Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Gastroenterology
Fecal Tumor Necrosis Factor-α and Calprotectin as Differential Diagnostic Markers for Severe Diarrhea of Small Infants
Kapel, N*; Roman, C†; Caldari, D†; Sieprath, F†; Canioni, D‡; Khalfoun, Y‡; Goulet, O†; Ruemmele, F M†
*Laboratory of Functional Coprology, Pitié-Salpétrière Hospital, Paris France; †Pediatric Gastroenterology Unit, Department of Pediatrics, Necker-Enfants Malades Hospital, University René Descartes, Paris, France; and ‡Department of Pathology, Necker-Enfants Malades Hospital, University René Descartes, Paris, France
Received June 6, 2005; accepted July 8, 2005.
Address correspondence and reprint requests to Dr. F. M. Ruemmele, Pediatric Gastroenterology Unit, Hôpital Necker-Enfants Malades, INSERM EMI0212, Necker Faculty of Medicine, University René Descartes, Paris V, 149 Rue de Sèvres, F-75743 Paris, Cedex 15, France. (e-mail: firstname.lastname@example.org).
Objective: The etiology of early-onset diarrhea of neonates and small infants that persists despite bowel rest is heterogeneous. Two different categories of disorders presenting with diarrhea in the first weeks of life can be distinguished: constitutive intestinal epithelial disorders (microvillus atrophy [MVA] or epithelial dysplasia [ED]) and immune-inflammatory disorders, (autoimmune enteropathy [AIE] or inflammatory colitis [IC]). We aimed to evaluate in a prospective manner the use of fecal inflammatory markers in the differential diagnosis of severe persistent diarrhea.
Material and Patients: Twenty-five patients (17 males) were enrolled in this study (median age 8 months). Fourteen children had a constitutive enterocyte disorder (group 1: MVA = 8, ED = 6), and 11 patients had an immuno-inflammatory disease (group 2: AIE = 5, IC = 6). Stool samples were collected at the time of diagnosis and stored at −80° until tumor necrosis factor (TNF)-α and calprotectin were measured by enzyme-linked immunoadsorbent assay.
Results: No significant differences in age at onset of diarrhea or in stool volumes were observed between both groups. In group 1, fecal TNF-α was undetectable/normal in 14 of 14 children, whereas group 2 showed dramatically elevated TNF-α levels (mean 3,104, range 237-18,078 pg/g) in 8 of 11 patients. Similarly, calprotectin levels were undetectable/normal in 14 of 14 patients in group 1 and highly raised in 11 of 11 patients in group 2 (median 1,145, range 375-3,095 μg/g), P < 0.01. Under therapy, these inflammatory parameters normalized.
Conclusions: Determination of fecal inflammatory markers is a simple method helping to distinguish constitutive from immuno-inflammatory etiologies of severe persistent diarrhea. These data also suggest that constitutive enterocyte disorders are not accompanied by an inflammatory mucosal reaction.
Severe early-onset neonatal diarrhea that persists despite bowel rest most often results in a tremendous, rapidly life-threatening clinical situation (1). An immediate and precise diagnosis is indispensable for optimal management without losing any valuable time. Over the last few years, major advances were made, allowing for identification of different causes of severe protracted diarrhea in neonates and small infants (1,2). Whereas in the past, this clinical picture was also called intractable diarrhea of infancy because its origins remained unexplained and no therapeutic concepts were available, several new treatment strategies emerged for a subgroup of patients (1,3). To date, two main types of persistent diarrhea with onset within the first weeks or months of life can be distinguished: constitutive intestinal epithelial disorders, such as microvillus atrophy (MVA) or epithelial dysplasia (ED), and immune-inflammatory disorders, such as autoimmune enteropathy (AIE) or inflammatory colitis (IC) (3-5).
Constitutive intestinal epithelial cell disorders are classically characterized by secretory diarrhea with onset within the first weeks of life or later as atypical forms, whereas intestinal immune-inflammatory diseases can manifest at any time point during the first months of life with an extremely variable presentation (3). Therefore, the clinical picture of a neonate or infant presenting with major secretory diarrhea persisting despite complete bowel rest on total parenteral nutrition (TPN) alone does not allow the attribution to a distinct etiology, and complementary examinations are mandatory to rapidly obtain a definitive diagnosis.
An easy approach in the diagnostic work-up of intestinal disorders is the measurement of fecal parameters. In inflammatory disorders, an increase in fecal tumor necrosis factor (TNF)-α levels was shown to closely correlate with the inflammatory activity of the intestinal mucosa (6). More recently, fecal calprotectin has been proposed as a reliable marker of the presence of malignant or inflammatory diseases in the gastrointestinal tract in adults and children (7-10). Calprotectin is a 36.5 kDa calcium and zinc binding protein found in human neutrophil granulocytes. In contrast with TNF-α, calprotectin is highly stable in the feces for up to 7 days at room temperature because its calcium bound form resists any proteolytic degradation and heat (11).
In the present study, we analyzed the diagnostic value of fecal parameters in neonates and infants presenting with severe diarrhea persisting on bowel rest. In children with an immune-inflammatory disease, fecal TNF-α and calprotectin levels were highly increased, whereas they remained undetectable/normal in constitutive epithelial disorders. In the present series, fecal calprotectin measurements had a 100% negative predictive value, with negative measurements indicating a primary enterocyte disorder.
PATIENTS AND METHODS
This study enrolled 25 children in a prospective manner at the time of definitive diagnosis, 14 with enterocyte disorders, that is, 6 with ED (median age 8 months, range 6-21) and 8 with MVA (median age 4 months, range 3-20), 5 with AIE (median age 5 months, range 2-7), and 6 with IC (median age 9 months, range 1-18). The definitive diagnosis (MVA, ED, AIE, or IC) was based on standard endoscopic and histologic criteria, and all children with an unclear diagnosis were excluded from this study. Histologic analyses were performed on hematoxylin-eosin and periodic acid Schiff stained sections. In parallel, six age-matched normal healthy controls (median age 9 months, range 2-18 months) were included to revalidate the normal reference values for calprotectine.
For each child, approximately 5 g of feces was collected and stored immediately at −80°C until assayed. Only correctly stored samples were analyzed. Immediately before analysis, frozen stool samples were thawed at room temperature. Fecal TNF-α was determined using an enzyme-linked immunoadsorbent assay (ELISA) kit adapted for fecal samples (TNF-α EASIA, Biosource, Fleurus, Belgium) as previously described (12). With use of this methodology, the limit of detection is 30 pg/g of stools. Fecal calprotectin concentration was determined using a commercial ELISA (Calprest, Eurospital, Italy) with a limit of detection of 15 μg/g of stools, according to the instructions of the manufacturer. In normal control cohorts of children, TNF-α levels are considered normal if they are inferior to 90 pg/g (95% confidence interval [CI]) and calprotectin values remain below 300 μg/g (95% CI) during the first year of life (13,14).
Results are given as median and ranges. Data were analyzed using the StatView statistical software package for Windows (Cary, NC). Comparisons between the results obtained in the different groups were performed using Mann-Whitney U test.
In all 25 patients, the definitive diagnosis causing severe diarrhea was based on complete endoscopic and subsequent histologic analysis. Only children with a clear-cut diagnosis were enrolled, allowing the attribution to one of the four subgroups (ED and MVA [based on the morphologic analysis showing epithelial tufts (ED) and enlarged PAS positive bands (MVA)] or AIE and IC [based on the presence of positive anti-enterocyte and anti-AIE 75 kDA antibodies along with a major mononuclear cell infiltration of the mucosa and the occurrence of epithelial cell apoptosis (AIE) (3) and the inflammatory mononuclear infiltrate of the mucosa with crypt abscesses and destruction in IC]).
Onset of diarrhea was within the first 3 months for all except one girl with AIE who presented at the age of 5 months (Table 1). There was no significant difference in age at onset of diarrhea between constitutive epithelial or immune-inflammatory disorders (Table 1). However, there was a tendency toward an earlier onset of digestive symptoms in children with MVA (i.e., within the first 3 weeks of life in 7/8 patients). As shown in Table 1, no significant differences in stool volumes on bowel rest were observed between the four different pathologies. The volume of diarrhea varied markedly within one subgroup (interindividual variation), and also intra-individually, with variations up to 100% on another day.
In contrast with the onset of diarrhea and stool volumes, fecal inflammatory parameters significantly differed between the four groups. TNF-α was measured in all 25 children at the time of diagnostic work-up. Similar to the control group, TNF-α was undetectable in six of six children with ED and seven of eight children with MVA. In only one boy with MVA% TNF-α level of 33 pg/g were measured (Fig. 1). In children with AIE, however, TNF-α levels were increased to median levels of 1,660 (range 237-3,082) pg/g; similarly in children with IC, TNF-α levels were tremendously enhanced (median 3,200 pg/g, range 1,450-18,078 pg/g). In two patients with IC and two patients with AIE, fecal TNF-α was not detectable, despite highly elevated calprotectin values in the same specimen. This is probably because of the rapid proteolytic degradation of TNF-α. Therefore, the difference between primary epithelial (group 1) and immune-inflammatory disorders (group 2) did not reach statistical significance (P = 0.068) (Fig. 1).
In parallel, fecal calprotectin levels were measured in all 25 children. It was under detection limit in 12 of 14 children with constitutive epithelial disorders and within normal limits (62 and 136 pg/g) in 1 child with ED and another child with MVA, respectively. The age-matched control children of 2 and 6 months had calprotectin levels of 241 and 156 μg/g, respectively. In contrast, high calprotectin levels were observed in all children with AIE (median 460 μg/g, range: 375-1,192 μg/g) and all children with IC (median 1,206 μg/g, range 510-3,095 μg/g). These levels were significantly higher compared with children with constitutive epithelial disorders (P < 0.01 for AIE and IC) (Fig. 2). The sensitivity of fecal calprotectin measurement in the differential diagnosis between constitutive epithelial (group 1) or immune-inflammatory disorders (group 2) causing severe diarrhea was 100% with 100% specificity a negative predictive value of 100%.
Longitudinal follow-up analyses in six of nine patients with immune-inflammatory disorders showed a rapid decrease of fecal markers in all patients upon onset of anti-inflammatory and immunosuppressive therapy (Fig. 2). Clinical remission was paralleled by a complete normalization of fecal calprotectin in all patients. The one patient who came into partial remission on initiation of treatment showed a clear increase of fecal inflammatory markers, which remained elevated.
The pathogenesis of early-onset severe diarrhea is complex. To date, at least four different clearly distinct etiologies are recognized that cause major and persistent diarrhea in neonates and small infants. The onset is most often tremendous, and all children require TPN for initial stabilization (1,15). Whereas no curative treatment options can be proposed for children suffering from constitutive epithelial disorders such as ED or MVA, children with AIE or IC can often be stabilized by anti-inflammatory and immunosuppressive medication (3). Therefore, a rapid and noninvasive test discriminating these different entities is most helpful in the initial work-up. In the present study, we observed that mode and time of onset of diarrhea does not help in the differential diagnosis; however, the simple measurement of fecal inflammatory parameters, such as TNF-α or calprotectin, allowed us to distinguish with 100% specificity between immune-inflammatory and constitutive epithelial disorders. This marked difference does not account for dilution of the fecal fluids secondary to the massive diarrhea because stool volumes are comparable in both groups, epithelial and immune-inflammatory.
Elevated fecal calprotectin concentrations (but not TNF-α) have been previously described during the first months of life compared with older children or adults (13,14). These increased calprotectin values have been related to an enhanced permeability of the gut during the first weeks of age, which may be associated with increased migration of granulocytes into the gut lumen. This inflammatory reaction might be in response to the initial colonization of the gut. In the following patients the process may be completely down-regulated. In patients with ED, an altered epithelial barrier, probably secondary to the deformation of the epithelium, was discussed. It is interesting to observe normal and even low calprotectin values in our patients with ED, which does not further support the hypothesis of an altered epithelial barrier function in ED causing chronic mucosal inflammation in ED. We did not observe any increase in inflammatory fecal parameters in ED (data not shown) either at the onset of disease or within the first months of evolution.
Calprotectin and TNF values were dramatically increased in neonates and small infants with immune-inflammatory disorders. These data reflect the accumulation of calprotectin-rich granulocytes and mononuclear cells within the mucosa, confirming the correlation of calprotectin levels with mucosal inflammation (16). Because TNF-α is highly susceptible to degradation (17), calprotectin measurement might be preferable in the diagnostic work-up of diarrhea and more appropriate to the clinical setting. In summary, routine measurement of fecal inflammatory parameters in neonates and infants presenting with severe secretory diarrhea persisting despite bowel rest proved to be useful in the differential diagnosis stratifying constitutive intestinal epithelial disorders from immune-inflammatory disorders, the latter being highly responsive to anti-inflammatory or immunosuppressive regimens. Therefore, the mere presence of inflammatory parameters in the stool indicates the need for early endoscopy and subsequent histologic analysis with the hope for a causal therapy. The negative predictive value of measuring fecal inflammatory parameters is 100% with absent inflammatory fecal parameters orientating toward constitutive epithelial disorders, which to date are not responsive to any medical therapy and require long-term parenteral nutrition or intestinal transplantation (15).
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