To the Editor: Increased local mucosal production of tumor necrosis factor-α (TNF-α) was reported, especially in inflammatory bowel disease, and it was shown that TNF-α concentrations in stool correlated with disease activity (1,2). Tumor necrosis factor-α in stool seems to come from two sources: from leakage across the epithelium after local synthesis in the lamina propria (3) and from neutrophils and macrophages containing TNF-α passing into the lumen and releasing their cytoplasmic cytokine on lysis (1).
The role of cytokines in the pathogenesis of enteric infections is still not known. On the basis of observations in inflammatory bowel disease and of the findings of nondetectable proinflammatory cytokine concentrations in stools in pathogen-negative diarrhea and in stools without diarrhea (2), we hypothesized that detectable TNF-α and interleukin-6 (IL-6) concentrations in stool may simply reflect their involvement in the pathogenesis of gastroenteritis and may provide a simple way to differentiate the type of gastroenteritis in children when microbiologic diagnosis is impossible or delayed.
Stools of 63 children with diarrhea or enteritis were collected. However, pathogenic infectious agents were definitely diagnosed in only 27 of them. Thus, 27 stool samples from patients with rotavirus (n = 17) and bacterial gastroenteritis (n = 10) were included in this preliminary study. Bacterial pathogens were Shigella flexneri (n = 2), Shigella sonnei (n = 3), Salmonella enteritidis (n = 2), and Salmonella typhi murium (n = 3). An enzyme-linked immunosorbent assay (ELISA) (Enzygnost Rotavirus, Behring, Germany) was used in the detection of rotavirus.
In addition to diarrhea 6 to 15 times a day, patients with rotavirus gastroenteritis had slightly elevated fever (82%), vomiting (58%), and moderate dehydration (23%), but no leukocytes or blood in stools. Patients with bacterial gastroenteritis had highly elevated fever (>38.5 °C) (70%), vomiting (50%), mild or moderate dehydration (40%), leukocytosis (80%), positive C-reactive protein (60%), and blood and leukocytes in the stools (70%).
Stools (10-20 g) were collected in sterile containers and weighed. Samples were then suspended in an equal volume of sterile phosphate-buffered saline (PBS) (1 g stool in 1 ml PBS) and centrifuged at 20,000g. Supernatant was stored at -70 °C until assay. Tumor necrosis factor-α and IL-6 were measured by ELISA and commercially available kits (Amersham, UK). The minimum detectable concentrations of TNF-α and IL-6 by this method are 0.1 pg/ml. The assays have no cross reactivity with the other cytokines. Inter- and intraassay variables were less than 10%. Samples were examined in duplicate. Results are presented as picograms per gram of stool. The significance of differences were calculated with the Mann-Whitney test.
Median TNF-α concentrations per gram of stool in children with rotavirus gastroenteritis and bacterial gastroenteritis were 10.37 pg/g (range 4.17-254 pg/g) and 129.17 pg/g (range 4.69-508 pg/g), respectively (Table 1), and there was a significant difference between the two groups (p < 0.05). Tumor necrosis factor-α concentrations were detectable in stools of all patients. A total of six samples (three in each group) showed nondetectable IL-6 levels. IL-6 concentrations in stools of children with bacterial gastroenteritis (median, 109.3 pg/g; range, nondetectable to 328 pg/g) were significantly higher than in stools of those with rotavirus gastroenteritis (median, 3.29 pg/g, range, nondetectable to 16.58 pg/g; p < 0.05; Table 1). In contrast, TNF-α and IL-6 in stools showed no significant difference (p > 0.05) between patients with Shigella and Salmonella gastroenteritis.
Local production of TNF-α and IL-6 has been shown in Shigella dysenteriae and Shigella flexneri by demonstrating these cytokines in the stools (2,4). In De Silva et al., (2) these cytokines were not detected in stools of patients with rotavirus gastroenteritis. In an in vitro study, no effect of rotavirus for TNF-α, IL-1β and IL-6 production was observed (5).
Although Shigella and Salmonella species and rotavirus have similar pathophysiology (6,7), it was found that bacterial agents cause higher amounts of local production of proinflammatory cytokines. However, according to our data, it is not possible to find a particular level of cytokines showing viral or bacterial etiology, because of widely overlapping concentrations in both groups and very high standard deviations. In addition, in contrast to De Silva et al.'s results (2), in the rotavirus group, TNF-α and IL-6 were detectable in 100% and 82% of stool samples, respectively.
In conclusion, it is obvious that there is an involvement of locally produced proinflammatory cytokines in the pathogenesis of viral and bacterial gastroenteritis.
Necil Kukukculer; Suat Caglayan
Ege University; Faculty of Medicine; Department of Pediatrics; Izmir, Turkey
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2. De Silva DGH, Mendis LN, Sheron N, et al. TNF-alpha in stool as a marker of intestinal inflammation (letter). Lancet
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