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Medycyna Doświadczalna i Mikrobiologia 2019, 71(2-4): 97-108

Molekularna diagnostyka preparatów histopatologicznych jako uzupełnienie badań mikrobiologicznych gruźlicy
[Molecular diagnostics of histopathological samples as a complement to microbiological diagnosis of tuberculosis]

S. Brzezińska, J. Dziadura, A. Zabost, M. Klatt, M. Szołkowska, E. Augustynowicz-Kopeć

Streszczenie
Molekularne techniki wykorzystujące hybrydyzację kwasów nukleinowych są coraz częściej stosowane w diagnostyce gruźlicy. Charakteryzują się one wysoką czułością i swoistością. Celem podjętych badań była ocena  przydatności metod molekularnych w diagnostyce gruźlicy w oparciu o badania próbek materiału klinicznego utrwalonych w parafinie. Przeprowadzona została genetyczna analiza DNA izolowanego z próbek materiałów tkankowych uzyskanych od 45 chorych z podejrzeniem gruźlicy. Określono korelację pomiędzy wynikiem uzyskanym w badaniu genetycznym a wynikami badania mikrobiologicznego przeprowadzonego na próbkach uzyskanych od tej grupy chorych.

ABSTRACT
Introduction: Methods of conventional microbiology, which constitute the gold standard of diagnostics, are necessary in case of detection of mycobacteria in fresh materials. The limited scope of the traditional methods analytical capabilities does not allow for retrospective diagnosis. Molecular methods, based on techniques involving nucleic acid hybridization are becoming widely used in the diagnosis of tuberculosis. These techniques provide the opportunity to perform a quick analysis of the material without the time-consuming stage of culture. Systems using molecular biology techniques in diagnostics show high sensitivity and specificity. The aim of the dissertation is to assess the usefulness of diagnostic molecular methods based on the detection of nucleic acids in paraffin fixed histopathological probes, taken from patients with suspected tuberculosis.
Materials and Methods: This dissertation presents the results of an analysis of tissue materials obtained from 45 patients with suspected tuberculosis. In histopathological ex98 amination the probes show changes suggesting the possibility of an ongoing tuberculosis.
The indications for diagnosis of tuberculosis infection include necrotic changes, granulation tissue, fibrosis and other changes indicating the development of the inflammatory process within the tissue from which the material was collected. Paraffin-fixed tissue probes are subjected to molecular diagnosis using the BD ProbeTec ET system. Clinical materials are analyzed by standard microbiological methods used in the diagnosis of tuberculosis.
Results: Molecular analysis of 45 paraffin-fixed materials showed presence of Mycobacterium tuberculosis complex in 22 cases, in 12 of them Mycobacterium was detected in microbiological methods. Analysis showed 54% agreement between the positive results in the molecular methods of paraffin preparations and microbiological diagnostics of clinical samples.
Conclusions: 1. The use of genetic methods enables quick and detailed diagnostics of infections caused by M. tuberculosis complex, which allows for the supplementation of microbiological and histopathological diagnostics. 2. Use of molecular methods to confirm the presence of Mycobacterium tuberculosis complex in paraffin-fixed materials is important also for patients whose diagnosis based solely on histopathology and were not microbiologically tested.

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