ORIGINAL ARTICLE
Meta-analysis: Fecal Calprotectin for Assessment of Inflammatory Bowel Disease Activity Jin-Feng Lin, MD, Jin-Min Chen, MD, Jun-Hua Zuo, MD, Allen Yu, BS, Zhu-Jun Xiao, MD, Fei-Hong Deng, MD, Biao Nie, MD, PhD, and Bo Jiang, MD, PhD
Background: Fecal calprotectin (FC) is a promising biomarker for diagnosis of inflammatory bowel disease (IBD). However, the utility of FC for assessment of IBD activity has yet to be clearly demonstrated. The aim of our study was to evaluate the diagnostic accuracy of FC for differentiating between patients with active IBD and those in remission.
Methods: We systematically searched the databases Medline, Web of Science, Cochrane Library, and EMBASE for eligible studies from December 2013 or earlier that evaluated activity in ulcerative colitis (UC) and Crohn’s disease (CD). A hierarchical summary receiver operating characteristic model was performed to calculate the area under the curve to evaluate the overall diagnostic accuracy. The sensitivities and specificities of each commonly applied cutoff value were pooled using a random effects model.
Results: We included 13 studies (744 patients with UC and 727 with CD) in the final analysis. The area under the curve values were 0.89 (95% confidence
interval, 0.86–0.92), 0.93 (0.89–0.97), and 0.88 (0.83–0.93) in the IBD, UC, and CD groups, respectively. For the IBD group at a cutoff value of 50 mg/g, the pooled sensitivity was 0.92 (0.90–0.94) and specificity 0.60 (0.52–0.67). For a cutoff value at 100 mg/g, the pooled sensitivity was 0.84 (0.80–0.88) and specificity was 0.66 (0.59–0.73). For a cutoff value at 250 mg/g, the pooled sensitivity was 0.80 (0.76–0.84) and specificity was 0.82 (0.77–0.86).
Conclusions: The FC test is a reliable marker for assessing IBD disease activity and may have greater ability to evaluate disease activity in UC than CD. (Inflamm Bowel Dis 2014;20:1407–1415) Key Words: inflammatory bowel disease, Crohn’s disease, ulcerative colitis, fecal calprotectin, diagnostic accuracy
T
he incidence and prevalence of both ulcerative colitis (UC) and Crohn’s disease (CD) is increasing worldwide. UC and CD, the major forms of idiopathic inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract that have been empirically defined by clinical, pathological, endoscopic, and radiological features. The courses of these diseases are characterized by episodes of flare-ups alternating with periods of remission.1–4 Determination of inflammatory activity is essential to tailoring therapy and predicting prognosis. Symptoms of colonic inflammation are clearly manifested but unspecific and poorly correlate with mucosal inflammation.5–7 Serum biomarkers Received for publication March 13, 2014; Accepted March 24, 2014. From the Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China. The authors have no conflicts of interest to disclose. J.-F. Lin and J.-M. Chen contributed equally to this work. B. Nie and B. Jiang were co-corresponding authors of the article. Supported by the NanFang Hospital Fund for Distinguished Young Scholars, Grant 2013JQ03. Reprints: Biao Nie, MD, PhD and Bo Jiang, MD, PhD, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhoudadaobei 1838, Guangzhou, Guangdong 510515, China (e-mail: [email protected]; [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000057 Published online 30 June 2014.
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such as C-reactive protein, erythrocyte sedimentation rate, and leukocytes reflect a summation of systemic host responses rather than being specific for intestinal inflammation.8 Endoscopy is currently considered the gold standard for evaluation of mucosal inflammation, and a number of endoscopic scoring systems exist to assess inflammatory activity in UC and CD.9,10 However, endoscopy is invasive, time consuming, expensive, and uncomfortable. Furthermore, an endoscopic approach carries risks of complications.11 Both patients and clinicians alike would benefit from a simple, reliable, and readily available test that enables them to recognize an imminent flare for timely intensification of treatment and better disease control. Fecal calprotectin (FC), a 36-kDa calcium and zincbinding protein, represents 60% of the cytosolic protein in the granulocytes.12 The amount of calprotectin in feces is therefore proportional to the amount of neutrophil migration from the inflamed bowel wall to the mucosa.13 Additionally, the FC concentration is stable for up to 7 days at room temperature and resistant to degradation.14 Compared with conventional serum markers, FC is a more promising marker for assessing intestinal activity with endoscopy as a reference standard. Although its utility has been extensively studied, its exact role has yet to be quantified. In this meta-analysis, we aim to assess the overall diagnostic accuracy of FC in assessing IBD inflammatory activity. www.ibdjournal.org |
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MATERIALS AND METHODS Literature Search We comprehensively and systematically searched the databases Medline, Web of Science, Cochrane Library, and EMBASE for eligible studies from December 2013 or earlier that evaluated activity in UC and CD. Language restrictions were not used. The search strategy used the following terms: (1) calprotectin: “calprotectin,” “leukocyte L1 antigen complex,” “calgranulin”; (2) IBD: “inflammatory bowel disease,” “IBD,” “ulcerative colitis,” “UC,” “crohn’s disease,” “CD,” “colitis,” “enteritis,” “intestinal inflammation.” References and reviews of related literature were searched manually.
Study Selection Articles were first screened by 2 independent reviewers (J.-F.L. and J.-M.C.) based on the title and abstract. The full text of a potentially eligible study was then assessed independently. Disagreements were resolved by discussion. A study was included if it met the inclusion criteria as follows: (1) the study evaluated FC for monitoring IBD activity; (2) an endoscopic scoring system was used as reference standard to assess inflammatory activity; (3) the study provided sufficient details to construct a 2-by-2 table. Studies were excluded if conducted in pediatric patients with IBD.
Data Extraction General information extracted from each study was as follows: first author, publication year, country, age range of study subjects, inclusion and exclusion criteria, and number of IBD subjects (subdivided into UC and CD). Clinical data included the details of the FC test (FC assay method, cutoff value), details regarding the reference standard, prevalence of endoscopy-positive cases in the study population (pretest probability), and 2-by-2 tables in the IBD, UC, and CD groups. When more than 1 cutoff value was reported in a study, we extracted all of them and their corresponding 2-by-2 tables. The microgram per gram unit was used for FC concentrations, except in 2 studies.15,16 One study15 used a previous FC assay kit with which FC was measured in micrograms per milliliter. We multiplied the data by a factor of 5, after confirming with the authors17 and the kit manufacturers (Calpro AS, Oslo, Norway). The author of another study,16 who was contacted through e-mail, claimed that the milligram per liter unit used in their study was equal to that of microgram per gram unit and that the sensitivity needed to be corrected from 79.2% to 76.6% due to a printing mistake. We also contacted corresponding authors through e-mail if further information was necessary for analysis.
recommended against using scales yielding a summary score because the interpretation of the summary score was problematic and potentially misleading.19 Two reviewers evaluated the checklist independently. Disagreements were resolved by consensus.
Patients’ Spectrum If the study consecutively recruited the diagnosed patients with IBD without signs of overt clinical relapse, we marked “yes” for the item. If the study recruited a group of healthy controls or a portion of patients with IBD with overt disease activity, we marked “no” for the item. If there was insufficient information available to make a judgment, we marked “unclear.”
Reference Standard The studies using endoscopy evaluation as reference standard were marked with “yes.” If the reference standard involved clinical indices or others, we marked “no” for the item. An unclear reference standard was marked “unclear.”
Appropriate Time Between Reference Standard and Index Test Collection of fecal samples shortly before the endoscopy examination was ideal. We marked “yes” for the studies that collected stool samples within 1 week before endoscopic examination. This time frame was acceptable because the inflammatory condition was unlikely to change within such a short period of time. A delay of over 1 week was considered unacceptable, and we marked “no” for the item. If information on timing of tests was not provided, the item was marked “unclear.”
Quality Assessment Study quality was assessed using the QUADAS (QUality Assessment of studies of Diagnostic Accuracy included in Systematic reviews) tool. Each item should be answered “yes,” “no,” or “unclear.” We chose all of the 11 items and followed the guidelines for scoring each of them included in the tool.18 We
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FIGURE 1. Flow chart of study selection.
Patients (UC/ CD) FC Assay
Test Results (Total/UC/CD)
Study
Country
Age (yr)
Total IBD
Cutoff (mg/g)
Sipponen et al29
Finland
19–70
106
0/106
PhiCal
Langhorst et al15
Germany
15–70
85
42/43
ELISA
50 100 200a 30
18–80
78
40/38
PhiCal
Schoepfer et al27
Switzerland
18–79
134
134/0
PhiCal
50a
Schoepfer et al28
Switzerland
18–85
140
0/140
PhiCal
Finland
18–69
126
0/126
PhiCal
The Netherlands
30–64
126
39/87
PhiCal
Turkey
49.7 6 10.7
60
60/0
PhiCal
Spain
18–85
146
146/0
ELISA
160
PhiCal
250a 50
ELISA
57a 100
ELISA ELISA —
250a 140 274 —
Vieira et al
af Bjokesten et al22 D’Haens et al23
Onal et al16 Lobaton et al24
26
Schoepfer et al
Nancey et al25
Switzerland
France
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31
Yamamoto et al Lobaton et al32 Total IBD
Japan Spain —
18–74
18–79
32 6 1.6 32–58 —
228
133
20 89 1471
228/0
55/78
0/20 0/89 744/727
100 50 70a 94a 100 250
99.5
CDEIS $ 3
Endoscopy-based classification of inflammation . 0 Mayo endoscopic score . 0/CDEIS $3 Rachmilewitz endoscopic activity index $ 4 SES-CD $ 3 SES-CD $ 3 Mayo endoscopic score . 0/SES-C $2 Rachmilewitz index $4 Mayo endoscopic score . 0 Modified Baron score $ 2 Rachmilewitz index $ 3/SES-CD $ 3 Rutgeerts score $ 2 CDEIS $ 3 —
Prevalence of Active Disease (%)
TP
FP
FN
TN
64 57 49 60/27/33
20 11 3 21/14/7
6 13 21 0/0/0
16 25 33 4/1/3
71
49/22/27 39
6/4/2 1
11/5/6 5
19/11/8 33
56
93
10
7
24
74
86 101 101 87 83 51/22/29
4 11 7 6 6 8/0/8
14 13 13 16 20 28/9/19
30 15 19 17 17 39/8/31
63
23
7
7
23
50
93
12
17
24
75
70 160
18 8
8 14
50 46
76
158 68/35/33
5 34/9/25
16 5/0/5
49 26/11/15
55
59/32/27 7 36 —
12/3/9 3 11 —
14/3/11 3 1 —
48/17/31 7 41 —
50 42 66
66
81 82
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a The cutoff value that showed the highest diagnostic accuracy in study of multiple cutoff values. CDEIS, Crohn’s disease index of severity; ELISA, enzyme-linked immunosorbent assay produced by unknown company (no mention in article); FN, false negative; FP, false positive; Phical, a quantitative enzyme linked immunosorbent assay produced by the Calprotectin Company; SES-CD, simple endoscopic score for Crohn’s disease; TN, ture negative; TP, ture positive.
Fecal Calprotectin for Assessment of IBD Activity
Brazil
240a 200
30
Reference Standard (UC/CD)
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TABLE 1. Study Characteristics of Included Studies
Lin et al
Partial Verification If all the study patients who received an FC test went on to receive verification of their disease status using the endoscopic evaluation, we marked “yes” for this item. If not, we marked it with “no.” If this information was not reported by the study, we marked it “unclear.”
Differential Verification If the same type of endoscopy was performed in all patients of a study to assess the inflammation condition, we marked “yes” for this item. If the choice of endoscopy varied between individuals in one study, we marked “no” for the item. If it was unclear whether different reference standards were used, then the item was marked “unclear.”
Index Test Incorporation If the reference standard was independent of the index test, then we marked “yes” for this item. In all the included studies in our meta-analysis, the FC test and endoscopic evaluation were independent of each other, so all of the items were marked with “yes.”
Index Test Results Blinded/Reference Standard Results Blinded These 2 items were used to check whether a double-blinded method was used in the study or not. If endoscopy performers were blinded for the results of index test or the technicians of FC test were blinded for the results of reference standard, then we marked “yes” for the item. If blinding was not used or was unclear, we marked “no” or “unclear” for the item, respectively.
Relevant Clinical Information Because the result of FC assay was generated by an objective measurement enzyme-linked immunosorbent assay,
FIGURE 2. Summary of the methodological assessment of the included studies basing on the Cochrane handbook. +, low risk; 2, high risk; ?, unclear.
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which was unaltered by clinical information, this item was marked “yes.”
Uninterpretable Results Reported In our study, because the number of results reported agreed with that of patients recruited in the primary studies, this item was marked “yes.”
Withdrawals Explained A “yes” indicated that clear outcomes were reported for all patients, including withdrawals and bad or missing results. A “no” meant that some of the patients who entered the studies did not complete the study and the withdrawals were not explained. If it was unclear how many patients entered and hence whether there were any withdrawals, this item was marked “unclear.”
Statistical Analysis The standard methods were used in this meta-analysis as recommended in the Cochrane handbook for systematic reviews of diagnostic test accuracy.20 The pretest probability of active disease, namely prevalence of active disease, was calculated according to the formula: reference-positive subjects/total patients. For the data analysis, we estimated both hierarchical summary receiver operating characteristic (HSROC) curves at different cutoff values and average operating points with each commonly applied threshold value, as they may complement each other in providing clinically useful summaries and powerful ways of detecting effects. An HSROC curve with 95% confidence region and 95% prediction region was performed to examine the interaction between sensitivity and specificity. Diagnostic odds ratio (DOR) and the area under the HSROC curve (AUC) were calculated to evaluate the diagnostic performance of FC over the IBD group and UC and CD subgroups. The DOR was calculated using the following formula: (sensitivity/[1 2 sensitivity])/ ([1 2 specificity]/specificity). A DOR of 1 indicates that the test cannot discriminate between patients with active and inactive IBD. A higher value indicates better test performance. AUC equals 1 for a perfect test and 0.5 for a completely uninformative test.20 Pooled sensitivity, specificity, and their corresponding 95% confidence intervals were calculated using a random effects model at each threshold. The heterogeneity was detected by a chi-square test or Q-statistic and Higgins I-squared statistic (I2). A P value of less than 0.1 was considered statistically significant heterogeneity for the chi-square or Q-statistics. The percentage of I2 represented the degree of heterogeneity. I2 percentages of 25%, 50%, and 75% indicated a low, moderate, and high degree of heterogeneity, respectively.21 The source of heterogeneity was explored using threshold analysis, meta-regression, and sensitivity analysis. Meta-regression covariate analysis included pretest probability, blinded design, and sample size. Sensitivity analysis was undertaken to assess the impact of a high pretest probability (pretest probability more than overall average pretest probability) and small sample studies (sample sizes ,100). Publication bias was assessed using Deeks’ test. P , 0.05 was considered to indicate
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statistically significant publication bias. We performed statistical analysis on Meta-Disc (version 1.4 for Windows; XI Cochrane, Colloquium, Barcelona, Spain) and Stata (version 12; Stata Corp., College Station, TX).
RESULTS Characteristics and Quality of the Included Studies As Figure 1 shows, 603 articles are available after the initial search. After reading the titles and abstracts and reviewing the full texts, 13 publications15,16,22–32 (1471 patients with IBD; 744 with UC and 727 with CD) were included in the final analysis. Two studies33,34 were excluded because their patients overlapped with the included study conducted by Sipponen et al.29 The clinical characteristics of these studies are listed in Table 1. All studies used a prospective study design and enrolled patients with diagnosed IBD. Ten of the studies were conducted in Europe and the other 3 were in Brazil, Turkey, and Japan. Three studies by Schoepfer et al26–28 and 2 studies by Lobaton et al24,32 performed analysis using different cohorts (confirmed by details of primary articles). In the 5 included studies,22,24,27–29 endoscopies and FC tests were performed more than once in 1 patient as independent samples. Yamamoto et al31 recruited patients with postoperative quiescent CD, af Bjorkestenf et al22 recruited patients with CD who received anti-TNF therapy and Onal et al16 recruited a number of clinical remission patients with UC for investigation. The rest of the 10 studies consecutively recruited patients referred for endoscopy. All the reference standards of included studies were based on endoscopy, including the Mayo endoscopic activity index, Rachmilewitz endoscopic activity index, Modified Baron Score, Endoscopy-based classification of inflammation in UC disease evaluation, Crohn’s disease index of severity, simple endoscopic score for Crohn’s disease, Rutgeerts score, and endoscopy-based classification of inflammation in CD. Figure 2 shows our opinions on each bias risk item for the included studies.
Diagnostic Accuracy
FIGURE 3. The HSROC plot for the utility of FC assay in assessing IBD, UC, and CD activity, with summary point, 95% confidence region, and 95% prediction region. The confidence region indicates the precision with estimation, which consists of the most likely values of true summary sensitivity and specificity. The prediction region predicts the true sensitivity and specificity of a future study. Individual study estimates are represented as circles, with size proportional to study weight. HSROC plot for IBD (A), HSROC plot for UC (B), HSROC plot for CD (C).
We chose the cutoff value that showed the highest diagnostic accuracy from each study and extracted their corresponding 2-by-2 tables to construct the HSROC model for evaluating IBD disease activity. The formula of diagnostic accuracy was ture positive + ture negative/total patients, with the higher values indicating better test performance. In Figure 3A, the HSROC curve of the IBD group is near the upper left corner, the DOR and AUC were 22.72 (14.24–36.25) and 0.89 (0.86–0.92), indicating a relatively high overall level of accuracy. The HSROC model generated a summary point represented by a dot, surrounded by a 95% confidence region and a 95% prediction region. The sensitivity and specificity of this point were 0.85 (0.82–0.87) and 0.81 (0.77–0.84). The Cochrane Q values detecting heterogeneity across studies are presented in Table 2. www.ibdjournal.org |
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TABLE 2. Different Groups IBD AUC (95% CI) Summary sensitivity (95% CI) Heterogeneitya (P, I2) Summary specificity (95% CI) Heterogeneitya (P, I2) Patients
0.89 0.85 55.20 0.81 23.10
(0.86–0.92) (0.82–0.87) (0.0000, 78.3%) (0.77–0.84) (0.0269, 48.1%) 1471
UC 0.93 0.88 15.07 0.82 20.91
(0.89–0.97) (0.85–0.91) (0.0351, 53.5%) (0.77–0.86) (0.0039, 66.5%) 744
CD 0.88 0.81 35.53 0.81 14.26
(0.83–0.93) (0.77–0.84) (0.0000, 77.5%) (0.76–0.86) (0.0752, 43.9%) 727
a Q-value. AUC, area under the curve; CI, confidence interval.
On the basis of the HSROC curve and pooled sensitivity and specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were pooled and applied to estimate posttest probability. The PLR and NLR were 3.92 (3.17–4.85) and 0.20 (0.14–0.28), respectively, in the IBD group. As Figure 4 shows, active IBD is confirmed in 66% (n ¼ 972) of the total included patients (n ¼ 1471). The use of the FC test changes the posttest probability of IBD activity, allowing us to illustrate the result in a more reasonable way: with a pretest probability of 66%, a positive FC test result increases the probability of active IBD to 90% and the negative FC result decreases the probability of active IBD to 26%. It is worth noting that the pretest probability of 66%
indicated that a relatively large proportion of recruited patients with IBD were endoscopically active. Of the 13 eligible studies, there were 8 studies evaluating UC and 9 evaluating CD. HSROCs were constructed for both of the diseases (Fig. 3B, C). The summary point of sensitivity, specificity, and AUC are also listed in Table 2. AUC of the UC group were higher than those of the CD group. Overall results suggested that the FC test appeared to have greater ability to evaluate disease activity in UC than in CD. The cutoff values for testing positive in the FC assay varied between studies, ranging from 30 to 274 mg/g. Pooled sensitivity and specificity were calculated at commonly applied thresholds (50, 100, and 250 mg/g). In 5 studies, the manufacturers’ recommended value (50 mg/g, including 30 mg/g) was used to obtain the following values: pooled sensitivity of 0.92, specificity of 0.60, PLR of 2.33, and NLR of 0.13. In 5 studies, 100 mg/g (including 99.5 mg/ g) was used as an adjusted cutoff value, and the results were sensitivity ¼ 0.84, specificity ¼ 0.66, PLR ¼ 2.95, and NLR ¼ 0.23. With cutoff values of 250 mg/g (including 200 and 274 mg/g) in 7 of the studies, the corresponding values were 0.80, 0.82, 4.17, and 0.22, respectively. The pooled estimates with 95% confidence interval and their corresponding heterogeneity values are shown in Table 3. As the cutoff value increases, sensitivity became lower and specificity became higher.
Heterogeneity Analysis
FIGURE 4. Fagan’s nomogram for showing posttest probability of IBD activity after FC-positive result (upper line) and FC-negative result (lower line).
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Our results were heterogeneous. We first explored heterogeneity through threshold analysis by finding the logarithm of truepositive rates of FC assay detection and the logarithm of falsepositive rates. We then performed a Spearman’s correlation between those 2 values. The coefficient was 0.113 (P ¼ 0.714), indicating that the threshold effect difference was not statistically significant. Secondly, a meta-regression model failed to demonstrate a statistically significant difference as well. Restricted by the number of available studies, we did not determine a reference standard in the meta-regression model. Lastly, sensitivity analysis showed that the result of large sample studies was stable, with summary DORs ranging from 22.72 to 22.75. In the low pretest probability (,66%)
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Fecal Calprotectin for Assessment of IBD Activity
TABLE 3. Different Cutoff Values 50 mg/g Sensitivity (95% CI) Heterogeneitya (P, I2) Specificity (95% CI) Heterogeneitya (P, I2) PLR (95% CI) Heterogeneitya (P, I2) NLR (95% CI) Heterogeneitya (P, I2) Patients
0.92 11.72 0.60 42.19 2.33 71.9 0.13 5.17
(0.90–0.94) (0.0195, 65.9%) (0.52–0.67) (0.0000, 90.5%) (1.18–4.61) (0.0000, 94.4%) (0.09–0.19) (0.2698, 22.7%) 693
100 mg/g 0.84 8.12 0.66 24.27 2.95 21.58 0.23 4.10
250 mg/g
(0.80–0.88) (0.0873, 50.7%) (0.59–0.73) (0.0001, 83.5%) (1.68–5.17) (0.0002, 81.5%) (0.18–0.29) (0.3931, 2.4%) 559
0.80 32.09 0.82 14.29 4.17 7.98 0.22 26.62
(0.76–0.84) (0.0000, 81.3%) (0.77–0.86) (0.0266, 58.0%) (3.15–5.52) (0.2399, 24.8%) (0.14–0.35) (0.0002, 77.5%) 763
a Q-value. CI, confidence interval.
studies, there was variation with summary DORs ranging from 22.72 to 20.02. These data showed that prevalence of active disease among the different study populations may have contributed to overall heterogeneity.
Publication Bias Although the funnel plot of publication bias showed some asymmetry due to the limited number of studies (Fig. 5), the Deeks’ test showed a statistically nonsignificant value (P ¼ 0.425), indicating no publication bias among the included studies.
DISCUSSION FC is an indirect indicator of intestinal mucosa condition. To date, 3 meta-analyses have shown that FC is useful for discriminating IBD from other diseases17,35 and predicting relapse of patients with IBD at remission.36 But IBD activity requires timely assessment by conventional endoscopy to aid clinicians in developing a personalized therapeutic regimen and predicting outcomes to avoid complications and death resulting from IBD. Furthermore, considering the complications and contraindications of endoscopy, clinicians and patients need a simple noninvasive
FIGURE 5. Deeks’ funnel plot for evaluating publication bias. It shows the correlation between the lnDOR and the effective sample size. Individual study estimates are represented as circles, with size proportional to study weight. www.ibdjournal.org |
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test to evaluate disease activity. Therefore, the role of FC assay in detecting IBD activity merits further evaluation. To our knowledge, this is the first meta-analysis of assessment of IBD activity using an FC assay. Our meta-analysis showed a pooled DOR of 22.72 and AUC of 0.89, indicating a relatively high level of overall accuracy in discriminating active from inactive IBD. DOR summarizes test accuracy in a single number, which makes it easy to use for metaanalysis, but it has little direct clinical relevance and can be difficult to apply directly in clinical practice.37 Compared with DOR, sensitivity and specificity may be more meaningful for clinicians. As shown in Table 3, we summarized the pooled statistics of 3 commonly applied thresholds. Although those statistics were objectively illustrated, the optimal cutoff value may be different depending on their use as either a triage tool or as the final assessment for active disease; clinicians should use those data soundly according to the proper clinical scenario. We suggest 50 mg/g as a screening cutoff value for further endoscopy examination in clinical practice, with specificity of 60% and pooled sensitivity of 92%, which is the highest sensitivity among the 3 commonly applied cutoff values. This suggestion will result in 8% of endoscopy-positive patients being unable to receive timely intensive treatment, but the numbers are acceptable when FC is introduced as a screening test. However, 40% of endoscopynegative patients will undergo an unnecessary invasive procedure when this low cutoff is used. Ultimately, when contemplating escalating therapy if there is active disease, higher specificity will be desired. According to our meta-analysis, we suggest the cutoff value at 250 mg/g as a confirmed test to contemplate escalating therapy with pooled sensitivity of 80% and specificity of 82%, the highest specificity among the 3 commonly applied cutoff values. Clinicians should be aware, however, that 18% of those without active disease would be identified as false positives and receive excessive treatment. Meanwhile, treatment will be delayed in 20% of patients with active IBD. In this meta-analysis, the result suggested that the diagnostic performance of the FC assay in the UC group appeared to be superior to that in the CD group. However, we cannot exclude that in some patients with CD, some intestinal lesions of the gastrointestinal tract were not reached by ileocolonoscopy. Patients with high FC levels but apparently low ileocolonoscopy disease activity may need further examination to assess the condition of the upper gastrointestinal tract and/or small bowel. In addition, we cannot exclude selection bias that may play a role here. Severe disease activity in UC manifested by gross blood in the stools is easier to recognize for both clinicians and patients than severe disease activity in CD, which is subtle in presentation. This meta-analysis successfully avoided the risk of partial verification bias through excluding studies that did not apply an endoscopy index as a reference standard. However, this metaanalysis has several limitations. First, the study was limited in part by a large portion of patients with active IBD (overall pretest probability, 66%) being enrolled in the primary studies because most of the studies were conducted in tertiary centers. This
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spectrum bias limited the usage of FC test in primary cares with a lower activity probability. Furthermore, some studies25–27,29 might include a considerable proportion of patients with overt clinical relapse and therefore have overestimated the accuracy of the FC assay. More multicenter large sample studies and strict patient access systems are required to precisely investigate the diagnostic accuracy of the FC test. Second, although we used endoscopic evaluation as reference standard, a consensus between endoscopic scoring systems is still absent for both UC and CD. The nonuniform endoscopic scoring systems may be the reason for the heterogeneous data. Due to the limited number of available studies, we did not perform meta-regression with respect to a reference standard. Third, we could not reach most of the corresponding authors for further information about clinical characteristics of patients, hindering us from investigating sources of heterogeneity by disease severity (e.g., mild, moderate, or severe disease) and disease location (e.g., distal or proximal involvement). During quality assessment, data extraction, and analysis process, we encountered many obstacles, highlighting the methodological flaws in the current studies. It is hoped that more widespread multicenter large samples and implementation of the Standards for the Reporting of Diagnostic Accuracy studies will enable readers to directly extract desired information. These can be emphasized as factors that should be considered and improved in future studies in the area. In conclusion, our results indicate that FC is a simple, reliable, and readily available test to measure IBD activity, appearing to have greater ability to evaluate disease activity in UC than in CD.
ACKNOWLEDGMENTS The authors would like to thank Dr. Ýbrahim Koral Önal (Department of Gastroenterology, Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey) for providing additional information.
REFERENCES
1. Ponder A, Long MD. A clinical review of recent findings in the epidemiology of inflammatory bowel disease. Clin Epidemiol. 2013;5:237–247. 2. Assadsangabi A, Lobo AJ. Diagnosing and managing inflammatory bowel disease. Practitioner. 2013;257:13–18, 12. 3. Fiasse R, Denis MA, Dewit O. Chronic inflammatory bowel disease: crohn’s disease and ulcerative colitis [in French]. J Pharm Belg. 2010;1:1–9. 4. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434. 5. Baars JE, Nuij VJ, Oldenburg B, et al. Majority of patients with inflammatory bowel disease in clinical remission have mucosal inflammation. Inflamm Bowel Dis. 2012;18:1634–1640. 6. Crama-Bohbouth G, Pena AS, Biemond I, et al. Are activity indices helpful in assessing active intestinal inflammation in Crohn’s disease? Gut. 1989;30:1236–1240. 7. Gomes P, du Boulay C, Smith CL, et al. Relationship between disease activity indices and colonoscopic findings in patients with colonic inflammatory bowel disease. Gut. 1986;27:92–95. 8. Foell D, Wittkowski H, Roth J. Monitoring disease activity by stool analyses: from occult blood to molecular markers of intestinal inflammation and damage. Gut. 2009;58:859–868.
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9. Stange EF, Travis SP, Vermeire S, et al. European evidence-based Consensus on the diagnosis and management of ulcerative colitis: definitions and diagnosis. J Crohns Colitis. 2008;2:1–23. 10. Van Assche G, Dignass A, Panes J, et al. The second European evidencebased consensus on the diagnosis and management of Crohn’s disease: definitions and diagnosis. J Crohns Colitis. 2010;4:7–27. 11. Gisbert JP, McNicholl AG, Gomollon F. Questions and answers on the role of fecal lactoferrin as a biological marker in inflammatory bowel disease. Inflamm Bowel Dis. 2009;15:1746–1754. 12. Vermeire S, Van Assche G, Rutgeerts P. Laboratory markers in IBD: useful, magic, or unnecessary toys? Gut. 2006;55:426–431. 13. Schoepfer AM, Trummler M, Seeholzer P, et al. Discriminating IBD from IBS: comparison of the test performance of fecal markers, blood leukocytes, CRP, and IBD antibodies. Inflamm Bowel Dis. 2008;14:32–39. 14. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol. 1992;27:793–798. 15. Langhorst J, Elsenbruch S, Koelzer J, et al. Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices. Am J Gastroenterol. 2008;103:162–169. 16. Onal IK, Beyazit Y, Sener B, et al. The value of fecal calprotectin as a marker of intestinal inflammation in patients with ulcerative colitis. Turk J Gastroenterol. 2012;23:509–514. 17. von Roon AC, Karamountzos L, Purkayastha S, et al. Diagnostic precision of fecal calprotectin for inflammatory bowel disease and colorectal malignancy. Am J Gastroenterol. 2007;102:803–813. 18. Reitsma JB, Rutjes AWS, Whiting P, et al. Chapter 9: assessing methodological quality. In: Deeks JJ, Bossuyt PM, Gatsonis C, eds. Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy Version 1.0.0. The Cochrane Collaboration; 2009. Available at: http://srdta.cochrane.org/. 19. Whiting P, Harbord R, Kleijnen J. No role for quality scores in systematic reviews of diagnostic accuracy studies. BMC Med Res Methodol. 2005;5:19. 20. Macaskill P, Gatsonis C, Deeks JJ, et al. Chapter 10: analysing and presenting results. In: Deeks JJ, Bossuyt PM, Gatsonis C, eds. Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy Version 1.0. The Cochrane Collaboration; 2010. Available at: http://srdta.cochrane.org/. 21. Higgins JP, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ. 2003;327:557–560. 22. af Bjorkesten CG, Nieminen U, Turunen U, et al. Surrogate markers and clinical indices, alone or combined, as indicators for endoscopic remission in anti-TNF-treated luminal Crohn’s disease. Scand J Gastroenterol. 2012;47:528–537. 23. D’Haens G, Ferrante M, Vermeire S, et al. Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis. 2012;18:2218–2224.
Fecal Calprotectin for Assessment of IBD Activity
24. Lobaton T, Rodriguez-Moranta F, Lopez A, et al. A new rapid quantitative test for fecal calprotectin predicts endoscopic activity in ulcerative colitis. Inflamm Bowel Dis. 2013;19:1034–1042. 25. Nancey S, Boschetti G, Moussata D, et al. Neopterin is a novel reliable fecal marker as accurate as calprotectin for predicting endoscopic disease activity in patients with inflammatory bowel diseases. Inflamm Bowel Dis. 2013;19:1043–1052. 26. Schoepfer AM, Beglinger C, Straumann A, et al. Fecal calprotectin more accurately reflects endoscopic activity of ulcerative colitis than the Lichtiger Index, C-reactive protein, platelets, hemoglobin, and blood leukocytes. Inflamm Bowel Dis. 2013;19:332–341. 27. Schoepfer AM, Beglinger C, Straumann A, et al. Ulcerative colitis: correlation of the Rachmilewitz endoscopic activity index with fecal calprotectin, clinical activity, C-reactive protein, and blood leukocytes. Inflamm Bowel Dis. 2009;15:1851–1858. 28. Schoepfer AM, Beglinger C, Straumann A, et al. Fecal calprotectin correlates more closely with the simple endoscopic score for Crohn’s disease (SES-CD) than CRP, blood leukocytes, and the CDAI. Am J Gastroenterol. 2010;105:162–169. 29. Sipponen T, Savilahti E, Kolho KL, et al. Crohn’s disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn’s disease activity index and endoscopic findings. Inflamm Bowel Dis. 2008;14:40–46. 30. Vieira A, Fang CB, Rolim EG, et al. Inflammatory bowel disease activity assessed by fecal calprotectin and lactoferrin: correlation with laboratory parameters, clinical, endoscopic and histological indexes. BMC Res Notes. 2009;2:221. 31. Yamamoto T, Shiraki M, Bamba T, et al. Faecal calprotectin and lactoferrin as markers for monitoring disease activity and predicting clinical recurrence in patients with Crohn’s disease after ileocolonic resection: a prospective pilot study. United Eur Gastroenterol J. 2013;1:368–374. 32. Lobaton T, Lopez-Garcia A, Rodriguez-Moranta F, et al. A new rapid test for fecal calprotectin predicts endoscopic remission and postoperative recurrence in Crohn’s disease. J Crohns Colitis. 2013;7:e641–e651. 33. Sipponen T, Savilahti E, Karkkainen P, et al. Fecal calprotectin, lactoferrin, and endoscopic disease activity in monitoring anti-TNF-alpha therapy for Crohn’s disease. Inflamm Bowel Dis. 2008;14:1392–1398. 34. Sipponen T, Bjorkesten CG, Farkkila M, et al. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn’s disease treatment. Scand J Gastroenterol. 2010;45:325–331. 35. 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. 36. Mao R, Xiao YL, Gao X, et al. Fecal calprotectin in predicting relapse of inflammatory bowel diseases: a meta-analysis of prospective studies. Inflamm Bowel Dis. 2012;18:1894–1899. 37. Deeks JJ. Systematic reviews in health care: systematic reviews of evaluations of diagnostic and screening tests. BMJ. 2001;323:157–162.
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Meta-analysis: Fecal Calprotectin for Assessment of Inflammatory Bowel Disease Activity Jin-Feng Lin, MD, Jin-Min Chen, MD, Jun-Hua Zuo, MD, Allen Yu, BS, Zhu-Jun Xiao, MD, Fei-Hong Deng, MD, Biao Nie, MD, PhD, and Bo Jiang, MD, PhD
Background: Fecal calprotectin (FC) is a promising biomarker for diagnosis of inflammatory bowel disease (IBD). However, the utility of FC for assessment of IBD activity has yet to be clearly demonstrated. The aim of our study was to evaluate the diagnostic accuracy of FC for differentiating between patients with active IBD and those in remission.
Methods: We systematically searched the databases Medline, Web of Science, Cochrane Library, and EMBASE for eligible studies from December 2013 or earlier that evaluated activity in ulcerative colitis (UC) and Crohn’s disease (CD). A hierarchical summary receiver operating characteristic model was performed to calculate the area under the curve to evaluate the overall diagnostic accuracy. The sensitivities and specificities of each commonly applied cutoff value were pooled using a random effects model.
Results: We included 13 studies (744 patients with UC and 727 with CD) in the final analysis. The area under the curve values were 0.89 (95% confidence
interval, 0.86–0.92), 0.93 (0.89–0.97), and 0.88 (0.83–0.93) in the IBD, UC, and CD groups, respectively. For the IBD group at a cutoff value of 50 mg/g, the pooled sensitivity was 0.92 (0.90–0.94) and specificity 0.60 (0.52–0.67). For a cutoff value at 100 mg/g, the pooled sensitivity was 0.84 (0.80–0.88) and specificity was 0.66 (0.59–0.73). For a cutoff value at 250 mg/g, the pooled sensitivity was 0.80 (0.76–0.84) and specificity was 0.82 (0.77–0.86).
Conclusions: The FC test is a reliable marker for assessing IBD disease activity and may have greater ability to evaluate disease activity in UC than CD. (Inflamm Bowel Dis 2014;20:1407–1415) Key Words: inflammatory bowel disease, Crohn’s disease, ulcerative colitis, fecal calprotectin, diagnostic accuracy
T
he incidence and prevalence of both ulcerative colitis (UC) and Crohn’s disease (CD) is increasing worldwide. UC and CD, the major forms of idiopathic inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract that have been empirically defined by clinical, pathological, endoscopic, and radiological features. The courses of these diseases are characterized by episodes of flare-ups alternating with periods of remission.1–4 Determination of inflammatory activity is essential to tailoring therapy and predicting prognosis. Symptoms of colonic inflammation are clearly manifested but unspecific and poorly correlate with mucosal inflammation.5–7 Serum biomarkers Received for publication March 13, 2014; Accepted March 24, 2014. From the Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China. The authors have no conflicts of interest to disclose. J.-F. Lin and J.-M. Chen contributed equally to this work. B. Nie and B. Jiang were co-corresponding authors of the article. Supported by the NanFang Hospital Fund for Distinguished Young Scholars, Grant 2013JQ03. Reprints: Biao Nie, MD, PhD and Bo Jiang, MD, PhD, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhoudadaobei 1838, Guangzhou, Guangdong 510515, China (e-mail: [email protected]; [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000057 Published online 30 June 2014.
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such as C-reactive protein, erythrocyte sedimentation rate, and leukocytes reflect a summation of systemic host responses rather than being specific for intestinal inflammation.8 Endoscopy is currently considered the gold standard for evaluation of mucosal inflammation, and a number of endoscopic scoring systems exist to assess inflammatory activity in UC and CD.9,10 However, endoscopy is invasive, time consuming, expensive, and uncomfortable. Furthermore, an endoscopic approach carries risks of complications.11 Both patients and clinicians alike would benefit from a simple, reliable, and readily available test that enables them to recognize an imminent flare for timely intensification of treatment and better disease control. Fecal calprotectin (FC), a 36-kDa calcium and zincbinding protein, represents 60% of the cytosolic protein in the granulocytes.12 The amount of calprotectin in feces is therefore proportional to the amount of neutrophil migration from the inflamed bowel wall to the mucosa.13 Additionally, the FC concentration is stable for up to 7 days at room temperature and resistant to degradation.14 Compared with conventional serum markers, FC is a more promising marker for assessing intestinal activity with endoscopy as a reference standard. Although its utility has been extensively studied, its exact role has yet to be quantified. In this meta-analysis, we aim to assess the overall diagnostic accuracy of FC in assessing IBD inflammatory activity. www.ibdjournal.org |
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MATERIALS AND METHODS Literature Search We comprehensively and systematically searched the databases Medline, Web of Science, Cochrane Library, and EMBASE for eligible studies from December 2013 or earlier that evaluated activity in UC and CD. Language restrictions were not used. The search strategy used the following terms: (1) calprotectin: “calprotectin,” “leukocyte L1 antigen complex,” “calgranulin”; (2) IBD: “inflammatory bowel disease,” “IBD,” “ulcerative colitis,” “UC,” “crohn’s disease,” “CD,” “colitis,” “enteritis,” “intestinal inflammation.” References and reviews of related literature were searched manually.
Study Selection Articles were first screened by 2 independent reviewers (J.-F.L. and J.-M.C.) based on the title and abstract. The full text of a potentially eligible study was then assessed independently. Disagreements were resolved by discussion. A study was included if it met the inclusion criteria as follows: (1) the study evaluated FC for monitoring IBD activity; (2) an endoscopic scoring system was used as reference standard to assess inflammatory activity; (3) the study provided sufficient details to construct a 2-by-2 table. Studies were excluded if conducted in pediatric patients with IBD.
Data Extraction General information extracted from each study was as follows: first author, publication year, country, age range of study subjects, inclusion and exclusion criteria, and number of IBD subjects (subdivided into UC and CD). Clinical data included the details of the FC test (FC assay method, cutoff value), details regarding the reference standard, prevalence of endoscopy-positive cases in the study population (pretest probability), and 2-by-2 tables in the IBD, UC, and CD groups. When more than 1 cutoff value was reported in a study, we extracted all of them and their corresponding 2-by-2 tables. The microgram per gram unit was used for FC concentrations, except in 2 studies.15,16 One study15 used a previous FC assay kit with which FC was measured in micrograms per milliliter. We multiplied the data by a factor of 5, after confirming with the authors17 and the kit manufacturers (Calpro AS, Oslo, Norway). The author of another study,16 who was contacted through e-mail, claimed that the milligram per liter unit used in their study was equal to that of microgram per gram unit and that the sensitivity needed to be corrected from 79.2% to 76.6% due to a printing mistake. We also contacted corresponding authors through e-mail if further information was necessary for analysis.
recommended against using scales yielding a summary score because the interpretation of the summary score was problematic and potentially misleading.19 Two reviewers evaluated the checklist independently. Disagreements were resolved by consensus.
Patients’ Spectrum If the study consecutively recruited the diagnosed patients with IBD without signs of overt clinical relapse, we marked “yes” for the item. If the study recruited a group of healthy controls or a portion of patients with IBD with overt disease activity, we marked “no” for the item. If there was insufficient information available to make a judgment, we marked “unclear.”
Reference Standard The studies using endoscopy evaluation as reference standard were marked with “yes.” If the reference standard involved clinical indices or others, we marked “no” for the item. An unclear reference standard was marked “unclear.”
Appropriate Time Between Reference Standard and Index Test Collection of fecal samples shortly before the endoscopy examination was ideal. We marked “yes” for the studies that collected stool samples within 1 week before endoscopic examination. This time frame was acceptable because the inflammatory condition was unlikely to change within such a short period of time. A delay of over 1 week was considered unacceptable, and we marked “no” for the item. If information on timing of tests was not provided, the item was marked “unclear.”
Quality Assessment Study quality was assessed using the QUADAS (QUality Assessment of studies of Diagnostic Accuracy included in Systematic reviews) tool. Each item should be answered “yes,” “no,” or “unclear.” We chose all of the 11 items and followed the guidelines for scoring each of them included in the tool.18 We
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FIGURE 1. Flow chart of study selection.
Patients (UC/ CD) FC Assay
Test Results (Total/UC/CD)
Study
Country
Age (yr)
Total IBD
Cutoff (mg/g)
Sipponen et al29
Finland
19–70
106
0/106
PhiCal
Langhorst et al15
Germany
15–70
85
42/43
ELISA
50 100 200a 30
18–80
78
40/38
PhiCal
Schoepfer et al27
Switzerland
18–79
134
134/0
PhiCal
50a
Schoepfer et al28
Switzerland
18–85
140
0/140
PhiCal
Finland
18–69
126
0/126
PhiCal
The Netherlands
30–64
126
39/87
PhiCal
Turkey
49.7 6 10.7
60
60/0
PhiCal
Spain
18–85
146
146/0
ELISA
160
PhiCal
250a 50
ELISA
57a 100
ELISA ELISA —
250a 140 274 —
Vieira et al
af Bjokesten et al22 D’Haens et al23
Onal et al16 Lobaton et al24
26
Schoepfer et al
Nancey et al25
Switzerland
France
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31
Yamamoto et al Lobaton et al32 Total IBD
Japan Spain —
18–74
18–79
32 6 1.6 32–58 —
228
133
20 89 1471
228/0
55/78
0/20 0/89 744/727
100 50 70a 94a 100 250
99.5
CDEIS $ 3
Endoscopy-based classification of inflammation . 0 Mayo endoscopic score . 0/CDEIS $3 Rachmilewitz endoscopic activity index $ 4 SES-CD $ 3 SES-CD $ 3 Mayo endoscopic score . 0/SES-C $2 Rachmilewitz index $4 Mayo endoscopic score . 0 Modified Baron score $ 2 Rachmilewitz index $ 3/SES-CD $ 3 Rutgeerts score $ 2 CDEIS $ 3 —
Prevalence of Active Disease (%)
TP
FP
FN
TN
64 57 49 60/27/33
20 11 3 21/14/7
6 13 21 0/0/0
16 25 33 4/1/3
71
49/22/27 39
6/4/2 1
11/5/6 5
19/11/8 33
56
93
10
7
24
74
86 101 101 87 83 51/22/29
4 11 7 6 6 8/0/8
14 13 13 16 20 28/9/19
30 15 19 17 17 39/8/31
63
23
7
7
23
50
93
12
17
24
75
70 160
18 8
8 14
50 46
76
158 68/35/33
5 34/9/25
16 5/0/5
49 26/11/15
55
59/32/27 7 36 —
12/3/9 3 11 —
14/3/11 3 1 —
48/17/31 7 41 —
50 42 66
66
81 82
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a The cutoff value that showed the highest diagnostic accuracy in study of multiple cutoff values. CDEIS, Crohn’s disease index of severity; ELISA, enzyme-linked immunosorbent assay produced by unknown company (no mention in article); FN, false negative; FP, false positive; Phical, a quantitative enzyme linked immunosorbent assay produced by the Calprotectin Company; SES-CD, simple endoscopic score for Crohn’s disease; TN, ture negative; TP, ture positive.
Fecal Calprotectin for Assessment of IBD Activity
Brazil
240a 200
30
Reference Standard (UC/CD)
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TABLE 1. Study Characteristics of Included Studies
Lin et al
Partial Verification If all the study patients who received an FC test went on to receive verification of their disease status using the endoscopic evaluation, we marked “yes” for this item. If not, we marked it with “no.” If this information was not reported by the study, we marked it “unclear.”
Differential Verification If the same type of endoscopy was performed in all patients of a study to assess the inflammation condition, we marked “yes” for this item. If the choice of endoscopy varied between individuals in one study, we marked “no” for the item. If it was unclear whether different reference standards were used, then the item was marked “unclear.”
Index Test Incorporation If the reference standard was independent of the index test, then we marked “yes” for this item. In all the included studies in our meta-analysis, the FC test and endoscopic evaluation were independent of each other, so all of the items were marked with “yes.”
Index Test Results Blinded/Reference Standard Results Blinded These 2 items were used to check whether a double-blinded method was used in the study or not. If endoscopy performers were blinded for the results of index test or the technicians of FC test were blinded for the results of reference standard, then we marked “yes” for the item. If blinding was not used or was unclear, we marked “no” or “unclear” for the item, respectively.
Relevant Clinical Information Because the result of FC assay was generated by an objective measurement enzyme-linked immunosorbent assay,
FIGURE 2. Summary of the methodological assessment of the included studies basing on the Cochrane handbook. +, low risk; 2, high risk; ?, unclear.
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which was unaltered by clinical information, this item was marked “yes.”
Uninterpretable Results Reported In our study, because the number of results reported agreed with that of patients recruited in the primary studies, this item was marked “yes.”
Withdrawals Explained A “yes” indicated that clear outcomes were reported for all patients, including withdrawals and bad or missing results. A “no” meant that some of the patients who entered the studies did not complete the study and the withdrawals were not explained. If it was unclear how many patients entered and hence whether there were any withdrawals, this item was marked “unclear.”
Statistical Analysis The standard methods were used in this meta-analysis as recommended in the Cochrane handbook for systematic reviews of diagnostic test accuracy.20 The pretest probability of active disease, namely prevalence of active disease, was calculated according to the formula: reference-positive subjects/total patients. For the data analysis, we estimated both hierarchical summary receiver operating characteristic (HSROC) curves at different cutoff values and average operating points with each commonly applied threshold value, as they may complement each other in providing clinically useful summaries and powerful ways of detecting effects. An HSROC curve with 95% confidence region and 95% prediction region was performed to examine the interaction between sensitivity and specificity. Diagnostic odds ratio (DOR) and the area under the HSROC curve (AUC) were calculated to evaluate the diagnostic performance of FC over the IBD group and UC and CD subgroups. The DOR was calculated using the following formula: (sensitivity/[1 2 sensitivity])/ ([1 2 specificity]/specificity). A DOR of 1 indicates that the test cannot discriminate between patients with active and inactive IBD. A higher value indicates better test performance. AUC equals 1 for a perfect test and 0.5 for a completely uninformative test.20 Pooled sensitivity, specificity, and their corresponding 95% confidence intervals were calculated using a random effects model at each threshold. The heterogeneity was detected by a chi-square test or Q-statistic and Higgins I-squared statistic (I2). A P value of less than 0.1 was considered statistically significant heterogeneity for the chi-square or Q-statistics. The percentage of I2 represented the degree of heterogeneity. I2 percentages of 25%, 50%, and 75% indicated a low, moderate, and high degree of heterogeneity, respectively.21 The source of heterogeneity was explored using threshold analysis, meta-regression, and sensitivity analysis. Meta-regression covariate analysis included pretest probability, blinded design, and sample size. Sensitivity analysis was undertaken to assess the impact of a high pretest probability (pretest probability more than overall average pretest probability) and small sample studies (sample sizes ,100). Publication bias was assessed using Deeks’ test. P , 0.05 was considered to indicate
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Fecal Calprotectin for Assessment of IBD Activity
statistically significant publication bias. We performed statistical analysis on Meta-Disc (version 1.4 for Windows; XI Cochrane, Colloquium, Barcelona, Spain) and Stata (version 12; Stata Corp., College Station, TX).
RESULTS Characteristics and Quality of the Included Studies As Figure 1 shows, 603 articles are available after the initial search. After reading the titles and abstracts and reviewing the full texts, 13 publications15,16,22–32 (1471 patients with IBD; 744 with UC and 727 with CD) were included in the final analysis. Two studies33,34 were excluded because their patients overlapped with the included study conducted by Sipponen et al.29 The clinical characteristics of these studies are listed in Table 1. All studies used a prospective study design and enrolled patients with diagnosed IBD. Ten of the studies were conducted in Europe and the other 3 were in Brazil, Turkey, and Japan. Three studies by Schoepfer et al26–28 and 2 studies by Lobaton et al24,32 performed analysis using different cohorts (confirmed by details of primary articles). In the 5 included studies,22,24,27–29 endoscopies and FC tests were performed more than once in 1 patient as independent samples. Yamamoto et al31 recruited patients with postoperative quiescent CD, af Bjorkestenf et al22 recruited patients with CD who received anti-TNF therapy and Onal et al16 recruited a number of clinical remission patients with UC for investigation. The rest of the 10 studies consecutively recruited patients referred for endoscopy. All the reference standards of included studies were based on endoscopy, including the Mayo endoscopic activity index, Rachmilewitz endoscopic activity index, Modified Baron Score, Endoscopy-based classification of inflammation in UC disease evaluation, Crohn’s disease index of severity, simple endoscopic score for Crohn’s disease, Rutgeerts score, and endoscopy-based classification of inflammation in CD. Figure 2 shows our opinions on each bias risk item for the included studies.
Diagnostic Accuracy
FIGURE 3. The HSROC plot for the utility of FC assay in assessing IBD, UC, and CD activity, with summary point, 95% confidence region, and 95% prediction region. The confidence region indicates the precision with estimation, which consists of the most likely values of true summary sensitivity and specificity. The prediction region predicts the true sensitivity and specificity of a future study. Individual study estimates are represented as circles, with size proportional to study weight. HSROC plot for IBD (A), HSROC plot for UC (B), HSROC plot for CD (C).
We chose the cutoff value that showed the highest diagnostic accuracy from each study and extracted their corresponding 2-by-2 tables to construct the HSROC model for evaluating IBD disease activity. The formula of diagnostic accuracy was ture positive + ture negative/total patients, with the higher values indicating better test performance. In Figure 3A, the HSROC curve of the IBD group is near the upper left corner, the DOR and AUC were 22.72 (14.24–36.25) and 0.89 (0.86–0.92), indicating a relatively high overall level of accuracy. The HSROC model generated a summary point represented by a dot, surrounded by a 95% confidence region and a 95% prediction region. The sensitivity and specificity of this point were 0.85 (0.82–0.87) and 0.81 (0.77–0.84). The Cochrane Q values detecting heterogeneity across studies are presented in Table 2. www.ibdjournal.org |
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TABLE 2. Different Groups IBD AUC (95% CI) Summary sensitivity (95% CI) Heterogeneitya (P, I2) Summary specificity (95% CI) Heterogeneitya (P, I2) Patients
0.89 0.85 55.20 0.81 23.10
(0.86–0.92) (0.82–0.87) (0.0000, 78.3%) (0.77–0.84) (0.0269, 48.1%) 1471
UC 0.93 0.88 15.07 0.82 20.91
(0.89–0.97) (0.85–0.91) (0.0351, 53.5%) (0.77–0.86) (0.0039, 66.5%) 744
CD 0.88 0.81 35.53 0.81 14.26
(0.83–0.93) (0.77–0.84) (0.0000, 77.5%) (0.76–0.86) (0.0752, 43.9%) 727
a Q-value. AUC, area under the curve; CI, confidence interval.
On the basis of the HSROC curve and pooled sensitivity and specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were pooled and applied to estimate posttest probability. The PLR and NLR were 3.92 (3.17–4.85) and 0.20 (0.14–0.28), respectively, in the IBD group. As Figure 4 shows, active IBD is confirmed in 66% (n ¼ 972) of the total included patients (n ¼ 1471). The use of the FC test changes the posttest probability of IBD activity, allowing us to illustrate the result in a more reasonable way: with a pretest probability of 66%, a positive FC test result increases the probability of active IBD to 90% and the negative FC result decreases the probability of active IBD to 26%. It is worth noting that the pretest probability of 66%
indicated that a relatively large proportion of recruited patients with IBD were endoscopically active. Of the 13 eligible studies, there were 8 studies evaluating UC and 9 evaluating CD. HSROCs were constructed for both of the diseases (Fig. 3B, C). The summary point of sensitivity, specificity, and AUC are also listed in Table 2. AUC of the UC group were higher than those of the CD group. Overall results suggested that the FC test appeared to have greater ability to evaluate disease activity in UC than in CD. The cutoff values for testing positive in the FC assay varied between studies, ranging from 30 to 274 mg/g. Pooled sensitivity and specificity were calculated at commonly applied thresholds (50, 100, and 250 mg/g). In 5 studies, the manufacturers’ recommended value (50 mg/g, including 30 mg/g) was used to obtain the following values: pooled sensitivity of 0.92, specificity of 0.60, PLR of 2.33, and NLR of 0.13. In 5 studies, 100 mg/g (including 99.5 mg/ g) was used as an adjusted cutoff value, and the results were sensitivity ¼ 0.84, specificity ¼ 0.66, PLR ¼ 2.95, and NLR ¼ 0.23. With cutoff values of 250 mg/g (including 200 and 274 mg/g) in 7 of the studies, the corresponding values were 0.80, 0.82, 4.17, and 0.22, respectively. The pooled estimates with 95% confidence interval and their corresponding heterogeneity values are shown in Table 3. As the cutoff value increases, sensitivity became lower and specificity became higher.
Heterogeneity Analysis
FIGURE 4. Fagan’s nomogram for showing posttest probability of IBD activity after FC-positive result (upper line) and FC-negative result (lower line).
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Our results were heterogeneous. We first explored heterogeneity through threshold analysis by finding the logarithm of truepositive rates of FC assay detection and the logarithm of falsepositive rates. We then performed a Spearman’s correlation between those 2 values. The coefficient was 0.113 (P ¼ 0.714), indicating that the threshold effect difference was not statistically significant. Secondly, a meta-regression model failed to demonstrate a statistically significant difference as well. Restricted by the number of available studies, we did not determine a reference standard in the meta-regression model. Lastly, sensitivity analysis showed that the result of large sample studies was stable, with summary DORs ranging from 22.72 to 22.75. In the low pretest probability (,66%)
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Fecal Calprotectin for Assessment of IBD Activity
TABLE 3. Different Cutoff Values 50 mg/g Sensitivity (95% CI) Heterogeneitya (P, I2) Specificity (95% CI) Heterogeneitya (P, I2) PLR (95% CI) Heterogeneitya (P, I2) NLR (95% CI) Heterogeneitya (P, I2) Patients
0.92 11.72 0.60 42.19 2.33 71.9 0.13 5.17
(0.90–0.94) (0.0195, 65.9%) (0.52–0.67) (0.0000, 90.5%) (1.18–4.61) (0.0000, 94.4%) (0.09–0.19) (0.2698, 22.7%) 693
100 mg/g 0.84 8.12 0.66 24.27 2.95 21.58 0.23 4.10
250 mg/g
(0.80–0.88) (0.0873, 50.7%) (0.59–0.73) (0.0001, 83.5%) (1.68–5.17) (0.0002, 81.5%) (0.18–0.29) (0.3931, 2.4%) 559
0.80 32.09 0.82 14.29 4.17 7.98 0.22 26.62
(0.76–0.84) (0.0000, 81.3%) (0.77–0.86) (0.0266, 58.0%) (3.15–5.52) (0.2399, 24.8%) (0.14–0.35) (0.0002, 77.5%) 763
a Q-value. CI, confidence interval.
studies, there was variation with summary DORs ranging from 22.72 to 20.02. These data showed that prevalence of active disease among the different study populations may have contributed to overall heterogeneity.
Publication Bias Although the funnel plot of publication bias showed some asymmetry due to the limited number of studies (Fig. 5), the Deeks’ test showed a statistically nonsignificant value (P ¼ 0.425), indicating no publication bias among the included studies.
DISCUSSION FC is an indirect indicator of intestinal mucosa condition. To date, 3 meta-analyses have shown that FC is useful for discriminating IBD from other diseases17,35 and predicting relapse of patients with IBD at remission.36 But IBD activity requires timely assessment by conventional endoscopy to aid clinicians in developing a personalized therapeutic regimen and predicting outcomes to avoid complications and death resulting from IBD. Furthermore, considering the complications and contraindications of endoscopy, clinicians and patients need a simple noninvasive
FIGURE 5. Deeks’ funnel plot for evaluating publication bias. It shows the correlation between the lnDOR and the effective sample size. Individual study estimates are represented as circles, with size proportional to study weight. www.ibdjournal.org |
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test to evaluate disease activity. Therefore, the role of FC assay in detecting IBD activity merits further evaluation. To our knowledge, this is the first meta-analysis of assessment of IBD activity using an FC assay. Our meta-analysis showed a pooled DOR of 22.72 and AUC of 0.89, indicating a relatively high level of overall accuracy in discriminating active from inactive IBD. DOR summarizes test accuracy in a single number, which makes it easy to use for metaanalysis, but it has little direct clinical relevance and can be difficult to apply directly in clinical practice.37 Compared with DOR, sensitivity and specificity may be more meaningful for clinicians. As shown in Table 3, we summarized the pooled statistics of 3 commonly applied thresholds. Although those statistics were objectively illustrated, the optimal cutoff value may be different depending on their use as either a triage tool or as the final assessment for active disease; clinicians should use those data soundly according to the proper clinical scenario. We suggest 50 mg/g as a screening cutoff value for further endoscopy examination in clinical practice, with specificity of 60% and pooled sensitivity of 92%, which is the highest sensitivity among the 3 commonly applied cutoff values. This suggestion will result in 8% of endoscopy-positive patients being unable to receive timely intensive treatment, but the numbers are acceptable when FC is introduced as a screening test. However, 40% of endoscopynegative patients will undergo an unnecessary invasive procedure when this low cutoff is used. Ultimately, when contemplating escalating therapy if there is active disease, higher specificity will be desired. According to our meta-analysis, we suggest the cutoff value at 250 mg/g as a confirmed test to contemplate escalating therapy with pooled sensitivity of 80% and specificity of 82%, the highest specificity among the 3 commonly applied cutoff values. Clinicians should be aware, however, that 18% of those without active disease would be identified as false positives and receive excessive treatment. Meanwhile, treatment will be delayed in 20% of patients with active IBD. In this meta-analysis, the result suggested that the diagnostic performance of the FC assay in the UC group appeared to be superior to that in the CD group. However, we cannot exclude that in some patients with CD, some intestinal lesions of the gastrointestinal tract were not reached by ileocolonoscopy. Patients with high FC levels but apparently low ileocolonoscopy disease activity may need further examination to assess the condition of the upper gastrointestinal tract and/or small bowel. In addition, we cannot exclude selection bias that may play a role here. Severe disease activity in UC manifested by gross blood in the stools is easier to recognize for both clinicians and patients than severe disease activity in CD, which is subtle in presentation. This meta-analysis successfully avoided the risk of partial verification bias through excluding studies that did not apply an endoscopy index as a reference standard. However, this metaanalysis has several limitations. First, the study was limited in part by a large portion of patients with active IBD (overall pretest probability, 66%) being enrolled in the primary studies because most of the studies were conducted in tertiary centers. This
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spectrum bias limited the usage of FC test in primary cares with a lower activity probability. Furthermore, some studies25–27,29 might include a considerable proportion of patients with overt clinical relapse and therefore have overestimated the accuracy of the FC assay. More multicenter large sample studies and strict patient access systems are required to precisely investigate the diagnostic accuracy of the FC test. Second, although we used endoscopic evaluation as reference standard, a consensus between endoscopic scoring systems is still absent for both UC and CD. The nonuniform endoscopic scoring systems may be the reason for the heterogeneous data. Due to the limited number of available studies, we did not perform meta-regression with respect to a reference standard. Third, we could not reach most of the corresponding authors for further information about clinical characteristics of patients, hindering us from investigating sources of heterogeneity by disease severity (e.g., mild, moderate, or severe disease) and disease location (e.g., distal or proximal involvement). During quality assessment, data extraction, and analysis process, we encountered many obstacles, highlighting the methodological flaws in the current studies. It is hoped that more widespread multicenter large samples and implementation of the Standards for the Reporting of Diagnostic Accuracy studies will enable readers to directly extract desired information. These can be emphasized as factors that should be considered and improved in future studies in the area. In conclusion, our results indicate that FC is a simple, reliable, and readily available test to measure IBD activity, appearing to have greater ability to evaluate disease activity in UC than in CD.
ACKNOWLEDGMENTS The authors would like to thank Dr. Ýbrahim Koral Önal (Department of Gastroenterology, Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey) for providing additional information.
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