Mycobacterium in Crohn’s: Something to ruminate about?

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Naser SA, Ghobrial G, Romero C, Valentine JF (Department of Molecular Biology and Microbiology and Biomedical Science Center, University of Central Florida, Orlando; Department of Medicine, University of Florida, Gainesville, Florida). Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 2004;364:1039–1044.

The causative factor(s) of Crohn’s disease remains a mystery nearly three quarters of a century since Crohn and colleagues published their landmark article on regional enteritis in 1932 (JAMA 1932;99:1323–1329). One theory that has waxed and waned over time has been the possible association with infection by Mycobacterium avium paratuberculosis (MAP) as either a primary infection responsible for the development of disease, or a secondary opportunistic infection that could perpetuate the cycle of inflammation and cytokine release.

This debate has been rekindled of late by an interesting controlled trial reporting a higher prevalence of MAP DNA in the peripheral blood of patients with Crohn’s disease (Lancet 2004;364:1039–1044). In this study, Naser and colleagues collected blood samples from 28 adult patients with Crohn’s disease, 9 with ulcerative colitis, and 15 patients with no known inflammatory bowel disease (IBD) (2 had colon cancer, 1 diverticulitis, and 1 gastroesophageal reflux; 11 were healthy controls). Information regarding patient age, gender, disease duration, location, activity, and type (penetrating, structuring, inflammatory) was collected, as well as concomitant immunosuppression.

The buffy coat from the blinded samples were cultured in Mycobacterial Growth Indicator Tubes (MGITs) and BACTEC bottles, incubated, and Ziehl-Neelsen smears prepared for acridine orange staining or immunostaining. Polymerase chain reaction (PCR) analysis of genomic DNA was also performed, with amplification by oligonucleotide primers derived from the MAP-unique IS900 DNA insertion sequence. Both the unique 398-bp fragment and a 298-bp internal nucleotide sequence were amplified, PCR products were subsequently purified and sequenced with BLAST, and alignment sequence analyses were performed.

The total group of patients was split evenly by gender, but 61% of the Crohn’s disease patients were female. Immunosuppressive drugs were used in 62% of the Crohn’s disease, 44% of the ulcerative colitis, and none of the non-IBD patients. MAP DNA was detected in 46% of the Crohn’s disease, 44% of the ulcerative colitis, and 20% of the non-IBD patients. After 12 weeks of incubation, 3 of the BACTEC cultures detected possible MAP growth, with PCR identifying MAP in all 3 BACTEC and 14 of 52 MGIT cultures (only 1 patient was positive by both methods). MAP was isolated in 50% of the Crohn’s disease, 22% of the ulcerative colitis, and 1 of the non-IBD patients (P = .0005 Crohn’s vs non-IBD).

Positive MAP PCR was verified by positive cultures in 11 of 13 Crohn’s disease patients, and of the 14 Crohn’s disease patients with positive cultures, 10 also tested positive by MAP PCR. One of the 4 PCR-positive ulcerative colitis patients was positive by culture; 1 culture-positive ulcerative colitis patient was PCR-negative. None of the 3 PCR-positive non-IBD patients had positive cultures.

The concurrent use of immunosuppressive agents did not correlate with developing a positive culture among the Crohn’s disease patients. Comparison of the MAP-specific IS 900 fragment sequences from 11 isolates revealed 9 different strains of MAP. The authors concluded that the higher incidence of viable MAP in patients with Crohn’s disease than in controls supports the theory that MAP may be a cause of Crohn’s disease.

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Comment 

One of the difficulties in determining the true relationship, if any, between MAP and Crohn’s disease is the difficulty in isolating and culturing the organism. Johne’s disease is a chronic enteritis in ruminants, characterized by granuloma formation, intestinal destruction, and involvement of adjacent lymphoid tissue (Immunol Cell Biol 1999;77:364–372, Annu Rev Microbiol 2004;58:329–363). The typical lesions are in the distal ileum and cecum, with a variety of inflammatory cells causing a transmural intestinal injury (Vet Pathol 1978;15:196–207). The etiologic agent has been proven to be MAP because it has been identified and isolated in cattle stricken with the disease, cultured, and then reinfected healthy animals, fulfilling Koch’s postulates (Cornell Vet 1933;23:1–15, Vet News 1914;11:79–81, Annu Rev Microbiol 2004;58:329–363). It has been hypothesized that MAP can be transmitted to humans through the ingestion of contaminated milk or water. The similarities between Johne’s disease and Crohn’s disease, both having granulomas, transmural inflammation, and a predisposition for the ileocecal area and surrounding lymph nodes, has naturally led to speculation that the agent may cause or perpetuate disease in susceptible individuals.

The identification of MAP is also a problem. One can look for evidence of the organism itself, through culture or DNA analysis, or for the host reaction (serology) against this agent (Clin Microbiol Rev 2001;14:489–512). Difficulties in culturing the slow-growing mycobacterium have been further confounded by the identification of acid-fast negative forms of MAP, which lack cell walls (J Clin Microbiol 1986;24:357–363, J Clin Microbiol 1993;31:1241–1245). Complicating the matter further is the use of the bacillus Calmette-Guérin (BCG) vaccine in many parts of the world, which is derived from Mycobacterium bovis, and may impact infection rates or detection (J Clin Microbiol 2000;38:4373–4381). Determination that an infectious agent such as MAP is causative in Crohn’s disease rather than simply acting as a secondary “infection” in a host with a compromised intestinal barrier is perhaps the biggest obstacle that MAP supporters have to overcome to convince a skeptical scientific community.

A review of the existing literature is equally confusing. A population-based case control study of seroprevalence of MAP measured by enzyme-linked immunosorbent assay (ELISA) in the University of Manitoba’s 2890 patient IBD registry and controls matched to the province’s health registry found no difference in rates between patients with Crohn’s disease (37.8%), ulcerative colitis (34.7%), and healthy controls (33.6%) or nonaffected siblings (34.1%) (J Clin Microbiol 2004;42:1129–1135). Furthermore, 32% of controls in a nested cohort of this study group had MAP-positive PCR on colonoscopy biopsy specimens (J Clin Microbiol 2003;41:4986–4990), not much different than the 26% PCR-positive rate reported in United Kingdom controls (J Clin Microbiol 2003;41:2915–23). Although other studies have shown higher rates of seropositivity against certain MAP antigens in Crohn’s disease patients than in controls, they have found poor correlation when comparing reactions against multiple MAP antigens (Clin Exp Immunol 1992;90:503–508). Whereas high rates of antibodies to the MAP p36 antigen in Crohn’s disease patients (Vet Microbiol 2000;77:497–504) suggest a possible pathogenic role of this organism, high rates of cross-reactivity of antibodies in Crohn’s patients have also been reported with other foreign antigens, such as Saccharomyces cerevisiae mannon (Am J Gastroenterol 2001;96:730–734). It is unlikely that simple serology, which cannot differentiate between past exposure and current infection, or exclude cross-reactivity of antibodies, will prove valuable in determining the causative role of MAP in Crohn’s disease.

Small uncontrolled trials have reported culturing MAP from patients with Crohn’s disease. Many investigations have concentrated on detection of MAP DNA by use of an IS900-specific PCR, an element unique to the subspecies. Using this technique, detection of MAP DNA was reported in the current study in 46% of biopsy samples from Crohn’s disease patients, 44% with ulcerative colitis, and 20% in non-IBD controls; other authors have reported rates as high as 92% of biopsy samples in Crohn’s disease patients vs 26% of non-IBD controls (J Clin Microbiol 2003;41:2915–2923, Lancet 2004;364:1039–1044). These results must be tempered against those from a study comparing the results of multiple diagnostic tests for MAP, whereby none of the 11 ELISA-positive cases or 34 PCR-positive cases tested positive by both tests (J Clin Microbiol 2000;38:4373–4381). Adding to the confusion is the finding that MAP is often present deeper than just in the mucosa, commonly in the lymph nodes, suggesting that mucosal biopsy specimens may test falsely negative, while resected bowel and lymph nodes from the same individual may test positive.

Although most published studies report higher rates of MAP (by serology, PCR, or culture) in patients with Crohn’s disease than in those with ulcerative colitis or noninfected controls, it is difficult to accept any claims of causality of disease, given the heterogeneous representations of Crohn’s disease, the numerous Crohn’s disease patients in these studies without any evidence of MAP by any type of testing, and variation of MAP “infection” rates in different populations worldwide (J Clin Microbiol 2004;5432–5433).

Claims that Crohn’s disease patients are victims of tainted dairy products or contaminated water are also met with skepticism. Because of the abundant consumption of dairy products worldwide and the unfortunate reality of consumption of water contaminated by bovine fecal material, one would expect high rates of Crohn’s disease in developed countries and an epidemic of Crohn’s disease in third-world countries if MAP was the etiologic agent. In contrast, incidence rates have been estimated at only 5.8/100,000 person-years both in the United States (Gastroenterology 1998;114:1161–1168) and in Norway (Scand J Gastroenterol 1996;31:355–361), and <1.0/100,000 person-years in various third-world countries (Epidem Rev 1986;8:60–91).

Perhaps the strongest argument against the MAP hypothesis is the success of the anti-tumor necrosis factor (TNF) drug infliximab in the treatment of Crohn’s disease (N Engl J Med 1997;337:1029–1035), coupled with the known dangers of treating patients with anti-TNF therapies who are infected with a different mycobacterium, M tuberculosis (N Engl J Med 2001;345:1098–1104). Antituberculous regimens have had mixed results in Crohn’s disease studies, sometimes outperformed by their placebo-arm, and have not been supported by meta-analysis (Am J Gastroenterol 2000;95:725–729, Evid Based Gastroenterol 2000;1:18–19).

Although regurgitation may be acceptable in the bovine herds susceptible to Johne’s disease, the medical community is at the point where studies must go beyond merely repeating the observation that MAP is more commonly found in patients with Crohn’s disease and must establish whether there is truly an infectious role of this agent.