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Sensitive and rapid diagnosis of pneumonia caused by Legionella species
Legionnaires' disease was first recognized during a pneumonia outbreak at the Legionnaires convention in Philadelphia in 1976. Investigators with the CDC isolated a novel, gram-negative bacillus, later named Legionella pneumophila. It is now widely recognized that Legionella pneumophila (and other members of the genus Legionella) cause Legionnaires' disease.
A positive PCR result for the presence of a specific sequence found within the Legionella 5S rRNA gene indicates the presence of a Legionella species DNA, which may be due to Legionella infection or environmental/water Legionella DNA in the specimen.
A negative PCR result indicates the absence of detectable Legionella DNA in the specimen, but does not rule-out legionellosis as false-negative results may occur due to inhibition of PCR, sequence variability underlying the primers and/or probes, or the presence of Legionella species in quantities less than the limit of detection of the assay.
This assay does not differentiate between the Legionella species. The assay is not recommended as a test of cure because nucleic acid may persist after successful treatment. False-positive results are theoretically possible if patient specimens are contaminated with Legionella DNA, which may occur since Legionella species are environmental organisms present in aquatic environments. The following uncommonly encountered species of Legionella are not detected by this assay: Legionella anisa, Legionella feeleii, Legionella maceachernii, Legionella parisiensis, and Legionella sainthelensi.
In a Mayo Clinic study, 153 archived respiratory specimens previously tested for Legionella species by direct fluorescence antibody (DFA) testing were extracted and tested using this PCR method. The PCR assay was 100% sensitive and 99.3% specific, in comparison to DFA. Additionally, 30 lung tissues and 30 pleural fluids were spiked with 3 of the most commonly isolated Legionella species. Spiking studies showed similar analytical sensitivity for PCR and the DFA method. The analytical sensitivity was less than 50 targets/20 microliter reaction. No cross-reactivity was observed when tested on a panel of respiratory pathogens or normal flora bacteria of the upper respiratory tract. Thirteen serogroups of Legionella pneumophila (Legionella pneumophila serogroups 1-12, 15/16) and 9 additional Legionella species (Legionella bozemanae, Legionella dumoffii, Legionella gormanii, Legionella jordanis, Legionella longbeachae, Legionella micdadei, Legionella oakridgensis, Legionella hackeliae, and Legionella wadsworthii) included in the panel were detected with the PCR method. The following uncommon species of Legionella are not detected by this assay: Legionella anisa, Legionella feeleii, Legionella maceachernii, Legionella parisiensis, and Legionella sainthelensi.
1. Cunningham SA, Sloan LM, Uhl JA, et al: Validation of a real-time PCR assay for the detection of Legionella species in respiratory samples. Abstracts of the Annual Meeting of the Association for Molecular Pathology, 2009 General Meeting, Kissimmee, FL, Nov. 19-22, 2009
2. Hayden RT, Uhl JR, Qian X, et al: Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture. J Clin Microbiol 2001;39(7):2618-2626
3. Diederen BM, Kluytmans JA, Vandenbroucke-Grauls CM, Peeters MF: Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice. J Clin Microbiol 2008;46(2):671-677
4. MacDonell MT, Colwell RR: The nucleotide sequence of the 5S rRNA from Legionella pneumophila. Nucleic Acids Res 1987;15(3):1335