Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immiinonuorescence 



INTERPRETATION OF RESULTS AND TEST LIMITATIONS 



In all clinical specimens, except for lung exudates (LE), the following criteria are used to 

 evaluate the test results: 



Result Report 



>25 strongly fluorescing bacteria/smear* FA + 



<25 strongly fluorescing bacteria/smear #'s only 



strongly fluorescing bacteria/smear FA - 



*In many lA-positive specimens, an average of 50 or more LDB per field ( 1 250X) may be observed. 



Positive FA staining should be reported as FA+ (group 1, 2, 3, or 4) depending on which 

 diagnostic conjugate gave positive FA staining. In LE, organisms are seldom numerous. Thus, the 

 observation of more than five brightly stained small rods morphologically typical of the LDB 

 constitutes a positive result in this type of specimen. 



Inteipreting the results of stained tissue scrapings, sections of Formalin-fixed lung, fresh 

 lung imprints, cultures, and pleural fluids is rather straightforward {3, 4). Organisms in culture are 

 usually longer rods than those seen in tissues. In older cultures, long filaments, swollen rods, and 

 other bizarre fomismay be seen. 



Interpreting the results of stained LE specimens such as expectorated sputa, transtracheal 

 aspirates, and those obtained by bronchial lavage or by bronchoscopy is more difficult. Tissue 

 cells and white blood cells may be highly autotluorescent. Bacteria such as staphylococci, dip- 

 lococci, and streptococci may fluoresce because of natural antibodies which are in the senmi of 

 the immunized rabbit. Frequently, many of the LDB are disintegrating from the effects of 

 cellular defense mechanisms. Ragged, swollen, atypical fomis and fluorescent debris are com- 

 monly observed. A smear stained with the negative control conjugate must be studied as carefully 

 as one which is presumed to be positive. Familiarity with the moiphology and staining charac- 

 teristics of the LDB is imperative if false-positive reports are to be avoided. For example, one of 

 23 strains oi Pseiidomuiias fliiorescens tested was brightly and specifically stained by the working 

 dilution of the serogroup 1 LDB conjugate. The fluorescent Pseiidomo}uis rods may, however, 

 appear wider than the LDB rods. The use of counterstains for sputum and tissue examination has 

 proved very helpful. 



Because of the size of the particles of tissue obtained by scraping Formalin-fixed lung, many 

 particles are lost from the slide during processing. The free bacteria and tissue cells will, however, 

 remain on the slide to give a good test substrate. 



Although isolation and characterization of the LDB is discussed elsewhere in this Manual, 

 we emphasize the necessity of attempting to culture these organisms from lung tissue, pleural 

 Ouid and LE of patients suspected of having the disease. Culture should be attempted even 

 though no LDB-like bacteria are seen by direct FA staining. The organisms may be present in 

 numbers too small to be detected by FA staining or new serogroups of the LDB may be 

 producing infection. All LDB isolates should be fully characterized culturally, serologically, and 

 physiologically to deteimine if they fit the accepted criteria for classification as the LDB. 



DISCUSSION 



Recently, Lattimer, Rhodes, and Cepil {8) commented as follows: "the degree of fluores- 

 cence in the I.E. A. and D.F.A. tests used for diagnosing Legionnaires" disease is dependent not 

 only on the presence of the bacterium but also on the materials from which the organism is 

 isolated (lung, egg yolk sac, or artificial media) and by the method used to prepare the antigen. 



Until a standard antigen with defined sensitivity and specificity is developed, serodiagnosis should 



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