Direct Detection of Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization

Jinxin Hu; Ajit P. Limaye; Thomas R. Fritsche; Matthias Horn; Stefan Juretschko; Romesh Gautom

doi:10.1128/9781555817985.ch42

Chapter 42 : Direct Detection of Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization

Authors: Jinxin Hu¹, Ajit P. Limaye¹, Thomas R. Fritsche¹, Matthias Horn², Stefan Juretschko³, Romesh Gautom³

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Affiliations: 1: Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA 98195-7110; 2: Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, 85350 Freising, Germany; 3: Public Health Laboratories, Washington State Department of Health, 1610 NE 150th Street, Shoreline, WA 98155

Content Type: Monograph

Publication Year: 2002

Category: Bacterial Pathogenesis

Book DOI: 10.1128/9781555817985

Chapter DOI: 10.1128/9781555817985.ch42

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Abstract:

In this chapter the authors report the application of fluorescence in situ hybridization (FISH) for the direct detection and identification of Legionella spp. and L. pneumophila from respiratory tract specimens of patients with pneumonia using published 16S rRNA-based probes specific for the genus Legionella (Leg705) and for L. pneumophila (LegPne1). To evaluate the usefulness of FISH as a clinical diagnostic method, the authors analyzed 160 respiratory samples (bronchoalvealar lavage (BAL), pleural fluid, sputum, and tissue) from patients suspected of having pneumonia caused by Legionella spp. Probes Leg705 and LegPne1, one labeled with fluorescein and the other labeled with Cy3, were added simultaneously to each well, except for the negative-control well, where hybridization buffer alone was added. Almost two-thirds of Legionella pneumonia cases identified were caused by either L. longbeachae or L. micdadei. Given this background, development of a series of probes specific for the predominant species may be of value for identification and epidemiologic purposes.

Citation: Hu J, Limaye A, Fritsche T, Horn M, Juretschko S, Gautom R. 2002. Direct Detection of Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization, p 221-224. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), Legionella. ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch42

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Direct Detection of Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization

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/content/10.1128/9781555817985.chap42.fig42-1

FIGURE 1

Microscopic examination of BAL specimen after hybridization with probe LegPnel that is specific for L. pnemophila. L. pneumophila was seen as a cluster of intracellular bacilli (arrowhead).

10.1128/9781555817985/fig42-1_thmb.gif

10.1128/9781555817985/fig42-1.gif

FIGURE 1

Microscopic examination of BAL specimen after hybridization with probe LegPnel that is specific for L. pnemophila. L. pneumophila was seen as a cluster of intracellular bacilli (arrowhead).

References

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1. Amann, R. I.,, W. Ludwig,, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143– 169.

2. Breiman, R. F.,, and J. C. Butler. 1998. Legionnaires' disease: clinical, epidemiological, and public health perspectives. Semin. Respir. Infect. 13: 84– 89.

3. Doebbeling, B. N.,, M. J. Bale,, F. P. Koontz,, C. M. Helms,, R. P. Wenzel,, and M. A. Pfaller. 1988. Prospective evaluation of the Gen-Probe assay for detection of legionellae in respiratory specimens. Eur. J. Clin. Microbiol. Infect. Dis. 7: 748– 752.

4. Edelstein, P. H.,, K. B. Beer,, J. C. Sturge,, A. J. Watson,, and L. C. Goldstein. 1985. Clinical utility of amonoclonal direct fluorescent reagent specific for Legionella pneumophila: comparative study with other reagents. J. Clin. Microbiol. 22: 419– 421.

5. Finegold, S. M. 1988. Legionnaires' disease— still with us. N. Engl. J. Med. 318: 571– 573.

6. Fraser, D. W.,, T. R. Tsai,, W. Orenstein,, W. E. Parkin,, H.J. Beecham,, R. G. Sharrar,, J. Harris,, G. F. Mallison,, S. M. Martin,, J. E. McDade,, C. C. Shepard,, and P. S. Brachman. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297: 1189– 1197.

7. Grimm, D.,, H. Merkert,, W. Ludwig,, K. H. Schleifer,, J. Hacker,, and B. C. Brand. 1998. Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe. Appl. Environ. Microbiol. 64: 2686– 2690.

8. Grimont, P. A.,, F. Grimont,, N. Desplaces,, and P. Tchen. 1985. DNA probe specific for Legionella pneumophila. J. Clin. Microbiol. 21: 431– 437.

9. Krimmer, V.,, H. Merkert,, C. von Eiff,, M. Frosch,, J. Eulert,, J. F. Lohr,, J. Hacker,, and W. Ziebuhr. 1999. Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization. J. Clin. Microbiol. 37: 2667– 2673.

10. Mahbubani, M. H.,, A. K. Bej,, R. Miller,, L. Haff, J. DiCesare,, and R. M. Atlas. 1990. Detection of Legionella with polymerase chain reaction and gene probe methods. Mol. Cell. Probes 4: 175– 187.

11. Manz, W.,, R. Amann,, R. Szewzyk,, U. Szewzyk,, T. A. Stenstrom,, P. Hutzler,, and K. H. Schleifer. 1995. In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology 141: 29– 39.

12. Plouffe, J. F.,, T. M. File, Jr.,, R. F. Breiman,, B. A. Hackman,, S. J. Salstrom,, B. J. Marston,, and B. S. Fields. 1995. Re-evaluation of the definition of Legionnaires' disease: use of the urinary antigen assay. Community Based Pneumonia Incidence Study Group. Clin. Infect. Dis. 20: 1286– 1291.

13. Trebesius, K.,, K. Panthel,, S. Strobel,, K. Vogt,, G. Faller,, T. Kirchner,, M. Kist,, J. Heesemann,, and R. Haas. 2000. Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation. Gut 46: 608– 614.

Tables

Direct Detection of Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization

/content/10.1128/9781555817985.chap42.tab42-1

TABLE 1

Detection limit of Legionella spp. using FISH with probe LegPnel compared with culture on BCYE agar

10.1128/9781555817985/tab42-1_thmb.gif

10.1128/9781555817985/tab42-1.gif

TABLE 1

Detection limit of Legionella spp. using FISH with probe LegPnel compared with culture on BCYE agar

/content/10.1128/9781555817985.chap42.tab42-2

TABLE 2

Comparison of culture, DFA test, and FISH in patients with legionellosis

10.1128/9781555817985/tab42-2_thmb.gif

10.1128/9781555817985/tab42-2.gif

TABLE 2

Comparison of culture, DFA test, and FISH in patients with legionellosis

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