The art of diagnosis has increasingly moved away from the clinician because of advances within other branches of medicine, which have conventionally been viewed as support services (e.g., microbiology, radiology, clinical biochemistry). In clinical biochemistry, the knowledge explosion in molecular biology has begun to alter the direction of service provision. Nevertheless, there remains a place for development of ideas and techniques along conventional lines to detect or characterize disease. Gastroenterologists caring for adults have been predictably slow to accept some of these developments and continue to rely heavily on direct visualization and sampling of the intestine, despite the fact that this only provides a static morphologic picture with little or no functional information. Pediatric gastroenterologists on the other hand do not have the same privilege of unrestricted use of invasive procedures.
The general philosophy underlying the development of noninvasive gastroenterological tests is to provide a reliable method for the assessment of intestinal physiology and function; the detection of intestinal disease either on an individual patient basis or as part of a screening program; the confirmation of diagnosis and monitoring response to therapy (e.g., gluten withdrawal and challenge in coeliac disease); to provide prognostic information; and to assess the etiology and pathogenesis of various intestinal diseases.
The prototype of such tests was the development of methods for the noninvasive assessment of intestinal permeability (1). These tests are now widely used for research purposes, but clinical acceptance has been slow because of perceived nonspecificity of the results, the complexity of test marker analysis, confusion over optimal test dose composition, the need for timed urinary collections, and the fact that symptomatic small bowel diseases are rare (2).
Analysis of fecal markers, however unpleasant this may seem, is a rapidly evolving field. This approach has the potential to provide information on the whole gastrointestinal tract or on the function of the pancreas or liver. Assessing markers of inflammation (or surrogate markers) in feces is of particular interest because many intestinal diseases have an inflammatory component that may be difficult to assess because of the location and patchy distribution of the disease. Fecal tests are, of course, not new. Initially there was direct microscopy of stool for neutrophil content, followed by chemical analysis for various compounds, osmolarity, among others. More recently, measurement of inflammatory markers (such as tumor necrosis factor α (TNF-α), neutrophil esterase, interleukins) has been advocated, but these methods suffer from the degradation of the markers within the intestine. Analysis of markers in whole-gut lavage improved diagnostic yield significantly (3), but the ingestion of large quantities of polyethylene glycol–based purgatives was problematic unless one lived in Edinburgh!
The introduction of indium-111-labeled white cells (and later technicium-99 labeling), which allows accurate localization and quantitation (applies to indium labeling only) of intestinal inflammation was a major landmark (4). However, this method, which involved complete 4-day fecal collections, was demanding on patients and was associated with substantial radiation exposure and cost, and its use has, therefore, largely been limited to a few research centers. Following on from this, an attempt was made to identify other neutrophil-specific markers in stool. Ideally, such a marker should not be subject to bacterial degradation, but, if degradation occurs, the measured epitope of the molecule should remain intact. Two such markers have been identified in human subjects: lactoferrin (5), which is found predominantly within neutrophil granules and can be measured by radioimmunoassay; and calprotectin. Calprotectin is a stable protein that accounts for about 60% of the neutrophil cytosolic protein and is quantitated by an enzyme-linked immunosorbent assay (ELISA) method (now commercially available) in stool extracts (6).
Initial validation in adults showed minimal bacterial degradation, a significant correlation between fecal excretion of calprotectin (daily excretions and single stool concentrations), and the excretion of neutrophils as assessed by the indium-111 white cell 4-day fecal excretion (7,8). There were also good correlations between fecal calprotectin and histopathologic disease activity in ulcerative colitis (9) and clinical disease activity in Crohn disease. As a screening test, it has high sensitivity in adult patients for the detection of active inflammatory bowel disease (IBD).
In this issue of the Journal of Pediatric Gastroenterology and Nutrition Bunn et al. (10) present their second “validation” article on the value of fecal calprotectin measurements in children. Previously, they had shown that the range of single-stool calprotectin concentrations in healthy children (aged 2–15 years) was 0.5 mg/L to 6.3 mg/L, comparable with the range in adults of 0.5 mg/L to 11 mg/L (95% confidence limits) (11). Elevated fecal calprotectin concentrations were evident in 92% of IBD patients with moderate clinical disease activity, and overall, there was a significant correlation (r = 0.61;P < 0.001) between calprotectin concentrations and clinical disease activity, comparable with the results in adults (8). The group has now taken a further step by correlating fecal calprotectin concentrations with accepted gold standards of “invasive” disease activity indices (histopathology and technicium-99 white cell scanning). Overall, the performance of the calprotectin technique was excellent and certainly correlated well with the results of the invasive tests.
Despite the limited data available in children, it is clear that the method has immense potential. If we look at the global potential of screening tests, one can immediately suggest a number of academically and clinically interesting studies involving the calprotectin method in children.
Assessment of Intestinal Physiology and Function
Assessment can be used to determine whether establishment of the normal comencal intestinal flora postpartum is associated with an inflammatory component, which may have relevance for “oral tolerance” and “autoimmunity.” Neonates exhibit increased intestinal permeability in the first few days of life, and it would be interesting to see whether this pre “gut closure” period is associated with intestinal inflammation.
Detection of Intestinal Disease
In adults, the calprotectin method had a sensitivity of 100% and a specificity of 97% in discriminating between Crohn disease and irritable bowel syndrome (8). In a group of 220 patients with a differential diagnosis of Crohn disease or irritable bowel syndrome and who were referred by general physicians to a gastroenterology outpatient clinic, measurement of fecal calprotectin could have avoided the performance of more than 200 colonoscopy, barium enema, or follow-through examinations. In children, the corresponding differential diagnoses might be between Crohn disease and recurrent abdominal pain. Children with recurrent abdominal pain, however, unlike adults with irritable bowel syndrome, have a high prevalence of increased intestinal permeability (12). If they additionally had evidence of intestinal inflammation, this would very be strong evidence against this syndrome being a psychosomatic or behavioral disorder.
It is important to note that the calprotectin technique is inflammation specific and not disease specific. In this context, it is of interest that increased intestinal permeability and inflammation are inter-related; one can be the cause of the other (13). A number of intestinal diseases in the pediatric population are associated with increased intestinal permeability, (2) and therefore, it is likely that these will also be associated with intestinal inflammation. Children with cystic fibrosis have increased intestinal permeability (14) and these children have recently been shown to have intestinal inflammation independent of fibrosing colonopathy (15). Children with infectious enteritis will almost certainly and predictably have increased fecal calprotectin concentrations, as will children taking nonsteroidal antiinflammatory drugs, those undergoing chemotherapy, immune compromised subjects, or those experiencing renal failure. However, the inflammation would be expected to be low grade, if one extrapolates the data from adults, and is easily differentiated from the 10-fold or greater inflammatory response seen in acute IBD. There remain a number of diseases in which intestinal permeability is increased (for instance, diabetes, malnutrition, liver disease, food allergy) but in which the degree of intestinal inflammation has not been established.
Monitoring Response to Therapy and to Provide Prognostic Information
In adults, and now evident in children, fecal calprotectin measurements provide an objective index of disease activity in IBD. In adult, fecal calprotectin concentrations decrease dramatically after successful treatment of Crohn disease with elemental diet, tumor necrosis factor antibodies, and corticosteroids (unpublished data). In asymptomatic adults with IBD, a high calprotectin concentration identifies those at significant risk of imminent clinical relapse of disease. This offers the possibility for targeted treatment of high-risk patients at an asymptomatic stage, which may be associated with fewer side effects than if a clinical relapse occurs. It also raises the wider question of whether we should treat the “inflammatory” component of IBD, irrespective of clinical symptoms, similar to what is done in patients with rheumatoid arthritis.
To Assess the Etiology and Pathogenesis of Various Intestinal Diseases
The role of intestinal inflammation in the pathogenesis of juvenile arthritis and spondylarthropathy is of major interest, as is the suggestion that childhood vaccinations are associated with intestinal inflammation, as suggested from the autistic enterocolitis theory. Both are eminently suitable areas for further studies on fecal calprotectin in the pediatric population.
This represents only a few examples of relevant research lines in the pediatric population. There is clearly scope for a number of other academically and clinically interesting studies involving fecal calprotectin measurements in children. Furthermore, as in adults, the technique has the potential for replacing invasive diagnostic screening tests for and it provides an objective disease activity index of intestinal inflammation in IBD. The test is exceedingly easy to carry out using as little as 100-mg portions of stool and, if the enzyme-linked measurements are batched ,the consumable cost, including the kit, is less than £20 per sample ($30 per sample). In our hospital-based gastroenterology outpatient practice, we routinely send out a collection pot and instructions to all new outpatients 4 weeks before they are seen. More than 90% of adult patients do the test without difficulty, and having the result at the first consultation has greatly facilitated treatment and investigation of these patients.
1. Travis S, Menzies IS. Intestinal permeability: functional assessment and significance. Clin Sci 1992; 82: 471–88.
2. Bjarnason I, Macpherson AJM, Hollander D. Intestinal permeability: An overview. Gastroenterology 1995; 108: 1566–81.
3. Handy LM, Ghosh S, Ferguson A. Investigation of neutrophil migration into the gut by cytology of whole gut lavage fluid. Eur J Gastroenterol Hepatol 1995; 7: 53–8.
4. Saverymuttu SH, Peters AM, Lavender JP, et al. Quantitative faecal indium-111 labelled leukocyte excretion in assessment of disease activity in Crohn's disease. Gastroenterology 1983; 85: 1333–9.
5. Guerrant RL, Araujo V, Soares E, et al. Measurement of fecal lactoferrin as a marker of fecal leukocytes. J Clin Microbiol 1992; 30: 1238–42.
6. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992; 27: 793–8.
7. Roseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of Indium-111-labelled granulocytes and Calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol 1999; 34: 50–4.
8. Tibble J, Teahon K, Thjodleifsson B, et al. A simple method for assessing intestinal inflammation in Crohn's disease. Gut 2000; 47: 506–13.
9. Roseth AG, Aadland E, Jahnsen J, et al. Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein. Digestion 1997; 58: 176–80.
10. Bunn SK, Bisset WM, Main MJ, et al. Fecal calprotectin: validation as a noninvasive measure of bowel inflammation in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2001; 33: 14–6.
11. Bunn SK, Bisset WM, Main MJ, et al. Fecal calprotectin as a measure of disease activity in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2001; 32: 171–7.
12. Van Der Meer SB, Forget PP, Heidendal GA. Small bowel permeability to 51Cr-EDTA in children with recurrent abdominal pain. Acta Paediatr Scand 1990; 79 (4): 422–6.
13. Bjarnason I, Macpherson AJS, Somasundaram S, et al. Non-steroidal anti-inflammatory drugs and Crohn's disease. In: Scholmeric J, Kruis W, Goebbell H, et al., eds. Inflammatory Bowel Diseases: Pathophysiology as Basis of Treatment.
Falk Symposium No 67. Lancaster: Kluwer Academic Publishers; 1993: 208–22.
14. Leclercq-Foucart J, Forget P, Sodoyez-Gouffaux F, et al. Intestinal permeability to 51CrEDTA in children with cystic fibrosis. J Pediatr Gastroenterol Nutr 1986; 5: 384–7.
15. Smyth RL, Croft NM, O'Hea U, et al. Intestinal inflammation in cystic fibrosis. Arch Dis Child 2000; 82: 394–9.