Introduction
Gastrointestinal (GI) complaints, such as abdominal pain, bloating, and diarrhea, are common symptoms in the general population and prevalent in the primary care setting, but may also be a presentation of various GI organic or functional diseases. Defining the underlying etiology is generally challenging and necessitates complementary diagnostic examinations, including blood tests, imaging, endoscopy and histology. The introduction of noninvasive diagnostic tools into clinical practice has been invaluable in the assessment of various GI symptoms and may ultimately decrease the use of invasive, complicated, expensive and potentially harmful procedures, such as endoscopy. In the last decade new markers have been extensively investigated with the aim of identifying a universal marker that can be introduced into clinical practice for primary care physicians and gastroenterologists. Fecal calprotectin (FC) appears to be the best candidate, as testing for levels of this protein has high accuracy, reproducibility and practicality, the test is easy to perform, and the results are simple to interpret. FC is a protein heterodimer (A100A8/A9) released by granulocytes, monocytes, macrophages and epithelial cells during an inflammatory response [1,2], with inflammatory cells accumulating in the mucosa. FC is subsequently released in the stool, from where it can be measured using a commercially available enzyme-linked immunosorbent assay, and it is resistant to metabolic degradation for up to 7 days at room temperature [3]. Roseth et al showed a good correlation between FC and the fecal excretion of indium-111-labeled granulocytes via scintigraphy in patients with inflammatory bowel disease (IBD) [4]. Several groups have investigated FC concentrations in healthy individuals and a cutoff of 50 μg/g for a positive test has been suggested, although a cutoff of 100 μg/g appears to have better accuracy for an IBD diagnosis [5], while factors such as type of assay and manufacturers’ recommendations should also be considered. Several factors affect FC levels, including acute GI infections, colon adenomas and malignancies, non-steroidal anti-inflammatory drugs, and age, since children under the age of 5 years have been found to have higher FC concentrations, probably due to higher intestinal permeability [6].
In this review we aim to present a concise outline of the current clinical experience of FC in non-IBD conditions of the GI tract commonly faced by the primary care physician, such as irritable bowel syndrome (IBS), microscopic colitis (MC), acute gastroenteritis (AGE), Clostridium difficile (C. difficile) infection (CDI), colorectal cancer (CRC), diverticular disease (DD), and celiac disease (CD), together with less common GI conditions (Tables 1 and 2). The role of FC in diagnosing and monitoring IBD has been extensively studied in recent years and there is a large body of data available in the literature; however, that subject is beyond the focus of this current review.
Table 1 Major findings of FC in various gastrointestinal conditions
Table 2 Summary of the studies reported on FC levels in in non-infectious gastrointestinal diseases
Role of FC in IBS
IBS and IBD share common symptoms, such as abdominal pain and diarrhea, thereby making a differential diagnosis challenging in clinical practice. A major concern for clinicians is misdiagnosing an organic disease as a functional one. FC is a validated biomarker for distinguishing between organic and functional cases, thus reducing the need for invasive tests, with all their associated negative consequences for patient safety and health system resources. Several systematic reviews and meta-analyses evaluating the role of FC in distinguishing between IBS and IBD have been published over the last decade. Von Roon et al performed a meta-analysis of 30 studies that included 5983 subjects investigated for various GI symptoms, including abdominal pain and changes in bowel habits. FC testing in all patients revealed a significant difference, with the mean value being 219 µg/g higher for IBD patients (P<0.001). A cutoff value of 50 µg/g had a sensitivity of 95% and a specificity of 91% in distinguishing between IBD and non-IBD subjects [7]. Waugh et al conducted a meta-analysis of 28 studies that analyzed the role of FC in diverse situations. In 730 patients from 7 studies, FC levels allowed a distinction to be made between IBS and IBD, with a sensitivity of 93% and specificity of 94% for a 50 μg/g cutoff value [8]. Buri et al found that an FC level >50 mg/g was capable of distinguishing between organic and non-organic GI conditions among 405 patients with heterogeneous gut symptoms who had been referred for colonoscopy, with a high sensitivity and specificity (84.4% and 94.5%, respectively) [9]. Similar results were also reported by Tibble et al in a study that included 602 patients with symptoms indicative of either IBS or IBD. FC levels in patients diagnosed with organic disease were found to be significantly higher than those in patients with non-organic disease, with a sensitivity and specificity of 89% and 79%, respectively, while the sensitivity of a positive ROME questionnaire for IBS was 85% with a specificity of 71% [10]. A study by Langhorst et al revealed that FC was higher in patients with active colitis (104 μg/g) compared to subjects who were ultimately diagnosed with IBS (19 μg/g) [11]. Consequently, due to the validated diagnostic accuracy of FC in differentiating between IBD and IBS, many physicians use levels of FC in clinical practice to screen for IBD in patients with symptoms of IBS in order to reduce the use of unnecessary investigations [12].
Since IBS is a multifactorial disease, low-grade inflammation is emerging as a potential mechanism that contributes to the pathogenesis of the disease, particularly in the post-infectious IBS subgroup. Therefore, theoretically, the implementation of FC as a surrogate marker of intestinal inflammation could represent an attractive tool for identifying IBS patients who may benefit from anti-inflammatory therapy. However, a randomized controlled study by Lam et al that tested the effect of the anti-inflammatory drug mesalazine in IBS patients yielded negative results [13]. A recent prospective study of 93 patients with IBS by Melchior et al aimed to characterize patients with increased FC levels using different factors, such as age, sex, disease subtype and disease severity, as well as diagnostic workup findings, such as breath tests, distension as measured by a rectal barostat, and assessment of inflammation in colonic biopsies. FC levels were found to be higher in one third of patients, and age was the only factor that correlated with this patient group. No other associations to clinical phenotype or workup findings, including inflammation in colonic biopsies, were correlated with increased FC levels [14]. Taken together, these findings indicate that FC has an important role in distinguishing between IBD and IBS, although its role in identifying IBS patients who may benefit from anti-inflammatory therapy requires further randomized controlled studies. Furthermore, it is important to note that, in patients with a high FC level diagnosed with IBS, another cause other than IBS should be suspected. Importantly, the patient’s clinical presentation and scenario are the most important parameters in decision making and diagnostic plan setting, especially when FC is only mildly elevated.
Role of FC in MC
MC is characterized clinically by watery diarrhea in old women and histopathologically by microscopic inflammation with abnormal sub-epithelial collagen deposition and lymphocyte infiltration. Generally, endoscopy shows a normal colon and biopsy is mandatory for establishing a diagnosis. The role of FC in MC was studied by Wildt et al in 21 patients with active MC, 12 patients with MC in remission, and 13 healthy subjects; the corresponding FC levels were 80 μg/g, 26 μg/g and 6.25 μg/g, respectively [15]. Patients with active MC had significantly higher levels of FC compared with the remission and control groups (P=0.025 and P=0.02, respectively). A more recent prospective study by Von Arnim et al measured FC levels in 23 patients with MC (16 in clinical remission and 7 with active disease) and compared the results to FC levels in patients with IBS. It was reported that FC levels were significantly higher in the active MC group compared with the remission and IBS groups (P<0.0001) [16].
The increased level of FC in MC is interesting because of the scarcity of neutrophils; however, FC could originate from macrophages and monocytes. Despite these results obtained to date, the role of FC in diagnosing and managing MC remains to be validated by future multicenter studies, although higher levels of FC appear to be a surrogate marker of active MC.
Role of FC in AGE
AGE is a prevalent clinical presentation in both the adult and pediatric populations in the primary care setting. Generally, these bouts of illness are self-limiting and do not require investigation or specific management. Nonetheless, some clinical scenarios—such as a severe clinical presentation and symptom onset in a hospitalized or immunocompromised patient, amongst others (high-risk patients)—may mandate evaluation, and in such cases it is critical to differentiate between viral and bacterial infections. Stool culture is considered the mainstay for diagnosing bacterial AGE, but the length of time needed to acquire the results and other practical limitations has led to other diagnostic tools being sought. The role of FC has been investigated in AGE and yielded some promising results. Shastry et al conducted a large prospective, multicenter study of 2383 patients who presented with AGE, with the aim of determining a correlation between bacterial AGE (diagnosed by stool culture) and FC levels. The results were impressive, with sensitivity and specificity of 83% and 87%, respectively, for FC levels in diagnosing bacterial infections [17]. These results have also been confirmed in adult populations, where FC levels were found to be higher during a bacterial infection and were correlated with disease severity [18,19]. However, the role of FC in the management of AGE is limited by the acute and self-limited nature of these conditions and more research is needed to better address the utility of FC in identifying bacterial infection in high-risk patients.
Role of FC in CDI
C. difficile is a potentially dangerous pathogen that causes nosocomial and community-acquired diarrhea, and is generally associated with morbidity and mortality. Treatment outcomes are related to infection severity; consequently, an optimal severity assessment is crucial for optimizing therapy and achieving the best outcomes. Kim et al measured FC levels in 80 patients with acute CDI; 50 patients had mild CDI and 30 had severe CDI, based on the CDI severity assessment score developed by Zar et al [20], and the results were compared with those in a control group of 71 patients. FC levels were found to be significantly higher in patients with severe CDI compared to mild CDI (P<0.001) and to the control group (P<0.001), and the implementation of FC levels as a predictive marker for assessing CDI severity was proposed [21]. Peretz et al confirmed these results and noted an additional interesting finding that higher FC levels were associated with virulent ribotype 027 C. difficile strains [22].
Role of FC in CRC
The role of FC in CRC diagnosis and screening was first examined in 1993 by Roseth et al, who found that levels of FC were significantly higher in 94.3% of patients with CRC [23]. A later study in 2001 by Tibble et al compared FC levels to fecal occult blood tests in 62 patients with CRC and reported that the sensitivity and specificity of FC levels for CRC were 79% and 72%, respectively [24]. A more recent study by a Hungarian group, who measured 5 biomarkers including FC in 95 patients with CRC, found a sensitivity of 77.8% and specificity of 70% for FC in the diagnosis of CRC [25]. However, 2 other studies have shown significantly lower sensitivity and specificity for FC in diagnosing CRC [26,27]. Therefore, the role of FC in CRC diagnosis and screening remains the subject of debate and requires further investigation, although CRC should be considered when there are GI symptoms accompanied by high FC levels.
Role of FC in DD
Given the inflammatory nature of DD and the lack of noninvasive biomarkers for diagnosing and monitoring the disease, FC is an attractive candidate warranting further investigation. Tursi et al measured FC levels in 48 patients with DD (16 with asymptomatic DD, 16 with symptomatic uncomplicated DD, and 16 with acute uncomplicated DD), and FC levels were compared to 16 IBS patients and 16 healthy controls. FC levels were found to be higher in patients with symptomatic uncomplicated DD (P<0.005), and in patients with acute uncomplicated diverticulitis (P<0.0005) compared with the healthy controls, IBS and asymptomatic DD patients. Moreover, when FC levels were assessed 8 weeks after treatment with mesalazine and rifaximin, they were completely normal in the groups with symptomatic uncomplicated DD and acute uncomplicated DD. It was concluded that FC levels may have a practical use in detecting inflammation and response to treatment in DD patients [28]. In addition, a further review study by Tursi et al found that FC >60 μg/g was correlated with acute complications of DD [29].
Role of FC in CD
Few studies have assessed FC levels in adult and pediatric patients with CD, and discordant results have been reported. Montalto et al measured FC levels in 28 adults with untreated CD and compared the results to FC levels in 30 healthy volunteers. Although FC levels were only slightly higher in CD patients compared with the healthy volunteers, a trend towards higher FC levels in CD was observed [30]. However, different results were obtained from a pediatric population in Turkey when 3 groups were studied: 31 children with untreated CD, 33 children with CD who adhered to a gluten-free diet, and 43 healthy controls. FC levels were found to be significantly higher in the untreated CD group compared to treated CD patients (P<0.001) and to the controls (P<0.001). An additional interesting finding was the significant decrease in FC levels reported after switching to a gluten-free diet in the untreated group (P<0.01). It was concluded that FC can assist in diagnosing CD and also in monitoring adherence to a gluten-free diet [31]. Another pediatric study by Ertiken et al found higher levels of FC in 29 newly diagnosed children with CD in comparison to 10 controls (P<0.04) [32]. Similarly, Capone et al measured FC concentrations in 50 newly diagnosed CD adults and compared the results to 50 controls. FC levels were examined in relation to clinical symptoms, histology findings and antibody levels for tissue transglutaminase, but a negative correlation was reported between FC levels and symptoms, histology and antibody levels; it was concluded that FC is irrelevant in CD workup and management [33]. Consequently, the role of FC in CD diagnosis and monitoring remains ambiguous, although CD should be considered as a cause of high FC levels in the pediatric population.
Role of FC in other GI diseases
The potential role of FC has been studied in many other GI conditions; however, most of these studies involved small numbers of patients and revealed inconclusive or contrary results. Levels of FC have been measured in patients with diarrhea following a hematopoietic stem cell transplant when they were suspected of developing GI graft versus host disease (GVHD), but contradictory results were reported by 2 different studies, although FC levels seemed to be higher in more advanced GVHD [34,35]. Pezzilli et al measured FC levels in patients with pancreatic disease, aiming to identify a possible link to intestinal inflammation, and found that 50% of the 20 patients with chronic pancreatitis had higher FC levels [36]. Khalid et al studied the potential of FC levels in diagnosing radiation enteritis in 59 patients undergoing pelvic radiotherapy for different malignancies. FC levels were measured at baseline and 5 weeks after treatment onset, but the increase was insignificant [37]. Raia et al studied the concentration of FC in 30 pediatric patients with cystic fibrosis; FC levels were reported to be significantly higher compared to controls (P<0.01) and significantly lower compared to 15 patients with IBD (P<0.001) [38]. Further studies have linked higher levels of FC to small intestinal bacterial overgrowth [39], food allergy, gastroesophageal reflux disease [40], hepatic encephalopathy in patients with liver cirrhosis [41], and iatrogenic causes, such as gastric bypass surgery [42].
Concluding remarks
Because of its positive attributes, the evaluation of FC as a biomarker in clinical practice for the diagnosis and monitoring of several GI disorders has yielded encouraging results. Its potential utilization is currently being investigated by many research groups from different fields and a significant body of data has emerged in recent years. Currently, the best available evidence in the literature supports its use in IBD diagnosis and monitoring, as well as in distinguishing between IBD and IBS. The increased level of FC in MC is interesting because of the scarcity of neutrophils. Despite these results obtained to date, the role of FC in diagnosing and managing MC remains to be validated by future multicenter studies, although higher levels of FC appear to be a surrogate marker of active MC. FC levels were found to be significantly higher in patients with severe CDI compared to mild CDI and to the control group, and the implementation of FC levels as a predictive marker for assessing and stratifying CDI severity has been proposed. FC levels may have a practical use in detecting inflammation and response to treatment in DD patients. In addition FC >60 μg/g was correlated with acute complications of DD. Knowledge is evolving concerning its potential application in various GI conditions, such as CD, CRC, GVHD and others, and further studies are required to address the many uncertainties which remain and to better define the use of FC levels in clinical practice.