Detection of Legionella DNA in clinical samples using real-time PCR
Abstract number: p974
Franzin L., Cabodi D., Bonfrate N., Tortorelli F.
Culture is the gold standard for the diagnosis of Legionella infection. However several days are required to obtain a positive result. DNA techniques are promising for rapid detection. In this study a real time (RT)-PCR system was evaluated for the direct detection of Legionella from clinical samples and compared to culture.
Culture and RT-PCR was performed on 78 respiratory samples (10 sputum, 44 bronchoalveolar lavage fluid, 19 bronchial aspirate, 5 lung tissue) from 76 patients with pneumonia. Legionella infection (LD) was confirmed in 23 patients by positivity to at least one of these tests: urine antigen detection (ELISA), serology (fourfold rise in antibody titres by indirect immunofluorescence and/or micro agglutination test) and culture. Aliquots of the untreated, heat-treated and acid-washed samples were plated on BCYE, BMPA and MWY. The plates were incubated at 37°C for 15 days. Blood samples from 11 symptomatic old patients involved in an LD outbreak were also examined. RT-PCR for L.pneumophila (Lp) and Legionella spp (L.spp) was performed after DNA extraction with iCycler and BIO-RAD reagents, according to a modified protocol.
Culture from respiratory samples was positive from 17 patients. Lp serogroup 1 was isolated from 15 subjects, Lp 3 from one and L.bozemanii from another. RT-PCR was positive in a total of 25 (32%) samples. 24/25 (96%) samples from LD patients were positive by RT-PCR, while 52/53 (98%) samples from patients without LD were negative. Discrepancy was observed in only one case (1.3%) for category of patients with and without LD. Three patients with proven LD was positive by RT-PCR for L. spp and negative for Lp, but one of these was culture positive for L.bozemanii. All culture positive respiratory samples were RT-PCR positive, except one where Lp3 was isolated. Concordance for respiratory samples was 97.4%. All blood culture samples were negative, while RT-PCR was positive from 4/6 outbreak patients with laboratory confirmed LD and from 5/5 patients with clinical, but not laboratory confirmed, LD.
Laboratory diagnosis LD is better achieved by combined urine antigen detection, serology and culture. Results of this study showed good concordance between the criterion based on traditional methods and RT-PCR. This new sensitive technique, that needs to be further evaluated, offers the advantage of a rapid diagnosis.
|Session name:||XXIst ISTH Congress|
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