IDENTIFICATION OF LEGIONELLA PNEUMOPHILA IN BRONCHOALVEOLAR LAVAGE FLUID SPECIMENS BY PCR

Background – Among the members of Legionellaceae, Legionella pneumophila is involved in more than 95% of cases of severe pneumonia. Isolation of the causative agent from bronchoalveolar lavage (BAL) fluid specimen is a delicate process and also time-consuming. Moreover, it has been shown that some Legionella strains may be viable but cannot be cultured. The serological diagnosis, which is usually determined by the immunofluorescent assay, may also be hindered, due to the delayed rise in Legionella antibody levels with the onset of illness. The aim of this study was to apply PCR method for specific identification of the L. pneumophila in the BAL fluid specimens. Method – In this study, 46 BAL fluid specimens were collected from patients suspected of having Legionnaires’ disease. These samples were kindly provided from educational hospitals of Hamadan and cultured on selective buffered charcoal-yeast extract agar (BCYE) and then tested with specific L. pneumophila PCR. In order to prepare the Legionella DNA, specimens were first treated by proteinase K. DNA was then extracted using the phenol-chloroform method. Specific primers used in this study were those targeting macrophage infectivity potentiator gene of the L. pneumophila. Results – The Legionella DNA was extracted from known strains and then PCR was optimized. The Legionella PCR sensitivity test showed 120 copies of chromosome DNA as a final detection limit. The specificity test did not produce a cross reaction for a range of respiratory pathogenic organisms except for L. pnuemophila. Forty-six BAL fluid specimens were cultured on BCYE medium to isolate the organisms and these were also tested by the PCR. Conclusion – Analysis of the results showed one positive for the culture and four for the specific Legionella PCR. The PCR set that was used showed that it is sensitive enough for identification of L. pneumophila to apply on BAL fluid specimens for the tested samples.