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PREVALENCE OF MYCOBACTERIUM AVIUM PARATUBERCULOSIS IN PEDIATRIC INFLAMMATORY BOWEL DISEASE
A Lee1, T Griffiths1, R Parab1, S Urbanski2, I Wrobel3, K Rioux11Department of Medicine, Division of Gastroenterology; 2Department of Pathology and Laboratory Medicine; 3Department of Pediatrics, Division of Gastroenterology, University of Calgary, Calgary, Alberta
Aims: Crohn's disease (CD) is the common label for a clinically heterogenous group of inflammatory conditions affecting the GI tract. The cause of CD is unknown, but each clinical phenotype likely has distinct immunogenetic, environmental, and microbial factors associated with it. For many years, Mycobacterium avium spp. paratuberculosis (Map) has been implicated in the pathogenesis of CD, but a causal role has not been proven. The strength of association between Map and CD varies widely between studies and it remains plausible that Map is etiologically important in only certain clinical subsets of the disease. The aim of this study was to determine the prevalence of Map in early-onset Crohn's disease.
Methods: Archival, formalin-fixed, paraffin-embedded (FFPE) terminal ileal and cecal biopsy specimens were used in these studies. The histopathology database at Alberta Children's Hospital was used to identify pediatric patients with granulomatous Crohn's ileocolitis (n=21) and ulcerative colitis (UC, n=12). Colonoscopy records identified healthy age- and gender-matched controls (n=9) who had normal endoscopic and histologic findings. Five micron sections were deparaffinized in xylene and DNA was extracted using the QIAamp DNA Micro Kit which was modified to include a bead beating step. IS900 nested PCR was performed in triplicate on each sample using the L/AV primer set according to Bull et al. (J Clin Microbiol 41:2915-23, 2003); 29 cycles were used in both the first round of amplification and in the subsequent nested PCR. The sensitivity of this PCR protocol was validated to a detection limit of 3.84 fg/ÁL of total DNA. FFPE tissue from bovine Johne's disease served as a positive control. IS900 positivity was determined in a blinded fashion. Data were compared using Fisher's exact test.
Results: In ileal biopsies, IS900 sequences were detected in 2 of 9 (22%) control subjects, 16 of 21 (76%) CD patients (p=0.001 vs. control), and 2 of 12 (16%) UC patients (p=NS vs. control). In cecal biopsies, IS900 amplicons were detected in 0 of 9 control subjects, 11 of 21 (52%) CD patients (p=0.011 vs. control) and 4 of 12 (33%) UC patients (p=NS vs. control). In CD patients, there was no correlation between disease activity and Map-positivity.
Conclusions: Map DNA was detected in archival FFPE endoscopic biopsy specimens. The presence of Map-specific DNA is strongly associated with pediatric granulomatous Crohn's disease, being particularly prevalent in ileal tissue. This clinical subset of CD may be useful for further studies to determine whether Map plays a causal role.