Indirect Inimunonuoresccncc Test tor Legionnaires' Disease 



D. Fluorescence Microscopy 



1 . Fluorescence intensities are read as follows: 



4+ = brilliant yellow-green staining of bacterial cells 



3+ = bright yellow-green staining 



2+ = definite but dim staining 



1+ = barely visible staining 



— = no staining, although dim autolluorescence of the cells may occur with 



some filter systems (see below) 



CAUTION: Disregard staining of filamentous cells and of cells on or adjacent 



to fluorescent yolk sac material. 



2. Microscope Assemblies 



We have found that the following give satisfactory results: Leitz Dialux 20 fluorescence 

 microscope equipped with an HBO-100 mercury incident light source; the Leitz I-cube 

 filter system (2 x KP490 and a 1-mm GG455 primary filter. TK5 10 dichroic beam-splitting 

 mirror and K515 secondary filter) or Leitz K-cube filter system (same as Lcube except 

 1-mm GG455 filter is replaced by a 2-mm GG475 or a K480 edge filter to reduce back- 

 ground tluorescence), 40 X dry objective, and 6.3 X eyepiece (magnification- 315 X). 

 Other microscope assemblies may also be satisfactory. 



E. Interjjretation* 



1. A 4-fold or greater increase in titer to >128 in sera obtained in the acute and convales- 

 cent phases of illness provides serological evidence of Legionnaires' disease. Optimal time 

 intervals from onset of illness to specimen collection appear to be <7 days for the acute- 

 and >21 days (but <6 weeks) for the convalescent-phase serum. 



2. A single or standing titer of >256 provides presumptive evidence of an LDB infection 

 at an undetermined time. Until more data are accumulated to show (a) how long a detect- 

 able level of antibody usually persists after infection with the LDB, and (b) tiie average 

 LDB antibody concentration in sera of apparently nomial individuals, the significance of 

 a single high titer will remain unknown. 



3. All other findings are considered negative. 



SELECTED REFERENCES 



1. Jones. Gilda L., G. Ann Hebert, and William B. Cherry. 1978. Fluorescent Antibody Techniques and Bac- 

 terial Applications. Center for Disease Control. Public Health Service, U.S. Department of Health, Education, 

 and Welfare, Atlanta. GA. 



2. McDade. J.E.. C.C. Shepard. D.W. Eraser. I.E. Tsai. M.A. Redus. VAR. Dovvdle. and Laboratory Investigation 

 Team. 1977. Legionnaires' disease. Isolation of a bacterium and demonstration ofits role in other respiratory 

 disease. New Engl. J. Med. 297:1197-1203. 



3. Tsai, T.E., and D.W. Eraser. 1978. The diagnosis of Legionnaires' disease. Ann. Intern. Med. 89:413-414. 



4. Wilkinson, H.W., B.J. Eikes, and D.D. Cruce. 1979. Indirect immunofluorescence test for serodiagnosis of 

 Legionnaires' disease: Evidence for serogroup diversity of Legionnaires" disease bacterial antigens and for 

 multiple specificity of human antibodies. J. Clin. Microbiol. 9:379-383. 



*Although these interpretations appear to be valid for serogroup 1 infections, they may not be vahd for those caused by members 

 of other serogroups. The serogroup to which the infecting strain belongs cannot be determined unequivocally by results of the 

 indirect IF test alone. 



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