|Year : 2013 | Volume
| Issue : 4 | Page : 336-341
A new cutoff value for fecal calprotectin level in differentiating functional from organic causes of chronic diarrhea
Shahira El-Etreby1, Hazam Hakim1, Maha Ragab1, Raghda Farag2, Ehsan Rizk3, Sahar Alsayed3, Hanaa Abdeen4
1 Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
4 Department of Biochemistry, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||05-Jun-2013|
|Date of Acceptance||06-Dec-2013|
|Date of Web Publication||1-Feb-2014|
Specialized Medical Hospital, Mansoura University, 35516
The gold standard to establish inflammatory bowel disease diagnosis remains in the hands of endoscopists and pathologists. A challenge is thus to distinguish symptoms of inflammatory bowel disease from that of irritable bowel syndrome.
Aim of this work
The aim of this study was to evaluate the clinical usefulness of fecal calprotectin level as a noninvasive marker in order to distinguish patients with diarrhea in need of intensified follow-up from those who do not need further workup.
Patients and methods
From a total of 150 patients presented with chronic diarrhea with or without bleeding per-rectum in the outpatient clinic of Specialized Medical Hospital, only 60 were involved in this study. Stool analysis and culture were carried out. Measurement of fecal calprotectin was done using the ELISA kit. Inflammatory biomarkers, such as erythrocyte sedimentation rate and C-reactive protein and perinuclear anti-neutrophil cytoplasmic autoantibodies (P-ANCA) and cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA), were tested. Full colonoscopy with histopathological examination was performed.
The frequencies of diseases on the basis of diagnostic colonoscopy and pathological examination were as follows: 19 patients with irritable bowel syndrome (31.67%), 41 patients with nonorganic gastrointestinal (GIT) disease versus organic GIT diseases (68.33%), 32 patients with ulcerative colitis (53.33%), two patients with Crohn's disease (3.33%), two patients with diverticulitis (3.33%), two patients with eosinophilic gastroenteritis (3.33%), and two patients with cancer colon (3.33%). There was a remarkable difference between organic GIT diseases versus nonorganic groups as regard fecal level of calprotectin (P < 0.001). Fecal calprotectin value of at least 350 μg/g with a receiver operating characteristic value of 0.931 [95% confidence interval (CI) 0.864-0.971] was diagnostic of ulcerative colitis with a sensitivity of 81.82% (95% CI 64.5-93), specificity of 85.19% (95% CI 66.3-95.8), positive predictive value of 86.32%, and negative predictive value of 80.39%.
Calprotectin, a fecal marker, is helpful as an adjunctive tool in overall evaluation of patients with nonspecific symptoms and as a diagnostic tool in those with inflammatory disease. It is less invasive than colonoscopy and can help to guide management in a more cost-effective manner.
Keywords: Calprotectin, diarrhea, functional, organic
|How to cite this article:|
El-Etreby S, Hakim H, Ragab M, Farag R, Rizk E, Alsayed S, Abdeen H. A new cutoff value for fecal calprotectin level in differentiating functional from organic causes of chronic diarrhea. Tanta Med J 2013;41:336-41
|How to cite this URL:|
El-Etreby S, Hakim H, Ragab M, Farag R, Rizk E, Alsayed S, Abdeen H. A new cutoff value for fecal calprotectin level in differentiating functional from organic causes of chronic diarrhea. Tanta Med J [serial online] 2013 [cited 2017 Oct 18];41:336-41. Available from: http://www.tdj.eg.net/text.asp?2013/41/4/336/126199
| Introduction|| |
The gold standard to establish inflammatory bowel disease (IBD) diagnosis remains in the hands of endoscopists and pathologists. Precise activity assessment of IBD is essential to determine the extent and severity of disease for optimized therapy  . Recent studies have identified mucosal healing on endoscopy as a key prognostic parameter in the management of IBD  . In addition, in IBD, many symptoms are similar to the functional noninflammatory disorder like irritable bowel syndrome (IBS). A challenge is thus to distinguish symptoms of IBD from IBS  . Because of the chronicity of these conditions and the early onset of symptoms in the majority of cases  , much effort has been made to identify noninvasive biomarkers able to select patients requiring invasive procedures and avoid unnecessary examinations.
Calprotectin is a 36-kDa calcium-binding and zinc-binding protein. It represents 60% of cytosolic proteins in neutrophils, which is a major player in gut intestinal inflammation. Calprotectin released from neutrophils after cell death or rupture induces apoptosis in other cells. As a result, levels of calprotectin from apoptotic neutrophils rise during cell activation and turn over under inflammatory conditions  . In normal individuals, most circulating neutrophils migrate through the mucosal membrane of the gut wall and undergo lysis within the gut lumen. This accounts for the median fecal levels of 2.0 mg/l seen in healthy controls. As it is resistant to colonic bacterial degradation and drug, and stable outside the body for days, fecal calprotectin is a marker of inflammation in IBD  .
| Aim of this work|| |
The aim of this study was to evaluate the clinical usefulness of fecal calprotectin level as a noninvasive marker in order to distinguish patients in need of an intensified follow-up from those who do not need further workup and to estimate the possible economic effects of a sequential testing strategy with fecal calprotectin to minimize colonoscopies.
| Patients and methods|| |
This prospective study comprised 150 patients who presented with chronic diarrhea with or without bleeding per-rectum at the gastroenterology clinic of the Specialized Medical Hospital, Mansoura University. All outpatients were referred to our tertiary hospital for colonoscopy from June 2011 to December 2012. Written informed consents were obtained from all participants before enrollment. The Ethical Scientific Committee of the Faculty of Medicine, Mansoura University approved this study.
Only 60 patients out of 150 completed the study; the remaining 90 were diagnosed with infectious colitis and responded well to antibiotics-guided stool culture.
All patients were subjected to (a) thorough historical evaluation with stress on frequency and consistency of diarrhea per day, along with the analysis of any associated symptoms, such as weight loss, fever, and bleeding per-rectum; (b) thorough physical examination with stress on pulse and temperature; and (c) laboratory investigations that included complete blood count [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum albumin], stool analysis and culture, P-ANCA and C-ANCA, cytomegalovirus (CMV) antibody [in ulcerative colitis (UC) cases], and measurement of fecal calprotectin.
Stool sample was taken into a dry, well-capped container and stored at −20°C until analysis.
Detection of fecal calprotectin (MRP 8/14) in stool was done using immune diagnostic AG ELISA kit (Bensheim, Germany).
Stool (100 mg) was suspended in 5 ml extraction buffer (diluted 1 : 2.5) using vortex and the suspension was centrifuged and diluted 1 : 50 [20 μl supernatant +980 μl wash buffer (diluted 1 : 10)]. Thereafter, 100 μl of this diluted supernatant and reconstituted standards and controls were added into respective wells, covered, and incubated for 1 h at 37°C on a horizontal mixer. After incubation, wells were washed five times, dried, and 100 μl of conjugate (diluted 1 : 100) was added to each well; then the plate was incubated for 1 h at 37°C on a horizontal mixer. After incubation, wells were washed five times, dried, and 100 μl of substrate was added; the plate was incubated for 10-20 min at room temperature, and then the reaction was stopped using 50 μl stop solution. The absorbance was determined with ELISA reader at 450 nm and the results were calculated by bloating standard curve using four-parameter algorithm. The median value of fecal calprotectin was 25 mg/g and considered positive if above 50 mg/g  .
Abdominal ultrasonography and barium enema were carried out in selected cases.
Full colonoscopy and endoscopic findings were collected by the endoscopists through a standardized questionnaire. According to this questionnaire, the following endoscopic diagnoses were considered: normal endoscopic signs, active UC, active Crohn's disease (CD), diverticula with peridiverticular inflammation, ischemic colitis, colorectal cancer or polyps, and miscellaneous diagnoses.
Endoscopic activity of UC and CD were evaluated by Truelove and Witt's scoring system and Crohn's Disease Endoscopic Index of Severity (CDEIS), respectively.
Patients with any of the following criteria were excluded from the study:
- diabetes mellitus,
- thyroid disorders,
- immunocompromised patients or patients on chemotherapy, and
- patients diagnosed with diarrhea-type IBS and already on treatment without flag-alarming symptoms.
The statistical analysis was conducted using the Statistical Package for Social Sciences (version 17.0 for Windows; SPSS Inc., Chicago, Illinois, USA). All quantitative variables were expressed as mean ± SD. Comparisons between the groups were performed using the Student's t-test whenever applicable. The qualitative or categorical variables were described as proportions. Proportions were compared using the χ2 -test or Fisher's exact test whenever applicable.
Correlations between fecal calprotectin and other variables were established using Pearson's or Spearman's rank correlation. Mann-Whitney U-test was used for the continuous ordinal data between two qualitative variables. Variables that achieved statistical significance with univariate analysis were included in multiple regression analysis to evaluate the independent factors associated with high fecal calprotectin. P values less than 0.05 were considered statistically significant.
| Results|| |
From a total of 150 patients presented with diarrhea with or without bleeding per-rectum in the outpatient clinic, only 60 did not respond to medical treatment and underwent specific laboratory investigations and diagnostic colonoscopy. The basic patient's characteristics of nonorganic gastrointestinal (GIT) disease versus organic GIT disease are mentioned in [Table 1]. IBS was more in female sex. There was statistically significant difference between both groups as regard decreased hemoglobin level and elevated white blood cell (WBC) count, although its value within normal ranges elevated ESR and CRP as markers of inflammation, and occurrence of bleeding per-rectum in organic group. A remarkable difference between organic GIT diseases versus nonorganic groups as regard fecal level of calprotectin was also observed.
|Table 1: Patient's characteristics of nonorganic gastrointestinal disease versus organic gastrointestinal disease|
Click here to view
From a total of 60 cases, 32 cases were diagnosed as UC (53.33%), the remaining cases were diagnosed as different entities: 19 patients with IBS (31.67%) and two cases of CD (3.33%), with their value for fecal calprotectin 1325 and 875 μg/g, respectively. Two cases were of diverticulitis (3.33%), with fecal calprotectin level 150 μg/g for one and 125 μg/g for the other. Eosinophilic gastroenteritis (3.33%) and cancer colon (3.33%) were represented with fecal calprotectin level of 237.5 and 1315 μg/g and 525 and 350 μg/g, respectively [Figure 1]. Patient's characteristics of UC are demonstrated in [Table 2].
There was a positive correlation between fecal calprotectin and chronicity of the disease (P < 0.01), male sex (P < 0.007), increased inflammatory markers such as ESR (P < 0.001) and CRP (P < 0.001), elevated WBC count (P < 0.01), increased pulse rate (P < 0.02), increased frequency of diarrhea (P < 0.001), occurrence of bleeding per-rectum (P < 0.009), more involvement of colon (P < 0.001), and finally with severity of UC (P < 0.001). In contrast, there was a negative correlation between fecal calprotectin and age of patients (P < 0.03), decreased hemoglobin level (P < 0.001), and serum albumin (P < 0.002; [Table 3]).
|Table 3: Correlation between fecal calprotectin and other parameters in the ulcerative colitis group|
Click here to view
[Table 4] demonstrates the linear regression analysis of fecal calprotectin to other parameters. High level of fecal calprotectin could predict presence of high WBCs (P = 0.01), low level of hemoglobin (P < 0.001), high ESR (P < 0.001) and CRP(P < 0.001), low serum albumin(P = 0.002), high pulse rate (P = 0.02), increased frequency of diarrhea (P < 0.001), occurrence of bleeding per-rectum (P < 0.001), more involvement of colon (P < 0.001), and more active disease (P < 0.001).
|Table 4: Regression analysis between fecal calprotectin and other parameters in the ulcerative colitis group|
Click here to view
Fecal calprotectin value of at least 350 μg/g with a receiver operating characteristic (ROC) value of 0.931 [95% confidence interval (CI) 0.864-0.971] was diagnostic of UC with sensitivity of 81.82% (95% CI 64.5-93), specificity of 85.19% (95% CI 66.3-95.8), positive predictive value (PPV) of 86.32%, and negative predictive value (NPV) of 80.39% [Table 5] and [Figure 2].
|Table 5: Accuracy of fecal calprotectin measurement in the diagnosis of ulcerative colitis|
Click here to view
| Discussion|| |
In this study, fecal calprotectin can significantly differentiate organic GIT diseases, including UC, from nonorganic disorders, including IBS patients (P < 0.001). In the IBDs, many symptoms are similar to the functional disorder IBS. A challenge is thus to distinguish symptoms of IBD from that of IBS. Jelsness-Jψrgensen et al.  concluded that calprotectin levels are elevated in subgroups of IBD patients who are in remission and have coexisting IBS-like symptoms. Fecal calprotectin can still differentiate inflammatory disease from functional bowel disorders. Comparison studies have found an overall diagnostic accuracy of 80-100% in IBD for calprotectin. However, elevated levels are found in both CD and UC, making it difficult to distinguish between these two diagnoses from these biomarkers alone  . In this study, patients with organic GIT diseases were significantly presented with high pulse rates, although within normal value; high ESR and CRP; high WBCs count; bleeding per-rectum; and high fecal calprotectin level.
Anemia is a frequent and serious complication in patients with IBD affecting about one-third of the patients. Although in many cases anemia correlates the clinical activity of the disease, many patients in remission have anemia because of iron, vitamin B 12 , and/or folic acid deficiency, which have serious consequences in the clinical status and quality of life of the patients  .
CRP is one of the many acute-phase proteins that increase in the serum of patients with acute-phase IBD. It is more sensitive in cases of CD than that of UC. So use of this marker alone to identify patients with symptoms compatible with IBD that undergo further evaluation would delay diagnosis for many cases  .
In this study, the mean of fecal calprotectin levels were 1325 μg/g in UC, 875 μg/g in CD, and 525 μg/g in cancer colon. Using ROC statistics, a cutoff value of 350 μg/g indicated the presence of UC with a sensitivity of 81.82% and a specificity of 85.179.5% (PPV 86.3% and NPV 80.39%).
In a recent study by Nancey et al.  , fecal calprotectin concentrations correlated closer with the endoscopic scores in UC (r = 0.75; P < 0.001). This study was in accordance with our result (r = 0.892; P < 0.001). Using cutoffs of 250 μg/g for fecal calprotectin, fecal markers had similar overall accuracies to predict endoscopic activity in patients with UC (88%), whereas accuracies of CRP were lower.
In a study by Dranga et al.  , 50 patients with UC were evaluated during the active disease. The correlation between the calprotectin values and lesions localization was analyzed, without finding any statistical differences. The data have shown a very strong correlation between the severity of the active disease, assessed through the UCDAI (Mayo) score, and the calprotectin value.
Our study revealed that fecal calprotectin level could be a predictor of increased WBC count, ESR, CRP, frequency of diarrhea, occurrence of bleeding per-rectum, more involvement of colon, and activity scoring system in UC patients.
Another study evaluated the diagnostic accuracy of fecal calprotectin against complete colonic and small bowel endoscopy in 83 adult patients referred for suspected IBD. In total, 40 patients were diagnosed with optimal cutoff of fecal calprotectin as 150 μg/g, providing 85% sensitivity and 81% specificity, with the area under ROC for fecal calprotectin significantly superior to that of CRP (0.87 vs. 0.53; P < 0.001)  .
A recent retrospective cost-minimization analysis further stressed the potential of fecal calprotectin as a screening tool, because it resulted in a 50% reduction in the estimated demand of colonoscopies when a 50 μg/g cutoff was used and a 67% reduction when the cutoff was doubled to 100 μg/g, with a cost avoidance of €1.57 million and €2.13 million, respectively , .
The wide range of different cutoff values and numbers of these studies makes it difficult to have a universal optimal cut point for fecal calprotectin with different sensitivities. On comparing results from different studies with our study, it is important to define the test-operating characteristic to compare cut points. Besides, because PPVs and NPVs are based on the prevalence of disease in different population and countries, it changes with wide change in the study population.
| Conclusion|| |
Calprotectin, a fecal marker, is helpful as an adjunctive tool in the overall evaluation of patients with nonspecific symptoms and as a diagnostic tool in those with inflammatory disease. It is less invasive than colonoscopy and can help to guide management in a more cost-effective manner.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
|1.||Neumann H, Vieth M, Neurath MF, Atreya R. Endocytoscopy allows accurate in vivo differentiation of mucosal inflammatory cells in IBD: a pilot study. Inflamm Bowel Dis 2013; 19:356-362. |
|2.||Neurath MF, Travis SP. Mucosal healing in inflammatory bowel diseases: a systematic review. Gut 2012; 6111:1619-1635. |
|3.||Jelsness-Jørgensen LP, Bernklev T, Moum B. Calprotectin is a useful tool in distinguishing coexisting irritable bowel-like symptoms from that of occult inflammation among inflammatory bowel disease patients in remission. Gastroenterol Res Pract 2013; 620-707. |
|4.||Caccaro R, D′Incà R, Pathak S, Sturniolo GC. Clinical utility of calprotectin and lactoferrin in patients with inflammatory bowel disease: is there something new from the literature? Expert Rev Clin Immunol 2012; 8:579-585. |
|5.||Sydora MJ, Sydora BC, Fedorak RN. Validation of a point-of-care desk top device to quantitate fecal calprotectin and distinguish inflammatory bowel disease from irritable bowel syndrome. J. Crohns Colitis 2012; 6:207-214 |
|6.||D′Haens G, Ferrante M, Vermeire S, Baert F, Noman M, Moortgat L, et al. Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis 2012; 18:2218-2224. |
|7.||Tibble J, Teahon K, Thjodleifsson B, Roseth A, Sigthoroson E, Bridgerrr S, et al. A simple method for assessing intestinal inflammation in Crohn′s disease. Gut 2000; 47:506-513 |
|8.||Lewis JD. The utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease. Gastroenterology 2011; 140:1817-1826. |
|9.||Hong ZW, Ren JA. Research progress of anemia associated with inflammatory bowel diseases. Zhonghua Wei Chang Wai Ke Za Zhi 2012; 15:644-647. |
|10.||Nancey S, Boschetti G, Moussata D, Cotte E, Peyras J, Cuerq C, 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. |
|11.||Dranga M, Dumitrescu G, Badea M, Blaj A, Mihai C, Prelipcean CC, et al. The semi-quantitative calprotectin rapid test: Is it useful in inflammatory bowel disease? Rev Med Chir Soc Med Nat Iasi 2012; 116:761-765. |
|12.||Jensen MD, Kjeldsen J, Nathan T. Fecal calprotectin is equally sensitive in Crohn′s disease affecting the small bowel and colon. Scand J Gastroenterol 2011; 46:694-700. |
|13.||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. |
|14.||Mindemark M, Larsson A. Ruling out IBD: estimation of the possible economic effects of pre-endoscopic screening with F-calprotectin. Clin Biochem 2012; 45:552-555. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]