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    The possible involvement of Mycobacterium avium ssp. paratuberculosis in the aetiology of Crohn's disease: a case control study in the Netherlands

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    Authors
    Herrewegh AAPM
    Overduin P
    Roholl PJM
    Gielis FK
    Robinson JE
    Mahmmod N
    Lieverse RJ
    Robijn RJ
    van der Zanden AGM
    van Soolingen D
    Series/Report no.
    RIVM rapport 230086002
    Type
    Report
    Language
    en
    
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    Title
    The possible involvement of Mycobacterium avium ssp. paratuberculosis in the aetiology of Crohn's disease: a case control study in the Netherlands
    Translated Title
    Mycobacterium avium ssp. paratuberculosis als mogelijke oorzaak van de ziekte van Crohn: resultaten van een patientenstudie in Nederland.
    Publiekssamenvatting
    De bacterie Mycobacterium avium ssp. paratuberculosis (Map) wordt beschouwd als een mogelijke oorzaak van de ziekte van Crohn (morbus Crohn, MC). In samenwerking met Gelre ziekenhuizen heeft het RIVM een onderzoek uitgevoerd naar het voorkomen van Map in darmbiopten van patienten met MC, patienten met Ulcerative Colitis (UC) en controlepersonen zonder darmontsteking. De aanwezigheid van Map in de darmbiopten werd onderzocht met behulp van kweekmethoden, een DNA amplificatiemethode (PCR) en een immunologische detectiemethode (immunoperoxidase kleuring, IP-kleuring). Met de PCR-methode werd Map aangetoond in 7% van de MC-patienten, 8% van de UC-patienten en 5% van de controlepersonen. Met de gecombineerde kweek- en PCR-methoden waren gemiddeld 25% van de MC-patienten, 7% van de UC patienten en 27% van de controlepersonen positief. Met behulp van de IP-kleuring werd Map aangetoond in 20% van de MC-patienten, 13% van de UC-patienten en 29% van de controlepersonen. De resultaten van de kweek- en PCR-methoden tonen geen significant verschil tussen de aanwezigheid van Map in MC-patienten vergeleken met controlepersonen. De resultaten van de IP-kleuring tonen zelfs een hoger percentage Map-positieve controlepersonen vergeleken bij MC-patienten. Onze resultaten tonen duidelijk aan dat Map zowel bij MC-patienten als bij UC-patienten en controlepersonen voorkomt. Naast de aanwezigheid van Map is ook gekeken naar de aanwezigheid van Chlamydia in de biopten. Op grond van IP en in situ hybridisatie kleuringen bleken Chlamydiae in grote aantallen aanwezig te zijn in de biopten van MC- en UC-patienten en nauwelijks aanwezig in de biopten van controle patienten.In conclusie: onze resultaten ondersteunen niet de hypothese dat Map direct betrokken is bij de ziekte van Crohn, maar sluiten ook niet uit dat Map een rol speelt bij het ontstaan van de ziekte van Crohn in een gevoelig deel van de populatie.

    A case control study was performed to investigate the possible role of Mycobacterium avium ssp. paratuberculosis (Map) in the aetiology of Crohn's disease (CD). Biopsy samples were collected from the ileum and colon of CD patients, Ulcerative Colitis (UC) patients and control persons. The biopsy samples were either cultured in MGIT and BACTEC medium, formaline-fixed, or immediately snap-frozen in liquid N2. The presence of Map bacteria in the cultures was determined using a nested PCR assay targeted to the conserved IS900 DNA-sequence of Map. 27% of CD patients, 6% of UC patients, and 28% of control persons were positive for MGIT-PCR. The BACTEC cultures resulted in a slightly smaller percentage of PCR positive patients, 22% for CD patients, 7% for UC patients and 25% for control persons. The presence of Map was further studied applying the IS900-PCR directly on DNA from frozen biopsy samples. 7% of CD patients, 8% of UC patients and 5% of control persons were PCR positive for Map. The presence of Map was also investigated in formalin-fixed biopsies samples using a hyperimmune antiserum to the M. avium-complex. With immunoperoxidase (IP) staining, M. avium-complex antigens were observed in biopsy samples from 20% of CD patients, 13% of UC patients and 29% of control persons. Surprisingly, these data show even a 50% higher presence of Map-antigens in control persons (29%) compared to CD patients (20%). Our data clearly show that Map is present both in IBD patients (CD and UC) and in control persons. The results of culturing and IP staining show a good correlation; with both techniques, an average of 23% of CD patients and 27% of control persons is positive for Map. Remarkably, the results of culturing and IP staining show an even higher prevalence of Map in control persons compared to CD patients. Moreover, only in a few instances patients were positive in two different tests (PCR, IP, MGIT and BACTEC), and even with both culturing assays almost no overlap in positive results is observed. This result indicates that the number of Map cells in human tissue is very low, and that the prevalence of Map in the population of IBD patients and of control persons is difficult to estimate. Unexpectedly, we obtained evidence that members of the Chlamydiales family are present in high amounts in most of the CD and UC patients, whereas they were of low incidence in control persons.In conclusion, our data do not support the hypothesis that Map is directly involved in Crohn's disease, but does not exclude that Map infection may play a role in the aetiology of Crohn's Disease for a susceptible subset of the population.
    Publisher
    Rijksinstituut voor Volksgezondheid en Milieu RIVM
    Dept of Medical Microbiology and Infectiouos Diseases Gelre Hospital
    BioMedics Scientific Consultancy
    Sponsors
    LNV/VWA
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