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Noninvasive, sensitive, and specific tools for early identification of chronic inflammatory bowel disease (IBD) are needed for clinical practice. The aim was to identify new noninvasive test combinations for characterization of IBD in children and adolescents by comparing serological responses to microbial antigens and fecal calprotectin, a new promising marker for intestinal inflammation.Our study included 73 children who underwent endoscopies because of suspicion of IBD. Their sera were tested for antibodies to the Pseudomonas fluorescens-associated sequence I2, a Bacteroides caccae TonB-linked outer membrane protein, OmpW, and anti-Saccharomyces cerevisiae (ASCA). Simultaneously, samples for fecal calprotectin measurements were obtained from 55 subjects.IBD was diagnosed in 60 patients (Crohn's disease [CD] in 18 patients, ulcerative colitis [UC] in 36, and indeterminate colitis [IC] in 6). Thirteen children had a non-IBD disease. Fecal calprotectin levels were elevated (≥100 μg/g) more frequently in IBD patients (89%, 39/44) compared to non-IBD cases (9%, 1/11, P < 0.001). ASCA antibodies in sera were detected in 67% (12/18) of patients with CD, in 14% (5/36) of the children with UC, and in 50% (3/6) of patients with IC. Seroreactivity for I2 was observed in 42% of the IBD patients, this frequency being higher than in non-IBD cases (7.7% seropositive; P = 0.025). Serum anti-I2 IgA levels (median absorbances) were higher in those with IBD compared to those without gut inflammation (P = 0.039). The combination of the measurements of fecal calprotectin and serological responses to microbial antigens (ASCA, I2, and OmpW) identified 100% of CD patients (sensitivity 100%, specificity 36%, positive predictive value [PPV] 66%, negative predictive value [NPV] 100%) and 89% of UC patients (sensitivity 89%, specificity 36%, PPV 77%, NPV 57%).Increased levels of serological responses to microbial antigens (ASCA, I2, and OmpW) and fecal calprotectin are evident in both CD and UC patients. The combination of these markers provides valuable, noninvasive tools for the diagnosis of IBD.