Detection of Legionnaires' Disease 



Bacteria in Clinical Specimens 



by 



Direct Immunofluorescence 



William B. Cherry and Roger M. McKinney* 



INTRODUCTION 



Direct fiuorescent antibody (FA) staining is useful for detecting Legionnaires' disease bac- 

 teria (LDB) in clinical specimens and in environmental samples. In early studies of the LDB, a 

 fluorescein isothiocyanate (FITC) conjugate of rabbit antibodies to the Knoxville 1 strain stained 

 all of 13 available isolates (i, 4). LDB isolates have since been obtained that do not stain with the 

 Knoxville 1 conjugate. Four serogroups of LDB have been recognized to date by direct FA 

 staining. Knoxville 1 , isolated by McDade from a postmortem lung specimen from a patient in 

 Knoxville, Tennessee, is representative of serogroup 1 ; Togus 1 , isolated by McDade from a 

 postmortem lung specimen from a patient in Togus, Maine, is representative of serogroup 2 (9); 

 Bloomington 2, an environmental isolate obtained from a creek in Bloomington, Indiana (2, 11), 

 is representative of serogroup 3; Los Angeles 1. obtained from a liunian lung specimen (5) is 

 representative of serogroup 4. Serogroup-specific FA conjugates for each of the four recognized 

 serogroups (70), and a polyvalent conjugate containing all four group-specific conjugates have 

 been prepared. The current practice in diagnostic direct FA staining at the Center for Disease 

 Control (CDC) is to first test clinical specimens by staining with the polyvalent reagent. If the 

 tissue is FA positive, the serogroup is determined by staining with each of the four group-specific 

 conjugates. Although the great majority of FA positive clinical specimens tested at the CDC have 

 reacted only to serogroup 1 conjugate, four clinical specimens have been found positive only with 

 serogroup 2 conjugate, three only with serogroup 3 conjugate, and two only with serogroup 4 

 conjugate. It is likely that additional serogroups of LDB will be recognized in the future. There- 

 fore, when isolates are obtained that have the growth characteristics and colonial appearance of 

 LDB but do not stain with polyvalent conjugate, further definitive tests for LDB such as bio- 

 chemical tests, gas4iquid chromatography of cellular fatty acids, and measurements of DNA 

 relatedness must be performed. 



Legionnaires' disease (LD) is naturally acquired by the respiratory route, and organs other 

 than the lung rarely appear to be involved. Therefore, this chapter deals almost exclusively with 

 detecting LDB in lung tissue or lung exudates. If all available clinical, epidemiological, micro- 

 biological, and serological data are considered in interpreting direct FA test results, they are 

 extremely valuable for indicating the scope of an outbreak and for establishing retrospective and 

 current diagnoses. 



*The contributions of Bertie Pittman, Patricia P. Harris, G. Ann Hebert, Berenice M. Thomason. LeRoy Tliacker, and Karen 

 Lewallen of the Immunofluorescence Section. Analytical Bacteriology Brancli, who participated in tlie development of the 

 methods described, are gratefully acknowledged. 



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