS Trust, Gastroenterology Malik, Ahmad; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Lee, Bee; Warwick Hospital, Gastroenterology Sung, Edmond; George Eliot Hospital, Gastroenterology Nwokolo, Chuka; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Waugh, Norman; University of Warwick, Warwick Evidence Arasaradnam, Ramesh; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology

<b>Primary Subject Heading</b>:

Gastroenterology and hepatology

Secondary Subject Heading: Diagnostics

Keywords: Inflammatory bowel disease < GASTROENTEROLOGY, Faecal Calprotectin, Intermediate, Outcomes

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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Clinical outcomes at 12 months and risk of IBD in patients with an

intermediate raised faecal calprotectin (FC): A “real world” view

Michael McFarlane1, Samantha Chambers

1, Ahmad Malik

1, Bee Lee

2, Edmond Sung

3,

Chuka Nwokolo1, Norman Waugh

5 and Ramesh Arasaradnam

1, 4

1Department of Gastroenterology, University Hospital Coventry & Warwickshire,

Coventry CV2 2DX, UK, 2Department of Gastroenterology, Warwick Hospital,

Warwick, CV34 5PP, UK, 3Department of Gastroenterology, George Eliot Hospital,

Nuneaton, CV10 7DJ, UK, 4Clinical Sciences Research Institute, University of

Warwick, Coventry CV2 2DX, UK, 5 Warwick Evidence, University of Warwick,

Coventry CV4 2DX, UK.

Correspondence to:

Dr R P Arasaradnam

Clinical Sciences Research Institute, University of Warwick,

Clifford Bridge Road,

Coventry CV2 2DX

[email protected]

Tel: 02476 966098

Fax: 02476 966090:

Running title: 12 month clinical outcomes for mildly elevated FCP

Key words: Faecal calprotectin, IBD, Intermediate, clinical outcomes

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Abstract

Objectives: A recent systematic review confirmed the utility of Faecal Calprotectin (FC)

in distinguishing organic (inflammatory bowel disease) from non-organic

gastrointestinal disease (irritable bowel syndrome). FC levels <50 mcg/g have a

negative predictive value >92% to exclude organic GI disease. Levels >250mcg/g

correlate with endoscopic IBD disease activity; sensitivity 90%. We aimed to determine

clinical outcomes in intermediate raised FC results (50-250 mcg/g).

Setting: Primary care general practices in Coventry and Warwickshire and three

secondary care hospitals

Participants: 443 FC results in adults (>16 years old) were reviewed from July 2012 -

October 2013. Clinical data was collected from hospital databases and General

Practitioners. Long term clinical data was available in 41 intermediate results.

Primary and secondary outcome measures: The number of new diagnoses of

inflammatory bowel disease (IBD), irritable bowel disease (IBS) and other diagnoses

for the intermediate group. The number referred and discharged from secondary care.

Results: A new IBD diagnosis was made in 19% (n=8) of intermediate results (1% of

normal and 38% of raised results). 5% (n=2) of intermediate results had known IBD in

remission. A new IBS diagnosis was made in 27% (n=11) of intermediate results whilst

34% (n=14) remained undiagnosed, although 8 of these were not referred to secondary

care.

Conclusions: FC testing remains useful in aiding diagnosis of organic GI conditions.

However, unlike negative and strongly positive FC results, intermediate FC results lead

to a mixture of diagnoses. The odds ratio of a new diagnosis of IBD for an intermediate

result compared to normal FC result was 26.6, whilst an intermediate FC result gave an

OR of 0.54 for a new IBS diagnosis compared to normal FC. For intermediate FC

results 1 in 3 patients remained in secondary care after 12 months with an OR of 3.6

compared to a normal FC result.

Strengths:

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• 12 month clinical follow up data of intermediate faecal calprotectin results

• A “real world” view of the utilisation of FC testing in primary and secondary

care – it is not being used entirely appropriately

• A “ real world” view of clinical outcomes – we don’t always find the answer

Limitations:

• Heterogenous data sources means data not as complete as could be e.g.

medication information incomplete

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1.0 Introduction

Calprotectin is a calcium binding protein, of the S100 family, found mainly in

neutrophils but also in other white blood cells (1). Inflammation results in neutrophil

activation and a subsequent release of calprotectin protein (2, 3). The use of faecal

calprotectin (FC) has particular interest as a non-invasive biomarker in the initial

screening and monitoring of patients with suspected or known inflammatory bowel

disease (IBD) (4). It is of particular use in the distinction between inflammatory GI

conditions such as IBD, from non-organic conditions such as irritable bowel syndrome

(IBS) (5). FC testing also has a growing role in the monitoring of IBD activity in

response to treatment, although long term data on the clinical advantages of this

approach is not yet available (6).

Current NICE and manufacturer guidelines for the cut of levels of FC in assays are that

levels of less than 50mcg/g of faeces suggest that there is no active inflammation

present within the GI mucosa (5, 7). NICE reported that for most of the studies that they

reviewed sensitivity and specificity were over 80%, where a cut-off of 50mcg/g was

used, and most positive and negative predictive values were 70-90% (7). One recent

study found that a cut-off of 50mcg/g gives a sensitivity and specificity of 88% and

78% respectively, with a negative predictive value of >92% to exclude organic GI

disease (8). A cut-off value of 100mcg/g has previously been suggested, and whilst this

increases sensitivity to 97%, specificity falls to 76%, with a negative predictive value of

97% and a positive predictive value of 75% (8). A cohort study involving consecutively

referred new patients with chronic diarrhoea proposed that a cut-off of 8mcg/g provides

near 99% sensitivity in detecting organic disease but at the cost of poor specificity (9).

In this study, no patients were diagnosed with IBD with FC levels of 50ug/g or less,

although this was a small study hence few patient overall with IBD. Another study

found that no patients with an FC result of <100mcg/g had IBD (10). A systematic

review of the use of FC, which informed the National Institute of Health and Care

Excellence diagnostic assessment group, found that most of the available evidence for

FC use in IBD is based on a cut-off value of 50mcg/g which reduces the number of false

negatives whilst maintaining cost effectiveness (5, 7).

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A FC value of greater than 250mcg/g has been demonstrated to correspond with

endoscopically and histologically active IBD. This cut-off level provides a sensitivity of

90% and specificity of 76% for excluding IBD and also determining if known disease

was quiescent (8). A recent meta-analysis into the utility of FC testing for monitoring

disease activity showed that for known IBD a cut-off level of 50mcg/g gave a pooled

sensitivity and specificity of 92% and 60% for determining if disease was quiescent, a

cut-off of 100mcg/g gave 84% and 66%, whilst cut-off of 250mcg/g gave 80% and 82%

respectively (11).

Despite the increasing number of studies involving utility of FC there has been limited

study on patients who have a FC value which falls within the “intermediate” group;

defined as levels of 50-250mcg/g of faeces. Whilst these patients may still have

underlying organic GI disease, current opinion suggests that the FC assay should be

repeated 4-6 weeks later to determine if inflammation is improving or worsening.

Although in reality an intermediate result may still be referred to secondary care if

clinical suspicion is high for underlying IBD.

The aim of this study was to determine the 12 month clinical outcomes of patients with

an intermediate raised FC value. There have been, to the best of our knowledge, no

published studies which look at clinical outcomes in this group of patients and

proportions remaining in secondary/tertiary care follow up.

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2.0 Materials and Methods

2.1 FC samples

FC test results from July 2012 to October 2013 in the University Hospital Coventry &

Warwickshire (UHCW) pathology database were reviewed. Tests were requested by

both primary care and secondary/tertiary care physicians from the Warwickshire region;

catchment population of 1 million. 495 patients were identified; 52 paediatric patients

(aged < 16 years) were excluded. Of the remaining, there were 365 (82%) with a normal

FC result (<50mcg/g), 48 (11%) with an intermediate result (50-250mcg/g) and 30 (7%)

with a high result (>250mcg/g). Outcome at 12 months were linked to index FC result.

We have not excluded patients with other GI conditions, organic or inorganic, as we

want to provide an insight into how FC testing is currently being used “in the wild”.

2.2 Clinical data collection

Clinical information was collected from review of the central base University Hospital

UHCW (tertiary catchment of 1m population) clinical results reporting system and the

corresponding systems in neighbouring district hospitals (Warwick and George Eliot

Hospitals). If no information was found then general practitioners (GPs) were contacted

for further details.

Due to the heterogenous nature of the data collection sources, clinical details such as

symptoms and medication which may affect FC values (e.g. NSAIDs or PPIs) were

often unavailable or poorly recorded, especially for the general practice patients. These

factors were not considered here due to the lack of incompleteness of the data.

No long term clinical information could be found for 7 individuals with an intermediate

raised result. This left a population of 41 patients.

2.3 FC sample analysis

Standardised laboratory protocol at UHCW for FC analysis was used. 100mg of stool

was weighed and dispensed into an analysis pot using a 10uL inoculation loop. As per

local protocol, first morning sample was requested. The exact weight was recorded and

5ml of extraction buffer added. The samples were then vortexed for 30 minutes in order

to ensure complete dissolution, and then centrifuged using an Eppendorf centrifuge. The

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supernatant was then removed for analysis using the Immunodiagnostik PhilCal ELISA

method.

2.4 Ethics

Ethical guidance was obtained from the Coventry and Warwick NRES Committee. As

this study was evaluating a test already in clinical practice, no further ethical approval

was required.

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3.0 Results

Our final population comprised 41 intermediate results. The mean age was 44 years (SD

19.0). There were 17 males (41%) and 24 females (59%). The mean FC for the

intermediate cohort was 130mcg/g (SD 58).

2 patients (4.8%) were known IBD patients under ongoing secondary care for their

condition. They were included as we wished to provide a “snapshot” of the usage of FC

testing in our centre, including that of already known IBD patients under surveillance.

28 (68%) of patients with an intermediate FC result were referred to secondary/tertiary

gastroenterology care by their primary care physicians, with 11 (27%) managed in

primary care. Only 1 patient had a repeat FC sample sent, this remained moderately

raised. The patient subsequently had normal investigations and was discharged.

29 (71%) of patients with an intermediate FC result went on to have a colonoscopy, 8

(20%) a CT scan and 7 (17%) an MRI. This would suggest that an intermediate result

does not reduce the number of colonoscopies or imaging investigations being done.

There were 8 new cases of IBD diagnosed (19%; SE6.1%) with FC levels ranging from

81 to 218mcg/g. All underwent colonoscopies. 3 (7.3%) were diagnosed with Crohn’s

colitis, 3 (7.3%) were diagnosed with UC and 2 (4.7%) with indeterminate colitis. 6

(15%) were diagnosed with other organic GI conditions, including bile salt

malabsorption, diverticular disease and carcinoid. 11 (27%; SE 7%) were diagnosed

with IBS. 14 (34%) remained undiagnosed, however, 8 (19%) were not referred to

secondary care by their GP. The reasons for this are unknown, their symptoms may

have settled spontaneously or the patient themselves may have declined referral. Of the

6 (15%) who were referred to secondary care, 3 (7.3%) were discharged after

investigation with no cause found for the raised FC, but symptoms having resolved and

3 (7.3%) were still under investigation for other causes of the intermediate FC result e.g.

small bowel bacterial overgrowth.

The odds ratio for a new diagnosis of IBD with an intermediate result compared to a

normal FC result was 26.6. The odds ratio of a new diagnosis of IBS with an

intermediate result compared to a normal FC result was 0.54. The odds ratio of

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remaining in secondary care 12 months after diagnosis with an intermediate FC result

was 3.6 compared to a normal FC result.

Twelve months after their initial FC test 14 (34%) of the cohort remained under

ongoing secondary/tertiary care for their conditions, with 14 (38%) discharged back to

their primary care physicians. The remainder were managed in primary care.

Subgroup analysis of the intermediate patients was conducted below and above

100mcg/g threshold. 18 (44%) of the intermediate group had FC <100mcg/g, whilst 23

(56%) had FC >100mcg/g. For the <100mcg/g group 14 (78%) were in primary care 12

months after index FC test compared with 13 (57%) of >100mcg/g group at the same

time point. 3 (17%) of <100mcg/g received a new diagnosis of IBD compared with 5

(22%) of >100mcg/g group. One patient in both the <100mcg/g group and the

>100mcg/g respectively were known IBD patients, with 2 (11%) and 3 (13%) patients

respectively being diagnosed with non-IBD organic GI conditions including bile acid

malabsorption, diverticular disease and rectal carcinoid. 28% of <100mcg/g cohort and

26% of >100mcg/g cohort were diagnosed with IBS. No statistical differences were

noted between these two groups. See table 2 for the subgroup analysis results.

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4.0 Discussion

Our study provides some insight into the “real world” use of FC testing in primary and

secondary care. An intermediate or raised FC result increases the probability of

remaining under secondary care 12 months after the index FC test - 34% of intermediate

results were still in secondary care 12 months after index FC, compared with 9.1% of

the normal group and 73% for the raised group. This reflects the increasing likelihood

of a diagnosis of IBD or another organic GI disease with intermediate or raised FC

results.

A new diagnosis of IBD was made in 19% of patients with an intermediate FC result,

compared with 1% for normal results and 37% for raised results. These findings suggest

that an initial intermediate FC result gives an OR of 26.6 for receiving a diagnosis of

IBD compared to a normal FC result and an OR of 3.6 for remaining in secondary care

12 months after index FC result for intermediate results compared to normal FC result.

This would seem to increase the evidence for suggesting that intermediate FC results

should undergo retesting 4-6 weeks later before referral to secondary care is made. Of

note a third in this group had no final diagnosis at 12 months, with most of these arising

from primary care. Unfortunately no repeat FC specimens were sent by primary care to

determine if the intermediate result was consistent, or transient.

Subgroup analysis of the intermediate group does not show a clinically significant

difference between the <100mcg/g group and the >100mcg/g. There were similar

numbers for: new IBD diagnoses made (17% (<100mcg/g) vs 22% (>100mcg/g); other

organic GI diseases (11% vs 13%) and IBS diagnoses (28% vs 26%). Therefore, based

on this study group and utility of the ELISA Phical methods, there is no suggestion that

the cut-off value for a normal FC result alters diagnostic yield if it were raised to

100mcg/g.

NICE guidelines on FC usage do not currently set an age cut-off for FC testing. The

NICE IBS guidelines uses an age cut-off >50 as a red flag for change in bowel habit.

Local guidelines state that FC testing should not be used over the age of 40 due to the

rising incidence of bowel cancer. Rather, such patients with a change in bowel habit or

diarrhoea should undergo endoscopic investigation. In this study we found that the

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mean age of patients undergoing FC testing was 39 years, with 40% older than 40 years

and 19% older than 50 years. Within the intermediate group 46% met local guidelines

on FC testing and 66% met NICE guidelines recommended red flag cut off of <50

years. The oldest patient to undergo FC testing was 89 years. This would suggest that

there needs to be greater education and awareness about the strengths and limitations of

FC testing in aiding with diagnosis or exclusion of organic GI diseases, principally IBD.

A limitation in this study is the heterogenous clinical data, which comprised both

primary and secondary care data, which, resulted in incomplete clinical details such as

symptoms and medications across all FC ranges. However, the objectives of this study

were to understand the way in which FC testing is currently being used in our region.

We have attempted to provide a “real world” snapshot of how FC testing is being used

in primary and secondary care settings with some not receiving a definitive diagnosis

The NICE appraisal did raise the issue of spectrum bias because most of the studies in

the assessment report (5) came from secondary care settings. Unfortunately the numbers

of patients in our study, and the lack of data on long-term outcomes on a substantial

number of those with normal results, make it impossible for us to analyse screening

parameters such as NPV, separately by source.

Further studies are needed to determine the long term clinical outcomes in patients

undergoing FC testing in both primary and secondary care, to aid interpretation of

intermediate raised FC results. It could encourage the use of repeat FC testing for

intermediate results, rather than immediate referral to secondary care for further

investigation. Better education of the indications, potential confounding drugs, and age

issues with FC testing would provide more efficient use of FC testing and would allow

for far clearer interpretation of the true meaning of intermediate results.

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Acknowledgements: Our thanks to the UHCW pathology department and the GP’s of

Coventry and Warwickshire for their co-operation with our study.

Contributions:

MM - co-ordinated project, collected and analysed data, wrote manuscript

SC - collected and analysed data, wrote manuscript

AM - collected data

BL - collected data

ES - collected data

CN - oversaw project, contributed to paper content

NW - analysed data, co-wrote manuscript

RA - co-ordinated project, co-wrote manuscript

Competing interests:

The authors have no competing interests to declare

Funding:

There is no funding to declare for this study

Data sharing statement:

Any further data required is available directly from the authors on request

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References

1. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil

dominating protein calprotectin in feces. A methodologic study. Scand J

Gastroenterol 1992;27:793–8.

2. Aomatsu T, Yoden A, Matsumoto K, et al. Fecal calprotectin is a useful marker

for disease activity in pediatric patients with inflammatory bowel disease. Dig

Dis Sci 2011;56:2372–7.

3. Stríz I, Trebichavský I. Calprotectin—a pleiotropic molecule in acute and

chronic inflammation. Physiol Res 2004;53:245–53.

4. van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of

patients with suspected inflammatory bowel disease: diagnostic meta-analysis.

BMJ 2010;341:c3369.

5. Waugh N, Royle P, Cummins E, et al. Faecal calprotectin testing for

differentiating amongst inflammatory and non-inflammatory bowel diseases:

systematic review and economic evaluation. Health Tech Assessment

2013;17:xv–xix, 1–211.

6. Sipponen T, Kärkkäinen P, Savilahti E, et al. Correlation of faecal calprotectin

and lactoferrin with an endoscopic score for Crohn’s disease and histological

findings. Aliment Pharmacol Ther 2008;28:1221–9.

7. NICE guidelines; Faecal calprotectin diagnostic tests for inflammatory diseases

of the bowel; DAP 12; June 2013.

8. Dhaliwal A, Zeino Z, Tomkins C, et al. Utility of faecal calprotectin in

inflammatory bowel disease (IBD): what cut-offs should we apply? Frontline

Gastroenterology 2014;0:1–6.

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9. Banerjee A, Srinivas M, Eyre R, Ellis R, Waugh N, Bardhan KD, Basumani P.

Faecal calprotectin for differentiating between irritable bowel syndrome and

inflammatory bowel disease: a useful screen in daily gastroenterology practice

Frontline Gastroenterol-100429. Epub Apr 2014. doi:10.1136/flgastro-2013-

100429

10. Koulaouzidis A, Douglas S, Rogers MA, Arnott ID, Plevris JN. Fecal

calprotectin: a selection tool for small bowel capsule endoscopy in suspected

IBD with prior negative bi-directional endoscopy. Scand J Gastroenterol.

2011;46(5):561-6. PMID: 21269246

11. Lin JF, Chen JM, Zuo JH,et al. Meta-analysis: fecal calprotectin for assessment

of inflammatory bowel disease activity. Inflamm Bowel Dis. 2014

Aug;20(8):1407-15. PMID: 24983982

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Table 1. Patient demographics and FC values per FC category of low, intermediate and

high over 12 months.

FC <50mcg/g FC 50-250mcg/g FC >250mcg/g

Mean Age (SD) 39 (15)

38 (13) 44 (19) 39 (16)

Male:Female (%) 36:64

35:65 42:58 35:65

Mean FC mcg/g

(SD)

22 (5.0) 130 (58) 784 (573)

Referred to

secondary care (n)

66.3% (138) 68.3% (28) 57.7%

Remain in

secondary care at 12

months post index

FC (n)

9.1% (19) 34.1% (14) 73.1% (15)

Diagnoses

Pre-existing IBD (n) 3.4% (7) 4.8% (2) 23% (6)

New diagnosis IBD

(n)

1.0% (2) 19% (8) 38.4% (10)

Other organic GI

diagnosis (n)

13% (27) 14.6% (6) 3.8% (1)

Diagnosis IBS (n) 40.4% (84) 26.8% (11) 7.7% (2)

Undiagnosed or

awaiting diagnosis

(n)

41.8% (87)

34.1% (14)

26.9% (7)

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Table 2. Outcome sub analysis of FC intermediate groups; <100 and >100 mcg/g.

FC< 100 (n=18) FC > 100 (n=23)

In primary care 12 months

post index FC (%)

78% (n=14) 57% (n=13)

New diagnosis of IBD 17% (n=3) 22% (n=5)

Known IBD 5.6% (n=1) 4.3% (n=1)

Other organic GI diagnosis 11% (n=2) 13% (n=3)

IBS diagnosis 28% (n=5) 26% (n=6)

others 38.4% 34.7%

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STROBE Statement—checklist of items that should be included in reports of observational studies

Item

No Recommendation

Title and abstract 1 (a) Indicate the study’s design with a commonly used term in the title or the abstract

Page 1

(b) Provide in the abstract an informative and balanced summary of what was done

and what was found Page 2

Introduction

Background/rationale 2 Explain the scientific background and rationale for the investigation being reported

Page 4

Objectives 3 State specific objectives, including any prespecified hypotheses

Page 5

Methods

Study design 4 Present key elements of study design early in the paper

Page 6

Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment,

exposure, follow-up, and data collection

Page 6

Participants 6 (a) Cohort study—Give the eligibility criteria, and the sources and methods of

selection of participants. Describe methods of follow-up

Case-control study—Give the eligibility criteria, and the sources and methods of

case ascertainment and control selection. Give the rationale for the choice of cases

and controls

Cross-sectional study—Give the eligibility criteria, and the sources and methods of

selection of participants

Page 6

(b) Cohort study—For matched studies, give matching criteria and number of

exposed and unexposed

Case-control study—For matched studies, give matching criteria and the number of

controls per case

Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect

modifiers. Give diagnostic criteria, if applicable

Page 6

Data sources/

measurement

8* For each variable of interest, give sources of data and details of methods of

assessment (measurement). Describe comparability of assessment methods if there

is more than one group

Page 6

Bias 9 Describe any efforts to address potential sources of bias

Page 11

Study size 10 Explain how the study size was arrived at

Page 6

Quantitative variables 11 Explain how quantitative variables were handled in the analyses. If applicable,

describe which groupings were chosen and why

NA

Statistical methods 12 (a) Describe all statistical methods, including those used to control for confounding

(b) Describe any methods used to examine subgroups and interactions

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(c) Explain how missing data were addressed

(d) Cohort study—If applicable, explain how loss to follow-up was addressed

Case-control study—If applicable, explain how matching of cases and controls was

addressed

Cross-sectional study—If applicable, describe analytical methods taking account of

sampling strategy

(e) Describe any sensitivity analyses

Page 6, 7 and 11

Continued on next page

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Results

Participants 13* (a) Report numbers of individuals at each stage of study—eg numbers potentially eligible,

examined for eligibility, confirmed eligible, included in the study, completing follow-up, and

analysed

(b) Give reasons for non-participation at each stage

(c) Consider use of a flow diagram

Page 8

Descriptive

data

14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information

on exposures and potential confounders

(b) Indicate number of participants with missing data for each variable of interest

(c) Cohort study—Summarise follow-up time (eg, average and total amount)

Page 8

Outcome data 15* Cohort study—Report numbers of outcome events or summary measures over time

Case-control study—Report numbers in each exposure category, or summary measures of

exposure

Cross-sectional study—Report numbers of outcome events or summary measures

Page 8

Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their

precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and

why they were included

(b) Report category boundaries when continuous variables were categorized

(c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful

time period

Page 8, 9

Other analyses 17 Report other analyses done—eg analyses of subgroups and interactions, and sensitivity

analyses

Page 9, 11

Discussion

Key results 18 Summarise key results with reference to study objectives

Page 10

Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision.

Discuss both direction and magnitude of any potential bias

Page 10, 11

Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity

of analyses, results from similar studies, and other relevant evidence

Page 10, 11

Generalisability 21 Discuss the generalisability (external validity) of the study results

Page 11

Other information

Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable,

for the original study on which the present article is based

Page 12

*Give information separately for cases and controls in case-control studies and, if applicable, for exposed and

unexposed groups in cohort and cross-sectional studies.

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Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and

published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely

available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at

http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is

available at www.strobe-statement.org.

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Clinical outcomes at 12 months and risk of inflammatory

bowel disease in patients with an intermediate raised faecal

calprotectin: A “real world” view

Journal: BMJ Open

Manuscript ID bmjopen-2016-011041.R1

Article Type: Research

Date Submitted by the Author: 23-Feb-2016

Complete List of Authors: McFarlane, Michael; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Chambers, Samantha; University Hospitals Coventry and Warwickshire

NHS Trust, Gastroenterology Malik, Ahmad; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Lee, Bee; Warwick Hospital, Gastroenterology Sung, Edmond; George Eliot Hospital, Gastroenterology Nwokolo, Chuka; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology Waugh, Norman; University of Warwick, Warwick Evidence Arasaradnam, Ramesh; University Hospitals Coventry and Warwickshire NHS Trust, Gastroenterology

<b>Primary Subject Heading</b>:

Gastroenterology and hepatology

Secondary Subject Heading: Diagnostics

Keywords: Inflammatory bowel disease < GASTROENTEROLOGY, Faecal Calprotectin, Intermediate, Outcomes


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Clinical outcomes at 12 months and risk of Inflammatory Bowel

Disease in patients with an intermediate raised faecal calprotectin: A

“real world” view

Michael McFarlane1, Samantha Chambers

1, Ahmad Malik

1, Bee Lee

2, Edmond Sung

3,

Chuka Nwokolo1, Norman Waugh

5 and Ramesh Arasaradnam

1, 4

1Department of Gastroenterology, University Hospital Coventry & Warwickshire,

Coventry CV2 2DX, UK, 2Department of Gastroenterology, Warwick Hospital,

Warwick, CV34 5PP, UK, 3Department of Gastroenterology, George Eliot Hospital,

Nuneaton, CV10 7DJ, UK, 4Clinical Sciences Research Institute, University of

Warwick, Coventry CV2 2DX, UK, 5 Warwick Evidence, University of Warwick,

Coventry CV4 2DX, UK.

Correspondence to:

Dr R P Arasaradnam

Clinical Sciences Research Institute, University of Warwick,

Clifford Bridge Road,

Coventry CV2 2DX

[email protected]

Tel: 02476 966098

Fax: 02476 966090:

Running title: 12 month clinical outcomes for mildly elevated FCP

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Key words: Faecal calprotectin, IBD, Intermediate, clinical outcomes

Abstract

Objectives: A recent systematic review confirmed the utility of Faecal Calprotectin (FC)

in distinguishing organic (inflammatory bowel disease) from non-organic

gastrointestinal disease (irritable bowel syndrome). FC levels <50 mcg/g have a

negative predictive value >92% to exclude organic GI disease. Levels >250mcg/g

correlate with endoscopic IBD disease activity; sensitivity 90%. We aimed to determine

clinical outcomes in intermediate raised FC results (50-250 mcg/g).

Setting: Primary care general practices in Coventry and Warwickshire and three

secondary care hospitals

Participants: 443 FC results in adults (>16 years old) were reviewed from July 2012 -

October 2013. Clinical data was collected from hospital databases and General

Practitioners. Long term clinical data was available in 41 patients (of 48). .

Primary and secondary outcome measures: The number of new diagnoses of

inflammatory bowel disease (IBD), irritable bowel disease (IBS) and other diagnoses

for the intermediate group. The number referred and discharged from secondary care.

Results: A new IBD diagnosis was made in 19% (n=8) of intermediate results (1% of

normal and 38% of raised results). 5% (n=2) of intermediate results had known IBD in

remission. A new IBS diagnosis was made in 27% (n=11) of intermediate results whilst

34% (n=14) remained undiagnosed, although 8 of these were not referred to secondary

care.

Conclusions: FC testing remains useful in aiding diagnosis of organic GI conditions.

However, unlike negative and strongly positive FC results, intermediate FC results lead

to a mixture of diagnoses. The odds ratio of a new diagnosis of IBD for an intermediate

result compared to normal FC result was 26.6, whilst an intermediate FC result gave an

OR of 0.54 for a new IBS diagnosis compared to normal FC. For intermediate FC

results 1 in 3 patients remained in secondary care after 12 months with an OR of 3.6

compared to a normal FC result.

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Strengths:

• 12 month clinical follow up data of intermediate faecal calprotectin results

• A “real world” view of the utilisation of FC testing in primary and secondary

care – it is not being used entirely appropriately

• A “ real world” view of clinical outcomes – we don’t always find the answer

Limitations:

• Heterogenous data sources means data not as complete as could be e.g.

medication information incomplete

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1.0 Introduction

Calprotectin is a calcium binding protein, of the S100 family, found mainly in

neutrophils but also in other white blood cells (1). Inflammation results in neutrophil

activation and a subsequent release of calprotectin protein (2, 3). The use of faecal

calprotectin (FC) has particular interest as a non-invasive biomarker in the initial

screening and monitoring of patients with suspected or known inflammatory bowel

disease (IBD) (4). It is of particular use in the distinction between inflammatory GI

conditions such as IBD, from non-organic conditions such as irritable bowel syndrome

(IBS) (5). FC testing also has a growing role in the monitoring of IBD activity in

response to treatment, although long term data on the clinical advantages of this

approach is not yet available (6).

Current NICE and manufacturer guidelines for the cut of levels of FC in assays are that

levels of less than 50mcg/g of faeces suggest that there is no active inflammation

present within the GI mucosa (5, 7). NICE reported that for most of the studies that they

reviewed sensitivity and specificity were over 80%, where a cut-off of 50mcg/g was

used, and most positive and negative predictive values were 70-90% (7). One recent

study found that a cut-off of 50mcg/g gives a sensitivity and specificity of 88% and

78% respectively, with a negative predictive value of >92% to exclude organic GI

disease (8). A cut-off value of 100mcg/g has previously been suggested, and whilst this

increases sensitivity to 97%, specificity falls to 76%, with a negative predictive value of

97% and a positive predictive value of 75% (8). A cohort study involving consecutively

referred new patients with chronic diarrhoea proposed that a cut-off of 8mcg/g provides

near 99% sensitivity in detecting organic disease but at the cost of poor specificity (9).

In this study, no patients were diagnosed with IBD with FC levels of 50ug/g or less,

although this was a small study hence few patient overall with IBD. Another study

found that no patients with an FC result of <100mcg/g had IBD (10). A systematic

review of the use of FC, which informed the National Institute of Health and Care

Excellence diagnostic assessment group, found that most of the available evidence for

FC use in IBD is based on a cut-off value of 50mcg/g which reduces the number of false

negatives whilst maintaining cost effectiveness (5, 7).

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A FC value of greater than 250mcg/g has been demonstrated to correspond with

endoscopically and histologically active IBD. This cut-off level provides a sensitivity of

90% and specificity of 76% for excluding IBD and also determining if known disease

was quiescent (8). A recent meta-analysis into the utility of FC testing for monitoring

disease activity showed that for known IBD a cut-off level of 50mcg/g gave a pooled

sensitivity and specificity of 92% and 60% for determining if disease was quiescent, a

cut-off of 100mcg/g gave 84% and 66%, whilst cut-off of 250mcg/g gave 80% and 82%

respectively (11). See appendix 1 for a tabulated form of the studies involving FC cut

offs for screening for IBD.

Despite the increasing number of studies involving utility of FC there has been limited

study on patients who have a FC value which falls within the “intermediate” group;

defined as levels of 50-250mcg/g of faeces. Whilst these patients may still have

underlying organic GI disease, current opinion suggests that the FC assay should be

repeated 4-6 weeks later to determine if inflammation is improving or worsening.

Although in reality an intermediate result may still be referred to secondary care if

clinical suspicion is high for underlying IBD. Our local guidance is in accordance with

these ranges (see figure 1).

The aim of this study was to determine the 12 month clinical outcomes of patients with

an intermediate raised FC value. There have been, to the best of our knowledge, no

published studies which look at clinical outcomes in this group of patients and

proportions remaining in secondary/tertiary care follow up.

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2.0 Materials and Methods

2.1 FC samples

FC test results from July 2012 to October 2013 in the University Hospital Coventry &

Warwickshire (UHCW) pathology database were reviewed. Tests were requested by

both primary care and secondary/tertiary care physicians from the Warwickshire region;

catchment population of 1 million. Outcome at 12 months were linked to index FC

result. We have not excluded patients with other GI conditions, organic or inorganic, as

we want to provide an insight into how FC testing is currently being used in real world

practice.

2.2 Clinical data collection

Clinical information was collected from review of the central base University Hospital

UHCW (tertiary catchment of 1m population) clinical results reporting system and the

corresponding systems in neighbouring district hospitals (Warwick and George Eliot

Hospitals). If no information was found then general practitioners (GPs) were contacted

for further details.

Due to the heterogenous nature of the data collection sources, clinical details such as

symptoms and medication which may affect FC values (e.g. NSAIDs or PPIs) were

often unavailable or poorly recorded, especially for the general practice patients. These

factors were not considered here due to the lack of incompleteness of the data.

2.3 FC sample analysis

Standardised laboratory protocol at UHCW for FC analysis was used. 100mg of stool

was weighed and dispensed into an analysis pot using a 10uL inoculation loop. As per

local protocol, first morning sample was requested. The exact weight was recorded and

5ml of extraction buffer added. The samples were then vortexed for 30 minutes in order

to ensure complete dissolution, and then centrifuged using an Eppendorf centrifuge. The

supernatant was then removed for analysis using the Immunodiagnostik PhilCal ELISA

method.

2.4 Ethics

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Ethical guidance was obtained from the Coventry and Warwick NRES Committee. As

this study was evaluating a test already in clinical practice, no further ethical approval

was required.

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3.0 Results

495 patients were identified; 52 paediatric patients (aged < 16 years) were excluded. Of

the remaining, there were 365 (82%) with a normal FC result (<50mcg/g), 48 (11%)

with an intermediate result (50-250mcg/g) and 30 (7%) with a high result (>250mcg/g).

Long term clinical data was available in 41 of the intermediate results. The mean age

was 44 years (SD 19.0). There were 17 males (41%) and 24 females (59%). The mean

FC for the intermediate cohort was 130mcg/g (SD 58).

2 patients (4.8%) were known IBD patients under ongoing secondary care for their

condition. They were included as we wished to provide a “snapshot” of the usage of FC

testing in our centre, including that of already known IBD patients under surveillance.

28 (68%) of patients with an intermediate FC result were referred to secondary/tertiary

gastroenterology care by their primary care physicians, with 11 (27%) managed in

primary care. Only 1 patient had a repeat FC sample sent, this remained moderately

raised. The patient subsequently had normal investigations and was discharged.

29 (71%) of patients with an intermediate FC result went on to have a colonoscopy,

with 8 (20%) also undergoing a CT scan and a further 7 (17%) undergoing an MRI.

Only 8 patients out of the 41 for whom clinical information was available did not

undergo any investigations. These patients were those who were not referred to

secondary care. This would suggest that an intermediate result does not reduce the

number of colonoscopies or imaging investigations being done.

The diagnoses of intermediate FC results were:

• 8 new cases of IBD diagnosed within the 41 patients (19%; SE6.1%) with FC

levels ranging from 81 to 218mcg/g. All underwent colonoscopies.

o 3 (7.3%) were diagnosed with Crohn’s Disease (Montreal Classification:

1 L2 (colonic); 2 L3 (ileocolonic)

o 3 (7.3%) were diagnosed with UC (Montreal Classification: 2 E1

(proctitis); 1 E2 (left sided)

o 2 (4.7%) with IBD unspecified .

• 6 (15%) were diagnosed with other organic GI conditions, including bile salt

malabsorption, diverticular disease and carcinoid.

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• 11 (27%; SE 7%) were diagnosed with IBS.

• 14 (34%) remained undiagnosed, however, 8 (19%) of these 14 were not

referred to secondary care by their GP. The reasons for this are unknown, their

symptoms may have settled spontaneously or the patient themselves may have

declined referral.

o Of the 6 undiagnosed patients (15%) who were referred to secondary

care:

� 3 (7.3%) were discharged after investigation with no cause found

for the raised FC, with their symptoms having resolved

� 3 (7.3%) were still under investigation for other causes of the

intermediate FC result e.g. small bowel bacterial overgrowth.

See table 1 and figure 2 for a further breakdown of these results.

The odds ratio for a new diagnosis of IBD with an intermediate result compared to a

normal FC result was 26.6. The odds ratio of a new diagnosis of IBS with an

intermediate result compared to a normal FC result was 0.54. The odds ratio of

remaining in secondary care 12 months after diagnosis with an intermediate FC result

was 3.6 compared to a normal FC result.

Twelve months after their initial FC test 14 (34%) of the cohort remained under

ongoing secondary/tertiary care for their conditions, with 14 (34%) discharged back to

their primary care physicians. The remainder were managed in primary care.

Subgroup analysis of the intermediate patients was conducted below and above

100mcg/g threshold. 18 (44%) of the intermediate group had FC <100mcg/g, whilst 23

(56%) had FC >100mcg/g. For the <100mcg/g group 14 (78%) were in primary care 12

months after index FC test compared with 13 (57%) of >100mcg/g group at the same

time point. 3 (17%) of <100mcg/g received a new diagnosis of IBD compared with 5

(22%) of >100mcg/g group. One patient in both the <100mcg/g group and the

>100mcg/g respectively were known IBD patients, with 2 (11%) and 3 (13%) patients

respectively being diagnosed with non-IBD organic GI conditions including bile acid

malabsorption, diverticular disease and rectal carcinoid. 28% of <100mcg/g cohort and

26% of >100mcg/g cohort were diagnosed with IBS. 38% of <100mcg/g and 35% of

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>100mcg/g groups did not receive a formal diagnosis. No statistical differences were

noted between these two groups. See table 2 for the subgroup analysis results.

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4.0 Discussion

Our study provides some insight into the “real world” use of FC testing in primary and

secondary care. An intermediate or raised FC result increases the probability of

remaining under secondary care 12 months after the index FC test - 34% of intermediate

results were still in secondary care 12 months after index FC, compared with 9.1% of

the normal group and 73% for the raised group. This reflects the increasing likelihood

of a diagnosis of IBD or another organic GI disease with intermediate or raised FC

results.

A new diagnosis of IBD was made in 19% of patients with an intermediate FC result,

compared with 1% for normal results and 37% for raised results. These findings suggest

that an initial intermediate FC result gives an OR of 26.6 for receiving a diagnosis of

IBD compared to a normal FC result and an OR of 3.6 for remaining in secondary care

12 months after index FC result for intermediate results compared to normal FC result.

This would seem to increase the evidence for suggesting that intermediate FC results

should undergo retesting 4-6 weeks later before referral to secondary care is made. Of

note a third in this group had no final diagnosis at 12 months, with most of these arising

from primary care. Unfortunately no repeat FC specimens were sent by primary care to

determine if the intermediate result was consistent, or transient.

Subgroup analysis of the intermediate group does not show a clinically significant

difference between the <100mcg/g group and the >100mcg/g. There were similar

numbers for: new IBD diagnoses made (17% (<100mcg/g) vs 22% (>100mcg/g); other

organic GI diseases (11% vs 13%) and IBS diagnoses (28% vs 26%). Therefore, based

on this study group and utility of the ELISA Phical methods, there is no suggestion that

the cut-off value for a normal FC result alters diagnostic yield if it were raised to

100mcg/g.

NICE guidelines on FC usage do not currently set an age cut-off for FC testing. The

NICE IBS guidelines uses an age cut-off >50 as a red flag for change in bowel habit.

Local guidelines state that FC testing should not be used over the age of 40 due to the

rising incidence of bowel cancer. Rather, such patients with a change in bowel habit or

diarrhoea should undergo endoscopic investigation. In this study we found that the

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mean age of patients undergoing FC testing was 39 years, with 40% older than 40 years

and 19% older than 50 years. Within the intermediate group 46% met local guidelines

on FC testing and 66% met NICE guidelines recommended red flag cut off of <50

years. The oldest patient to undergo FC testing was 89 years. This would suggest that

there needs to be greater education and awareness about the strengths and limitations of

FC testing in aiding with diagnosis or exclusion of organic GI diseases, principally IBD.

A limitation in this study is the heterogenous clinical data, which comprised both

primary and secondary care data, which, resulted in incomplete clinical details such as

symptoms and medications, including Non Steroidal Anti Inflammatories, across all FC

ranges. However, the objectives of this study were to understand the way in which FC

testing is currently being used in our region. We have attempted to provide a “real

world” snapshot of how FC testing is being used in primary and secondary care settings

with some not receiving a definitive diagnosis The NICE appraisal did raise the issue of

spectrum bias because most of the studies in the assessment report (5) came from

secondary care settings. Unfortunately the numbers of patients in our study, and the lack

of data on long-term outcomes on a substantial number of those with normal results,

make it impossible for us to analyse screening parameters such as NPV, separately by

source.

Further studies are needed to determine the long term clinical outcomes in patients

undergoing FC testing in both primary and secondary care, to aid interpretation of

intermediate raised FC results. It could encourage the use of repeat FC testing for

intermediate results, rather than immediate referral to secondary care for further

investigation. Better education of the indications, potential confounding drugs, and age

issues with FC testing would provide more efficient use of FC testing and would allow

for far clearer interpretation of the true meaning of intermediate results.

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Acknowledgements: Our thanks to the UHCW pathology department and the GP’s of

Coventry and Warwickshire for their co-operation with our study.

Contributions:

MM - co-ordinated project, collected and analysed data, wrote manuscript

SC - collected and analysed data, wrote manuscript

AM - collected data

BL - collected data

ES - collected data

CN - oversaw project, contributed to paper content

NW - analysed data, co-wrote manuscript

RA - co-ordinated project, co-wrote manuscript

Competing interests:

The authors have no competing interests to declare

Funding:

There is no funding to declare for this study

Data sharing statement:

No additional data available.

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References

1. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil

dominating protein calprotectin in feces. A methodologic study. Scand J

Gastroenterol 1992;27:793–8.

2. Aomatsu T, Yoden A, Matsumoto K, et al. Fecal calprotectin is a useful marker

for disease activity in pediatric patients with inflammatory bowel disease. Dig

Dis Sci 2011;56:2372–7.

3. Stríz I, Trebichavský I. Calprotectin—a pleiotropic molecule in acute and

chronic inflammation. Physiol Res 2004;53:245–53.

4. van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of

patients with suspected inflammatory bowel disease: diagnostic meta-analysis.

BMJ 2010;341:c3369.

5. Waugh N, Royle P, Cummins E, et al. Faecal calprotectin testing for

differentiating amongst inflammatory and non-inflammatory bowel diseases:

systematic review and economic evaluation. Health Tech Assessment

2013;17:xv–xix, 1–211.

6. Sipponen T, Kärkkäinen P, Savilahti E, et al. Correlation of faecal calprotectin

and lactoferrin with an endoscopic score for Crohn’s disease and histological

findings. Aliment Pharmacol Ther 2008;28:1221–9.

7. NICE guidelines; Faecal calprotectin diagnostic tests for inflammatory diseases

of the bowel; DAP 12; June 2013.

8. Dhaliwal A, Zeino Z, Tomkins C, et al. Utility of faecal calprotectin in

inflammatory bowel disease (IBD): what cut-offs should we apply? Frontline

Gastroenterology 2014;0:1–6.

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9. Banerjee A, Srinivas M, Eyre R, Ellis R, Waugh N, Bardhan KD, Basumani P.

Faecal calprotectin for differentiating between irritable bowel syndrome and

inflammatory bowel disease: a useful screen in daily gastroenterology practice

Frontline Gastroenterol-100429. Epub Apr 2014. doi:10.1136/flgastro-2013-

100429

10. Koulaouzidis A, Douglas S, Rogers MA, Arnott ID, Plevris JN. Fecal

calprotectin: a selection tool for small bowel capsule endoscopy in suspected

IBD with prior negative bi-directional endoscopy. Scand J Gastroenterol.

2011;46(5):561-6. PMID: 21269246

11. Lin JF, Chen JM, Zuo JH,et al. Meta-analysis: fecal calprotectin for assessment

of inflammatory bowel disease activity. Inflamm Bowel Dis. 2014

Aug;20(8):1407-15. PMID: 24983982

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Table 1. Patient demographics and FC values per FC category of low, intermediate and

high over 12 months.

FC <50mcg/g FC 50-250mcg/g FC >250mcg/g

Mean Age (SD) 39 (15)

38 (13) 44 (19) 39 (16)

Male:Female (%) 36:64

35:65 42:58 35:65

Mean FC mcg/g

(SD)

22 (5.0) 130 (58) 784 (573)

Referred to

secondary care (n)

66.3% (138) 68.3% (28) 57.7%

Remain in

secondary care at 12

months post index

FC (n)

9.1% (19) 34.1% (14) 73.1% (15)

Diagnoses

Pre-existing IBD (n) 3.4% (7) 4.8% (2) 23% (6)

New diagnosis IBD

(n)

1.0% (2) 19% (8) 38.4% (10)

Other organic GI

diagnosis (n)

13% (27) 14.6% (6) 3.8% (1)

Diagnosis IBS (n) 40.4% (84) 26.8% (11) 7.7% (2)

Undiagnosed or

awaiting diagnosis

(n)

41.8% (87)

34.1% (14)

26.9% (7)

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Table 2. Outcome sub analysis of FC intermediate groups; <100 and >100 mcg/g.

FC< 100 (n=18) FC > 100 (n=23)

In primary care 12 months

post index FC (%)

78% (n=14) 57% (n=13)

New diagnosis of IBD 17% (n=3) 22% (n=5)

Known IBD 5.6% (n=1) 4.3% (n=1)

Other organic GI diagnosis 11% (n=2) 13% (n=3)

IBS diagnosis 28% (n=5) 26% (n=6)

others 38.4% 34.7%

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Figure 1. Local Guidelines for GPs to determine the utility of FC in those with chronic

diarrhoea.

• Do NOT use FC for ?bowel cancer

• Do NOT use FC for ?infection

• DO give your patient a labelled stool pot; 2p sized amount of sample is sufficient

(solid stool is best)

• DO ask them to bring it to their local hospital/GP to send to BIOCHEMISTRY (not

microbiology) – stable at room temp

• DO fill in as much clinical information as possible, incl. symptoms and duration

• DO send a separate sample to microbiology for M, C&S unless infection has already

been excluded

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Figure 2. Flow chart of Intermediate Faecal Calprotectin (FC) diagnoses

BAD – Bile acid diarrhoea

DD – Diverticular disease

IBS – Irritable bowel syndrome

IBD – Inflammatory bowel disease

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Local Guidelines for GPs to determine the utility of FC in those with chronic diarrhoea. • Do NOT use FC for ?bowel cancer • Do NOT use FC for ?infection

• DO give your patient a labelled stool pot; 2p sized amount of sample is sufficient (solid stool is best) • DO ask them to bring it to their local hospital/GP to send to BIOCHEMISTRY (not microbiology) – stable at

room temp • DO fill in as much clinical information as possible, incl. symptoms and duration

• DO send a separate sample to microbiology for M, C&S unless infection has already been excluded 250x135mm (96 x 96 DPI)

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Figure 2. Flow chart of Intermediate Faecal Calprotectin (FC) diagnoses (BAD – Bile acid diarrhoea; DD – Diverticular disease; IBS – Irritable bowel syndrome; IBD – Inflammatory bowel disease)

250x135mm (96 x 96 DPI)

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Appendix 1. Table of previous studies into FC cut off values in IBD diagnosis.

Study authors FC cut off

value

Sensitivity Specificity Positive

Predictive

Value

Negative

Predictive

Value

Bannerjee et

al

8mcg/g 99% 51% 30% 100%

NICE 50mcg/g >80% >80% 70-90% 70-90%

Dhaliwal et al 50mcg/g 88% 78% - >92%

Dhaliwal et al 100mcg/g 97% 76% 75% 97%

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STROBE Statement—checklist of items that should be included in reports of observational studies

Item

No Recommendation

Title and abstract 1 (a) Indicate the study’s design with a commonly used term in the title or the abstract

Page 1

(b) Provide in the abstract an informative and balanced summary of what was done

and what was found Page 2

Introduction

Background/rationale 2 Explain the scientific background and rationale for the investigation being reported

Page 4

Objectives 3 State specific objectives, including any prespecified hypotheses

Page 5

Methods

Study design 4 Present key elements of study design early in the paper

Page 6

Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment,

exposure, follow-up, and data collection

Page 6

Participants 6 (a) Cohort study—Give the eligibility criteria, and the sources and methods of

selection of participants. Describe methods of follow-up

Case-control study—Give the eligibility criteria, and the sources and methods of

case ascertainment and control selection. Give the rationale for the choice of cases

and controls

Cross-sectional study—Give the eligibility criteria, and the sources and methods of

selection of participants

Page 6

(b) Cohort study—For matched studies, give matching criteria and number of

exposed and unexposed

Case-control study—For matched studies, give matching criteria and the number of

controls per case

Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect

modifiers. Give diagnostic criteria, if applicable

Page 6

Data sources/

measurement

8* For each variable of interest, give sources of data and details of methods of

assessment (measurement). Describe comparability of assessment methods if there

is more than one group

Page 6

Bias 9 Describe any efforts to address potential sources of bias

Page 11

Study size 10 Explain how the study size was arrived at

Page 6

Quantitative variables 11 Explain how quantitative variables were handled in the analyses. If applicable,

describe which groupings were chosen and why

NA

Statistical methods 12 (a) Describe all statistical methods, including those used to control for confounding

(b) Describe any methods used to examine subgroups and interactions

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(c) Explain how missing data were addressed

(d) Cohort study—If applicable, explain how loss to follow-up was addressed

Case-control study—If applicable, explain how matching of cases and controls was

addressed

Cross-sectional study—If applicable, describe analytical methods taking account of

sampling strategy

(e) Describe any sensitivity analyses

Page 6, 7 and 11

Continued on next page

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Results

Participants 13* (a) Report numbers of individuals at each stage of study—eg numbers potentially eligible,

examined for eligibility, confirmed eligible, included in the study, completing follow-up, and

analysed

(b) Give reasons for non-participation at each stage

(c) Consider use of a flow diagram

Page 8

Descriptive

data

14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information

on exposures and potential confounders

(b) Indicate number of participants with missing data for each variable of interest

(c) Cohort study—Summarise follow-up time (eg, average and total amount)

Page 8

Outcome data 15* Cohort study—Report numbers of outcome events or summary measures over time

Case-control study—Report numbers in each exposure category, or summary measures of

exposure

Cross-sectional study—Report numbers of outcome events or summary measures

Page 8

Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their

precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and

why they were included

(b) Report category boundaries when continuous variables were categorized

(c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful

time period

Page 8, 9

Other analyses 17 Report other analyses done—eg analyses of subgroups and interactions, and sensitivity

analyses

Page 9, 11

Discussion

Key results 18 Summarise key results with reference to study objectives

Page 10

Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision.

Discuss both direction and magnitude of any potential bias

Page 10, 11

Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity

of analyses, results from similar studies, and other relevant evidence

Page 10, 11

Generalisability 21 Discuss the generalisability (external validity) of the study results

Page 11

Other information

Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable,

for the original study on which the present article is based

Page 12

*Give information separately for cases and controls in case-control studies and, if applicable, for exposed and

unexposed groups in cohort and cross-sectional studies.

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Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and

published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely

available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at

http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is

available at www.strobe-statement.org.

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