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

doi:10.1128/JCM.39.7.2618-2626.2001

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Journal of Clinical Microbiology, July 2001, p. 2618-2626, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2618-2626.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

R. T. Hayden, J. R. Uhl, X. Qian, M. K. Hopkins, M. C. Aubry, A. H. Limper, R. V. Lloyd, and F. R. Cockerill^*

Mayo Clinic, Rochester, Minnesota

Received 16 November 2000/Returned for modification 11 January 2001/Accepted 22 March 2001

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionellaspecies directly from clinical specimens. This method uses theLightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.)and requires approximately 1 to 2 h to perform. Both a Legionellagenus PCR assay and Legionella pneumophila species-specific PCRassay were designed. A total of 43 archived specimens from 35patients were evaluated, including 19 bronchoalveolar lavage (BAL)specimens and 24 formalin-fixed, paraffin-embedded open lung biopsyspecimens. Twenty-five of the specimens were culture-positivefor Legionella (9 BAL specimens and 16 tissue specimens). BALspecimens were tested by LightCycler PCR (LC-PCR) methods andby a direct fluorescent antibody (DFA) assay, which detects L.pneumophila serogroups 1 to 6 and several other Legionella species.Tissue sections were tested by the two LC-PCR methods, by DFA,by an in situ hybridization (ISH) assay, specifically designedto detect L. pneumophila, and by Warthin-Starry (WS) staining.The results were compared to the "gold standard" method of bacterialculture. With BAL specimens the following assays yielded the indicatedsensitivities and specificities, respectively: Legionella genusdetection by Legionella genus LC-PCR, 100 and 100%; Legionellagenus detection by DFA assay, 33 and 100%; and L. pneumophiladetection by L. pneumophila species-specific LC-PCR, 100 and 100%.With open lung biopsy specimens the following assays yielded theindicated sensitivities and specificities, respectively: Legionellagenus detection by LC-PCR 68.8 and 100%; Legionella genus detectionby DFA assay, 44 and 100%; Legionella genus detection by WS staining,63 and 100%; L. pneumophila species-specific detection by LC-PCR,17 and 100%; and L. pneumophila species-specific detection byISH, 100 and 100%. The analytical sensitivity of both LC-PCR assayswas <10 CFU/reaction. LC-PCR is a reliable method for the directdetection of Legionella species from BAL specimens. The Legionellagenus LC-PCR assay could be performed initially; if positive,L. pneumophila species-specific LC-PCR could then be performed(if species differentiation is desired). The speed with whichthe LC-PCR procedure can be performed offers significant advantagesover both culture-based methods and conventional PCR techniques.In contrast, for the methods evaluated, culture was the best fordetecting multiple Legionella species in lung tissue. WS staining,Legionella genus LC-PCR, and L. pneumophila species-specific ISHwere useful as rapid tests with lungtissue.

^* Corresponding author. Mailing address: Division of Clinical Microbiology, Department of Pathology and Laboratory Medicine,Hilton Building, Mayo Clinic, 200 First St., S.W., Rochester,MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail:cockerill.franklin{at}mayo.edu.

Present address: St. Jude Children's Research Hospital, Memphis,Tennessee.

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