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Detection of Legionella DNA in clinical samples using real-time PCR

Abstract number: p974

Franzin  L., Cabodi  D., Bonfrate  N., Tortorelli  F.

Objectives: 

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.

Methods: 

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.

Results: 

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.

Conclusion: 

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 Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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