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PMID: 12843021 Published · ppublish English Evaluation Study Journal Article Research Support, Non-U.S. Gov't

Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease.

Journal of clinical microbiology ·Vol. 41 ·No. 7 ·2003-07-00 ·Pages 2915-23

Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, Hermon-Taylor J

Abstract

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

MeSH Terms
Adolescent Adult Base Sequence Biopsy Colon/microbiology Crohn Disease/microbiology Culture Media DNA Transposable Elements/genetics DNA, Bacterial/analysis Female Humans Ileum/microbiology Intestinal Mucosa/microbiology Male Middle Aged Molecular Sequence Data Mycobacterium avium subsp. paratuberculosis/classification,genetics,isolation & purification Paratuberculosis/microbiology Polymerase Chain Reaction/methods Sensitivity and Specificity Sequence Analysis, DNA
Chemicals
Culture Media DNA Transposable Elements DNA, Bacterial
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Bull Tim J
Department of Surgery, St. George's Hospital Medical School, London SW17 0RE, United Kingdom.
McMinn Elizabeth J
Sidi-Boumedine Karim
Skull Angela
Durkin Damien
Neild Penny
Rhodes Glenn
Pickup Roger
Hermon-Taylor John
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32 references, click to expand
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Article Info
Journal
Journal of clinical microbiology
Abbr.
J Clin Microbiol
ISSN
0095-1137
DOI
10.1128/JCM.41.7.2915-2923.2003
Published
2003-07-00
Pages
2915-23
Language
English
Region
United States
NLM ID
7505564
PMCID
PMC165291
Subset
IM
External Links
PubMed source DOI: 10.1128/JCM.41.7.2915-2923.2003 PMC Full Text View journal details
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