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06-Feb-2001
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Arch Hellen Med, 16(5), September-October 1999, 437-551
ORIGINAL PAPER
Application
of an innovative molecular method
for the detection and identification of mycobacteria
on archive paraffin embedded samples with histological evidence
of granulomatous inflammation
I. IKONOMOPOULOS,1
V. GORGOULIS,1 P. ZACHARATOS,1
A. KOTSINAS,1 N. GOUTAS,2 Ch. KITTAS1
1Department
of Histology-Embryology, University of Athens, Medical School
2Department of Histopathology, Evgenidio Hospital, Athens, Greece
OBJECTIVE The
aim of this study was to design a diagnostic procedure for the detection and
identification of some of the more important pathogenic mycobacteria, using
paraffin embedded tissue samples. This method would be applied for the diagnostic
study of cases of tuberculosis, sarcoidosis and Crohn’s disease.
METHOD A combination of a polymerase chain
reaction (PCR) and DNA hybridization assay, with three specifically designed
pairs of primers and a probe, were used for the detection and identification
of the Mycobacterium tuberculosis complex, Mycobacterium avium,
Mycobacterium intracellulare and Mycobacterium paratuberculosis.
RESULTS The method was applied directly on
paraffin embedded tissue samples and was shown to specifically detect the presence
of even a single bacterial cell. The application of the assay on selected material
from patients with Crohn’s disease and sarcoidosis, demonstrated the implication
of Mycobacterium paratuberculosis and Mycobacterium tuberculosis
complex respectively, in forms which could not be demonstrated by culture.
CONCLUSIONS The proposed method can rapidly detect and
identify some of the most important pathogenic mycobacteria distinguishing species
with great genetic similarity. The procedure can be applied directly on paraffin
embedded tissue samples for the diagnosis of tuberculosis but also for the study
of certain diseases with unknown etiology, such as sarcoidosis and Crohn’s disease.
Key words: Mycobacterium avium complex, Mycobacterium tuberculosis.