Indirect Immunofluorescence Test 

 for Legionnaires' Disease 



Hazel W. Wilkuisoii, Donna D. Cruce, Bonnie J. Fikes. 

 Lynn P. Yealy, and Carol E. Farshy 



INTRODUCTION 



The indirect immunofluorescence (IF) test for Legionnaires' disease (LD) has been modified 

 several times since it was first used by McDade et al. to provide serological evidence that an or- 

 ganism which had been isolated from patients with Legionnaires' disease was actually the causa- 

 tive agent of that disease. We now know that at least four different serogroups exist, with indi- 

 viduals responding immunologically in different ways to serogroup-specific antigenic determi- 

 nants. Some patients appear to respond to the Legionnaires' disease bacterium (LDB) with a 

 group-specific antibody response; others respond to antigenic determinants presumably common 

 to serogroups 1-4. Preliminary data obtained at the Center for Disease Control (CDC) suggest 

 that a polyvalent antigen (not yet in general use) can be used to screen sera for LDB antibodies. 

 Positive sera are then titrated against monovalent antigens. We now use heat instead of diethyl 

 ether to kill the organisms because ether extracts or destroys the serogroup 2 (Togus 1) antigen. 

 Further modifications of the test may be necessary as we gain more knowledge about the organ- 

 ism and about the immune response to the LDB. 



Perhaps because specific diagnostic criteria for LD have not been completely formulated, 

 expectations of sensitivity, specificity, and reproducibility have been unusually liigh for the LD 

 indirect IF test. Nevertheless, it is limited by the same immunologic parameters that affect 

 other serological tests. For example, repeat tests in which titers are obtained that vary by no 

 more than one 2-foid dilution are generally considered acceptable within limits of experimental 

 error. Therefore, paired sera should be tested simultaneously. A 4-fold rise in titer from the 

 acute to the convalescent phase of illness is usually considered the only solid serologic evidence 

 of infection. Some sera from patients with LD do not contain detectable antibodies against the 

 identified "infecting" strain of the LDB, whereas sera from some other patients infected with 

 dissimilar pathogens may contain antibodies that react with LDB, presumable because of sim- 

 ilarities of the heterologous organisms" antigenic structure or perhaps because of nonspecific 

 stimulation of the reticuloendothelial lymphocytic system. It is interesting that most of the 

 reported "cross-reactions" between LDB and other pathogens have not been confirmed. It 

 would be unusual, however, for serologic cross-reactions not to occur with antigenic mimicry 

 being so common in nature. No serologic test is lOO'^r sensitive or specific. For these reasons, 

 the indirect IF test should be used in conjunction with other diagnostic criteria. 



The indirect IF test for LD has the following components: (a) an antigen composed of 

 heat-killed, whole LDB cells, (b) appropriate dilutions of the human serum to be tested or of 

 a control serum, (c) rabbit antihuman conjugate [fluorescein isothiocyanate(FITC)-labeled rabbit 

 antibody with specificity for human immunoglobulins G and M|, and (d) an appropriately 

 equipped fluorescence microscope. 



