Last update:
06-Feb-2001

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. KITTAS^1
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.