Faecal calprotectin in differentiating between functional and organic bowel diseases

Results Patients with inflammatory bowel disease had faecal calprotectin levels of 379.2 ± 177.9 μg/g (mean ± SD); this was higher than in patients with functional bowel disease [(27.97 ± 15.2 μg/g); p : 0.004] and healthy controls [(21.64 ± 11.3 μg/g); p : 0.0002]. Patients with organic non-inflammatory bowel disease had faecal calprotectin levels of 273.4 ± 157.8 μg/g, which is higher than in patients with functional bowel disorders [(27.97 ± 15.2 μg/g); p : 0.002] and healthy controls [(21.64 ± 11.3 μg/g); p : 0.0001]. There was no statistically significant difference between faecal calprotectin in patients with functional bowel disease and healthy controls ( p : 0.264), and between both groups with organic bowel disease ( p : 0.312). Conclusion Faecal calprotectin is a sensitive non-invasive method which can be used to identify patients with organic bowel disorders. It is not, however, able to differentiate between different types of organic bowel diseases. Keywords Faecal calprotectin Functional bowel disease Organic bowel disease Inflammatory bowel disease Introduction Recurrent abdominal pain and bowel habit alterations are common symptoms in the general population. Functional bowel disorders (FBD) – and in particular irritable bowel syndrome (IBS) – affect 5–20% of the general population in industrialised countries and are the most common intestinal disorders in primary care visits and gastroenterology consultations [1] . In routine clinical practice, gastroenterologists often encounter the diagnostic challenge of differentiating between patients with IBS and those with organic pathology, in particular, inflammatory bowel disease (IBD). In addition, patients presenting with unexplained bloating, abdominal distress or chronic diarrhoea with or without malabsorption should undergo serologic tests (e.g. antigliadin and anti-tissue transglutaminase antibodies) to exclude the presence of coeliac disease [2] . Because this clinical discrimination remains problematic, many patients with IBS are extensively investigated with multiple diagnostic tests, including invasive endoscopic and/or radiological procedures, to make an accurate diagnosis [3] . The selection of patients who should undergo these procedures is one of the key points in the diagnosis, which should avoid the abuse of invasive and expensive tests as well as the underestimation of potentially harmful diseases [4] . When FBD is suspected, the clinical criteria can be combined with many non-invasive diagnostic tests to reach the proper diagnosis. Unfortunately, serological markers of inflammation, including ESR, CRP, leucocyte and platelet count, lack diagnostic sensitivity and specificity as they do not directly reflect the extent of local inflammation [5,6] . Further, the faecal occult blood test is of little value in detecting IBD and has a low sensitivity for diagnosing colorectal cancer, especially at an early stage [7] . During the last decade, different neutrophil-derived proteins excreted in faeces have emerged as biomarkers of intestinal inflammation. These include lactoferrin, neutrophil elastase and leucocyte esterase [8,9] . Among these, faecal calprotectin (FC) appears to be the most promising surrogate parameter [10–12] . Faecal calprotectin is a 36.5-kDa calcium and zinc binding protein that accounts for 60% of total proteins in the cytosol fraction of neutrophil granulocytes. Its molecular structure consists of a heterotrimer made up of two heavy chains and one non-glycosylated light chain [13,14] . Calprotectin appears to play a regulatory role in the inflammatory process, revealing both antimicrobial and antiproliferative capacities [15] . Under inflammatory conditions of the gastrointestinal tract there is an increase in mucosal permeability, with granulocytes and monocytes undergoing translocation into the intestinal mucosa. The subsequent activation and then death of these cells results in the release of large amounts of calprotectin, which is excreted in faeces [16] . Many studies have shown that there is an association between calprotectin levels and degree of inflammation; as a consequence, this could be used to monitor treatment response and to predict the risk of relapse [17,18] . In inflammatory bowel disease, FC level is closely correlated with the faecal excretion of 111 In-labelled leucocytes, considered the gold standard stool marker of intestinal inflammation [19] . Faecal calprotectin is eliminated intact in the faeces as it can resist metabolic degradation by bacterial enzymes and intestinal proteases [20,21] . This gives it an advantage for the screening of intestinal inflammation over other used markers, such as lactoferrin, neutrophil elastase, or leucocyte esterase [22] . An improved assay is available to determine calprotectin using ELISA [23] . Compared to the original assay, this method requires smaller samples and increases the performance of the extraction solution. It is a simple, accurate, sensitive, reproducible, and cheaper technique (FC assay costs about 20 US dollars per patient). With the original ELISA method a higher reference limit of 10 mg/L was considered, but with the improved method the limit was established at 50 μg/g of faeces. The value of faecal calprotectin assay in discriminating between functional and organic bowel disorders had been tested in Egyptian children but not in adult patients [24] . The aim of this study was to evaluate the sensitivity and diagnostic accuracy of FC assay at different cutoff values in discriminating between functional and organic gastrointestinal disorders in adult patients. Patients and methods The study included 39 patients who had been referred to the Internal Medicine Department in Cairo University Hospital for diagnostic differentiation of functional or organic bowel disorders. Patients were prospectively enrolled between November 2008 and August 2009. In addition, 10 healthy subjects (who were referred for colonoscopy screening due to family history of colorectal cancer) were included as a control group. The research objectives and procedures were explained to patients and control subjects; participants signed an informed consent form before inclusion. The principal inclusion criteria for the study were the presence of symptoms suggestive of organic pathology, including intense abdominal pain, chronic diarrhoea, weight loss and/or anorectal bleeding (IBS patients did not present with GI bleeding). The symptoms were present for at least six months and at the initial visit to the hospital it was necessary to do an endoscopic and/or intestinal radiological procedure to confirm or exclude organic bowel disorder. The exclusion criteria were the regular intake of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, and/or anticoagulants, or the concomitant presence of other non-gastrointestinal diseases, in particular, rheumatoid arthritis, other connective tissue inflammatory diseases or liver cirrhosis, as these diseases have been reported to have a possible influence on faecal calprotectin concentration. All the studied patients were subjected to the following: full medical history with thorough clinical examination; stool analysis and culture to exclude infections; complete blood picture, including ESR, CRP, serum albumin, ALT, AST, and serum bilirubin; abdominal ultrasound examination; and complete colonoscopy with intubation of the terminal ileum including multiple biopsies from the lesions for histopathological evaluation. Each patient was evaluated according to the ROME III criteria, which has been suggested as a guide for the clinical diagnosis of irritable bowel syndrome [25] . Faecal calprotectin assay A single stool sample was collected from each patient and stored in a suitable container at −20 °C until assayed for calprotectin. The faecal sample was delivered 3 days before colonoscopy. The stool sample was tested using the PhiCal ELISA test device, produced by Nycomed Pharma AS. Essentially, 5 g of faeces was mixed with 10 ml extraction solution (Tris buffered isotonic saline with 10 ml CaCl, pH 8.4) in a high speed homogeniser. The homogenate was then centrifuged for 20 min before the supernatant was harvested, and calprotectin levels were measured using an ELISA assay. The normal reference value was <15 μg/g and the manufacturer established the margin values between 50 and 100 μg/g. Statistical analysis The numeric data were presented as mean ± standard deviation and range. We followed the STARD checklist for studies of the diagnostic accuracy of tests. The sensitivity and specificity of the FC assay and its positive and negative predictive values were calculated by standard statistical methods. The Mann–Whitney U test was used to compare the faecal calprotectin values recorded in all the studied groups. We assumed a statistically significant difference when p < 0.05. Results The 39 patients were allocated into four groups after clinical, laboratory, imaging, endoscopic and pathological assessment. Among the 39 patients, 20 proved to have functional bowel disorders (IBS group). Nine patients had IBD (Ulcerative colitis 8 and 1 Crohn’s Disease) (IBD group), while 10 demonstrated organic (non-IBD) diagnosis (3 colorectal carcinoma, 2 intestinal polyps and 5 diverticulitis/diverticulosis). In addition, 10 subjects were included as a control group. Patients’ demographic details are shown in Table 1 . There was a non-significant difference ( p > 0.05) between patients and control subjects as regards mean age and sex distribution. Eighteen out of 20 patients with IBS (90%), 2 out of 9 with IBD (22.2%), and 2 out of 10 with organic (non-IBD) group (20%) fulfilled the ROME III criteria. Table 2 shows faecal calprotectin values in all studied groups. The IBD group had FC values significantly higher than both the IBS ( p = 0.004) and the control group ( p = 0.0002). FC levels were also significantly higher in the organic group of non-IBD patients as compared to both the IBS group ( p = 0.002) and the control group (0.0001). However, no significant difference in FC levels was found between the control and the IBS groups ( p = 0.264) or between IBD and organic non-IBD group ( p = 0.312). Fig. 1 represents the distribution of FC values in all the studied groups. In the control group, only one of 10 subjects had a calprotectin concentration of 52.1 μg/g (10%), and in the rest it was <50 μg/g. In the IBS group, only three patients (15%) with diarrhoea-predominant symptoms had calprotectin levels > 50 μg/g, and in the rest it was <50 μg/g. Calprotectin levels were >100 μg/g in 8 out of 10 patients (80%) in the organic group (non-IBD). In the IBD group, calprotectin levels were >100 μg/g in 7 out of 9 patients (77.8%); the lower values were 97.9 μg/g in a patient with UC mild activity and the other was 99.8 μg/g in a patient with CD. At a cut off value of 50 μg/g, FC had 100% sensitivity, 86% specificity with 82.6% positive predictive and 100% negative predictive value in diagnosing organic bowel diseases. At a cutoff value of 100 μg/g, it showed 78% sensitivity, 100% specificity with 100% positive predictive value and 88% negative predictive values in diagnosis of organic bowel diseases ( Table 3 ). Discussion Patients presenting to gastroenterologists with chronic abdominal pain or diarrhoea sometimes need extensive investigations to reach a final diagnosis. Colonoscopy with biopsy, the gold standard for IBD and other organic colonic disorders, is an invasive and expensive procedure. A normal colonoscopy can exclude the possibility of ulcerative colitis, however, if colonoscopy is normal and an organic disease (especially Crohn’s disease) is still suspected, even more expensive procedures as enteroscopy or capsule endoscopy can be used. Patients with warning signs – rectal bleeding, anaemia, weight loss, fever, family history of colon cancer, onset of first symptoms after 50 years of age and a major change in symptoms – need further evaluation including colonoscopy. In patients who meet the ROME criteria for diarrhoea predominant IBS and have no warning signs, the differential diagnosis should include coeliac disease, which should be excluded by serologic tests. If these tests are positive, gastroscopy with duodenal mucosal biopsy should be done [26] . This strategy has an acceptable cost only if the prevalence of coeliac disease is above 1% [27] . Accordingly, there is a need for a reliable, simple, cheap and non-invasive test to be used to discriminate patients with functional bowel disorders from those with organic bowel disorders in order to reserve the more invasive procedures to the later patient group. Our study showed that faecal calprotectin (FC) measurement is useful in discriminating patients with organic and function GIT disorders, supporting evidence of previous studies [28,29] . At a cutoff value of 50 μg/g, our results showed 100% sensitivity and 86% specificity with a positive predictive value of 82.6% and a negative predictive value of 100% in differentiating functional from organic GIT disorders. This means that a normal FCP almost excludes the presence of an organic bowel disease. These findings agree with previous results, which have suggested that FC has a good negative predictive value in excluding gut inflammation [11] . However, at a cutoff value of 100 μg/g the sensitivity and specificity of FC estimation in differentiating organic from function GIT disorders is 78% and 100% respectively, with a positive predictive value of 100% and a negative predictive value of 88%. As FC is a marker of intestinal inflammation and not disease specific, we found that it is significantly higher in patients with organic bowel diseases (IBD or non-IBD) compared to healthy controls or IBS patients. Among patients with organic bowel disease, IBD patients had significantly higher FC when compared to non-IBD patients, however, we could not determine a FC level that allows differentiation between both groups of patients. It has been shown that faecal calprotectin levels correlate with faecal excretion of 111 In-labelled granulocytes, the reference method for assessing intestinal inflammation [19] . Being cheap, easy to assess and without radiation hazards, it can be used to assess the severity of inflammation and response to treatment. In view of the high sensitivity and negative predictive value of FC assay in diagnosis of organic bowel diseases, we recommend that FC should be an integral component of the investigation of patients presenting with bowel disorders, especially diarrhoea predominant IBS. A normal FC precludes further investigation for organic bowel diseases. Serologic markers for IBD, i.e. ASCA and p-ANCA should be reserved for patients with high FC. In summary, we suggest that FC is a useful tool that can be used to discriminate between patients with functional and organic bowel disease, so that more invasive and expensive diagnostic procedures can be reserved for patients with a high probability of having organic bowel disease. 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