Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Immunonuorescence 



be considered as only suggestive for the diagnosis of Legionnaires' disease and definitive diagnosis 

 should be established by culture." In respect to the direct fluorescent antibody (D.F.A.) test, this 

 view places unjustified constraints on the application of the test and limits its proven value. Our 

 experience with large numbers of human and animal diagnostic specimens over the past 2 years 

 supports the reliability of the test if the methodology presented in this chapter is used. In the 

 hands of experienced workers, the specificity of the direct test is high. Failure to isolate LDB 

 from clinical materials that are positive by direct FA staining does not necessarily indicate 

 cross-reactivity. The best available media are probably still suboptimal for growth and the LDB 

 are quite susceptible to inhibition by contaminants. The organisms may be drug inhibited or they 

 may have lost viability during storage or handling of the specimens. If non-LDB are present and 

 responsible for positive staining, they must be more difficult to isolate than the LDB because we 

 have not encountered them during the examination of thousands of colonies from human and 

 guinea pig tissues and from sputum and environmental specimens. We isolated only one organism, 

 a single strain of Ps. fluorescens, that is antigenically related to the serogroup 1 LDB. It was 

 present as a contaminant in commercial Evans' blue counterstain. Twenty-two laboratory- 

 maintained strains of Ps. fluorescens were nonreactive with the group specific and polyvalent 

 LDB conjugates. Over 400 pure cultures representing 25 genera and 60 species and 54 unidenti- 

 fied cultures were negative. Lung tissues from a number of patients with pneumonia known to be 

 caused by other bacteria or fungi also were negative when tested with LDB conjugates. 



Undoubtedly the future will reveal some additional antigenic relationships between LDB 

 and other organisms. Certainly, every effort should be made to isolate and identify organisms 

 which are stained by the LDB conjugates. However, sufficient experience has been accumulated 

 attesting to the specificity of the direct FA test for the LDB to place the burden of proof of 

 nonspecificity on those who suggest otherwise. 



The fact remains that direct FA staining of LDB is still the only rapid diagnostic test for 

 these organisms and also the only specific laboratory test that can be used by clinicians for 

 guidance in instituting therapy. Autopsy and biopsy tissue can be screened for LDB more quickly 

 by the direct FA test than by pathological examination. In addition the direct FA test has a 

 dimension of serological specificity not possessed by the Dieterle silver impregnation stain and 

 other histological staining techniques used to demonstrate the organisms in tissue. 



PERFORMANCE CHARACTERISTICS 



1. The diagnostic dilution of an LDB polyvalent or monovalent conjugate should stain, at least 

 to a 3+ intensity, all known strains of LDB belonging to the serogroups (polyvalent) or to a 

 serogroup (monovalent). 



2. The corresponding control conjugate at its use dilution should not stain any of the stock 

 strains to more than a 1 + intensity. 



SUMMARY 



In this chapter we discuss the basic steps required for detection of the LDB by direct 

 immunofluorescence. The question of controls and the inteipretation of results and limitations of 

 the direct FA tests are also discussed. Questions related to specificity and sensitivity are treated 

 in some detail. An Addendum includes the preparation of vaccine for antiserum production, the 

 preparation of reagents, and a listing of LDB isolates by serogroup. 



Finally, the color plate (Figures 1-8) shows the appearance of the LDB in pure culture at 

 different ages, in Formalin-fixed tissue sections, in scrapings of wet Formalin-fixed tissue, and in 

 sputum from LD patients. 



97 



