Rapid Culturing and PCR Detection of Mycobacterium avium ss paratuberculosis
from Crohn's Disease Tissue
I.
Shafran, W. Fenster, C. Piromalli, C. Romero, D. Schwartz, D. Campbell, and S.
Naser
The
insertion sequence IS900 of Mycobacterium
avium ss paratuberculosis (M
para) has been used as a fingerprint-type marker for the identification of M para or M para-like
microorganisms in clinical specimens from patients with Crohn's disease (CD), a
chronic inflammatory bowel disease. However, amplification of IS900 in these
specimens does not confirm the virulent form of the bacterium. Culturing M
para from CD tissue has been reported but with limited success. This, in
part, is significantly related to the type of culture media used and the
integrity of the specimen. Amplification of the IS900 followed by DNA
hybridization from cultured tissue, provide accurate diagnosis of CD. In this
study, 59 tissue specimens (20 CD consisting of 7 resected tissue and 13 biopsy
and 39 controls consisting of biopsies from Ulcerative colitis and
non-inflammatory bowel disease patients) were homogenized, decontaminated and
inoculated into BACTEC bottles and MGIT media (radioactive and non-radioactive
methods, respectively). Aliquots of culture were analyzed by PCR assays specific
for M avium complex (MAC) and
M para (IS900). This was followed with hybridization using specific DNA
probes. Of the 20 CD specimens, 15(75%) were positive for MAC with 6 (30%)
confirmed for the IS900 as M para. Of
the 39 control specimens, none were positive with either assay. Our data confirm
previous studies reporting the association of M
para or M para-like microorganisms
in CD. Our data also suggest that M para may be isolated from tissue specimens, especially those
resected (if present) within 10 weeks in the MGIT media. Ultimately, this
provides a rapid culturing protocol for the diagnosis of CD especially at its
proliferative stage.
All content copyright ©1999 Dr. Ira Shafran, M.D.