Results of Multiple Diagnostic Tests for Mycobacterium avium subsp. paratuberculosis in Patients with Inflammatory Bowel Disease and in Controls

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Journal of Clinical Microbiology, December 2000, p. 4373-4381, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Results of Multiple Diagnostic Tests for Mycobacterium avium subsp. paratuberculosis in Patients with Inflammatory Bowel Disease and in Controls

Michael T. Collins,¹^,^* Gorm Lisby,² Claus Moser,³ Debra Chicks,⁴ Steen Christensen,⁵ Mark Reichelderfer,⁴ Niels Høiby,³ Bruce A. Harms,⁶ Ole Ø. Thomsen,⁵ Ulrik Skibsted,² and Vibeke Binder⁵

Department of Pathobiological Sciences, School of Veterinary Medicine,¹ and Department of Clinical Gastroenterology and Department of Medicine⁴ and Department of Surgery,⁶ Medical School, University of Wisconsin, Madison, Wisconsin, and Department of Clinical Microbiology, Rigshospitalet,³ and Department of Clinical Microbiology² and Department of Medical Gastroenterology,⁵ Herlev Hospital, Copenhagen, Denmark

Received 17 July 2000/Returned for modification 9 August 2000/Accepted 26 September 2000

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies todate have been relatively small and generally only used a singlediagnostic assay. The objective of the study was to reexaminethe association of M. avium subsp. paratuberculosis and CD usingmultiple diagnostic tests. Five methods were used to detect M.avium subsp. paratuberculosis infections in 439 inflammatory boweldisease (IBD) patients and 324 control subjects in the UnitedStates and Denmark. Most assays were adaptations of diagnostictests for this infection performed routinely on animals. PCR forIS900, a genetic element unique to M. avium subsp. paratuberculosis,was positive significantly more often on resected bowel and lymphnode tissues from CD patients (19.0%) and ulcerative colitis (UC)patients (26.2%) than from controls (6.3%) (P < 0.05). PositiveIS900 PCR results occurred more often in U.S. than in Danish IBDpatients, 32.0 versus 13.3% (P = 0.025). The majority of Danishpatients were bacillus Calmette-Guérin (Mycobacterium bovis BCG)vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas noneof the U.S. patients with IBD and only 2% of U.S. controls werevaccinated. Among Danish IBD patients, positive PCR findings werefour times more common among subjects who were not BCG vaccinated(33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture ofthe same tissues tested by PCR using modified BACTEC 12B mediumfailed to grow M. avium subsp. paratuberculosis from patientsor controls. U.S. CD patients had the highest serological evidence(enzyme-linked immunosorbent assay [ELISA] for serum antibodies)of M. avium subsp. paratuberculosis infection (20.7% of patientspositive) which was higher than for all UC patients studied (6.1%)or healthy controls (3.8%, P < 0.005). Among Danish patients alone,however, no significant differences in rates of ELISA-positiveresults among CD, UC, or control patients were found. For 181study subjects, both IS900 PCR and ELISA were performed. Although11 were ELISA positive and 36 were PCR positive, in no instancewas a patient positive by both tests, suggesting that these statesare mutually exclusive. Evaluation of cytokine-mediated immuneresponses of IBD patients was complicated by the influence ofimmunosuppressive therapy given most IBD patients. Gamma interferon(IFN- $gamma$ ) release by peripheral blood leukocytes after M. aviumpurified protein derivative PPD antigen stimulation showed significantlylower responses in CD patients than in UC patients or controlsin both U.S. (by ex vivo assay) and Danish (by in vitro assay)populations (P < 0.05). Interleukin-5 responses were not differentamong CD, UC, or control groups. Collectively, the PCR, ELISA,and IFN- $gamma$ tests for M. avium subsp. paratuberculosis togetherwith the unexpected observation that BCG vaccination influencedM. avium subsp. paratuberculosis detection, lead us to concludethat M. avium subsp. paratuberculosis, or some similarly fastidiousmycobacterial species, infects at least a subset of IBD patients.Whether the infection is primary (causal) or secondary, it maycontribute to the etiopathogenesis ofIBD.

^* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, 2015 LindenDr. West, Madison, WI 53706-1102. Phone: (608) 262-8457. Fax:(608) 265-6463. E-mail: mcollin5{at}facstaff.wisc.edu.

Journal of Clinical Microbiology, December 2000, p. 4373-4381, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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