DJM HALDANE, R PEPPARD, RK SUMARAH. Direct immunofluor escenc e for t he d iagnosis of legionellosis. Can J Infect Dis 1993;4(2):101-104. Culture and direct immunofluorescent m icroscopy (DFA) results for Legionellapneumophila were reviewed over a two-year period. In the first year, a positive result was defined as having at least one morphologically typical fluorescing organism. In the second year. a positive was defined as at least five typical fluorescing organisms. Despite these stricter criteria and other measures to reduce the possibility of reagent contamination, there was no statistically significant difference in the sensitivity or specificity of the DFA in the two years for sputa, deep specimens or overall. Of 37 sputum specimens from infected patients, 16 were positive on DFA. Thirty-two of38 positive patients were detected by sputum culture. DFA can provide rapid diagnostic information but cannot be used to rule out the diagnosis. Sputum is a useful specimen for the initial laboratory investigation of patients with legionellosis. de donner des renseignements diagnostiques rapides. mais elle ne peut etre utilisee pour eliminer les diagnostics. L&apos;expectoration est un prelevement utile pour le premier examen de laboratoire des patients atteints de legionellose

FCSLT Robert K Peppard

J M David

MB Frc Haldane

MSc Sumarah

Ruth Pc

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ORIGINAL ARTICLE 
Direct immuno-
fluorescence for the 
diagnosis of legionellosis 
DAVID JM HALDANE, MB, FRCPC, RUTH PEPPARD, FCSLT, ROBERT K SUMARAH, MSc 
DJM HALDANE, R PEPPARD, RK SUMARAH. Direct immunofluorescenc e for the diagnosis of legionellosis. 
Can J Infect Dis 1993;4(2):101-104. Culture and direct immunofluorescent microscopy (DFA) results for 
Legionellapneumophila were reviewed over a two-year period. In the first year, a positive result was defined 
as having at least one morphologically typical fluorescing organism. In the second year. a positive was 
defined as at least five typical fluorescing organisms. Despite these stricter criteria and other measures to 
reduce the possibility of reagent contamination, there was no statistically significant difference in the 
sensitivity or specificity of the DFA in the two years for sputa, deep specimens or overall. Of 37 sputum 
specimens from infected patients, 16 were positive on DFA. Thirty-two of38 positive patients were detected 
by sputum culture. DFA can provide rapid diagnostic information but cannot be used to rule out the 
diagnosis. Sputum is a useful specimen for the initial laboratory investigation of patients with legionellosis. 
Key Words: Immunofluorescence, Legionella, Sensitivity. Specificity 
L'immunofluorescence directe dans le diagnostic de Ia legionellose 
REsUME: Les resultats de culture et de microscopie par immunofluorescence directe (DFA) concernant 
Legionella pneumophila ont ete passes en revue sur une periode de deux ans. Pour Ia premiere annee. un 
resultat positif a ete defini par au moine un micro-organisme fluorescent morphologiquement typique. Dans 
Ia deuxieme annee, un resultat positif a ete defini par au moins 5 micro-organismes fluorescents 
morphologiquement typiques. Malgre ces criteres plus stricts et d'autres mesures visant a reduite Ia 
possibilite d'une contamination des reactifs. il n'a pas ete observe de difference statistiquement significative 
dans Ia sensibilite ou specificite de Ia microscopie en DFA au cours des deux ans, concernant les 
expectorations, les prelevements profonds ou !'ensemble des prelevements. Sur 37 echantillons d'expecto-
ration provenant de patients infectes, 16 etaient positifs a l'examen microscopique en DFA. Trente-deux 
des 38 patiente positifs avaient ete detectes par culture d'expectoration. La microscopie en DFA est capable 
de donner des renseignements diagnostiques rapides. mais elle ne peut etre utilisee pour eliminer les 
diagnostics. L'expectoration est un prelevement utile pour le premier examen de laboratoire des patients 
atteints de legionellose. 
Department of Microbiology. Victoria General Hospita~ Halifax, Nova Scotia 
Correspondence and reprints: Dr DJM Haldane, Department of Microbiology, Victoria General Hospital, 5788 University Avenue. 
Halifax, Nova Scotia B3H 1 VB. Telephone (902) 428-2392 
Receivedfor publication September 5, 1991. Accepted June 16. 1992 
CAN J INFECT DIS VOL 4 No 2 MARCH/ A PRIL 1993 101 
HALDANE ef a/ 
LEG/ONELLA PNEUMOPHILA IS A SIGNIFICANT CAUSE OF' NOSO-comial pneumonia. Early recognition and treat-
ment is important. and therefore rapid means of diag-
nosis are desirable . Culture is the most sensitive 
technique. but results may not be available for up to 
nine days (1). Serology is of use as a retrospective 
means of diagnosis. Antigen detection is possible using 
latex agglutination (2). radioimmunoassay (3). enzyme-
linked immunosorbent assay (4), nucleic acid probe (5) 
and immunofluorescence microscopy (6). A direct im-
munofluorescent staining method (DFA) has been used 
for many years in our laboratory. in combination with 
culture. This paper reviews our experience with culture 
and immunofluorescent staining oflegionella using two 
sets of criteria to define a positive result for the DFA. 
METHODS 
In the first year of the study (January to December 
1987) 515 specimens were submitted for legionella DFA 
and culture from 353 patients. In the second year 
(1988) there were 538 specimens from 385 patients 
(Table 1). Specimens were cultured on buffered char-
coal yeast extract agar with alpha-ketoglutarate 
(BCYE), and on two selective media: BCYE supple-
mented with cefamandole (4 mg/L), polymyxin B 
(80.000 u/L) and anisomycin (80 mg/L); and BCYE 
with polymixin (40.000 u/L), anisomycin (80 mg/L) and 
vancomycin (0.5 mg/L) (Gibco, Wisconsin) (7). In addi-
tion, a sheep blood agar plate was used to assess other 
flora as routine sputum cultures are performed else-
where in the laboratory. Fluid specimens were concen-
trated by centrifugation, and tissues were homogenized 
and diluted 1:100 and 1:10,000 in sterile water prior to 
culture. The cultures were incubated at 37°C in 5% 
carbon dioxide and examined daily using a stereo-
microscope for up to one week. Isolates were identified 
using direct immunofluorescence and the inability to 
grow on blood agar. 
DFA was performed using a polyclonal fluorescein 
isothiocyanate (F'ITC)-labelled rabbit immunoglobulin 
directed against L pneumophila serogroup 1, Knoxville 
strain (Centers for Disease Control, Georgia). Slides 
were stained in accordance with the manufacturer's 
instructions. Briefly, slides were prepared in a safety 
cabinet, air dried, heat and formalin fixed, and stained 
with the antisera using laboratory prepared reagents. A 
negative control with F'ITC-labelled nonspecific rabbit 
antisera was included with each set of slides to be 
stained. A positive slide had at least one brightly fluo-
rescing apple green rod seen in the test slide (8). with 
no fluorescing rods seen in the negative control. DFA 
was performed without knowledge of the culture result 
of the specimen. 
Turn around times for culture were measured 
retrospectively by review of laboratory records. and 
defined as the number of days from the day of receipt 
in the laboratory unW the day the final report was 
102 
TABLE 1 
Number and types of specimens received for direct 
immunofluorescent assay and culture of legionella 
1987 (n=353) 1988 (n=385) 
383 408 
Bronchial wash 94 108 
Tissue 30 18 
Sterile fluid 7 4 
Post mortem lung swab 0 
Total 515 538 
n Number of patients 
issued. All positive reports were telephoned to the pa-
tient's unit. DFA was performed on the day of receipt or 
on the next working day if the specimen was received 
outside normal working hours. 
In the second year, methods for culture remained the 
same. DFA was performed using reagents prepared 
with commercial steam sterilized water (sterile water for 
irrigation USP, Baxter Corp). as previous cultu res of 
faucets in the laboratory had been positive for L pneu· 
mophila. A positive result was defined as having at least 
five typically staining organisms per slide (9). 
Staining procedures with DFA reagents were re-
viewed to ensure positive slides were separated from 
others. The reagents, including the formalin, the swabs 
used and sterile water were culture-negative for le-
gionella. DFA to detect viable but nonculturable organ-
isms was also negative. Reagent container design was 
changed to minimize the danger of airborne contamina-
tion of reagents. 
Specimen results were analyzed as either sputa or 
deep specimens, which included bronchial washes, tis-
sues, pleural fluids and a post mortem lung swab. 
Culture was used as the reference technique. Speci-
mens giving positive DFA results with negative cultures 
were interpreted as being false positive. The DFA re-
sults were analyzed to calculate sensitivity, specificity, 
positive and negative predictive values, and efficiency 
relative to culture. The results for each year were sta-
tistically analyzed using Fisher's exact test for low 
numbers or the l test. 
RESULTS 
During the first year there were 27 culture-positive 
specimens from 23 patients. In the second year there 
were 19 culture-positive specimens from 16 patients. 
The number of discrepant results is shown in Table 2. 
Over the two years of the study, 38 patients were 
identified with infection with L pneumophila serogroup 
1. A further patient's cultures yielded Legionella boze-
manii; the DFA was negative as expected. Thirty-two of 
these 38 positive patients (84%) had positive sputum 
cultures. These cases occurred sporadically throughout 
the period, and no point source of infection was impli-
cated. 
CAN J INFECT DIS VOL 4 No 2 MARCH/ APRIL 1993 
The mean of the tum around times for sputum 
culture was five days (95% confidence interval, 4.45 to 
5.54) and for bronchial wash specimens was 5.4 days 
(95% confidence interval, 3.93 to 6.92). 
The analysis of the performance of the DFA relative 
to culture is summarized in Table 3. There was no 
statistically significant difference in the sensitivity, 
specificity, predictive values or efficiency between the 
two years. 
DISCUSSION 
The legionella DFA has been shown previously to be 
a specific rapid test for the diagnosis of legionellosis 
(10). Our results confirm this finding. The DFA was 
specific for both sputum and deep specimens. 
False positive results may arise either because of 
other species of microorganisms cross-reacting with 
the antisera used, or from environmental L pnewno-
phila contaminating the specimen. The similar specifi-
cities for sputum and deep specimens, despite the 
oropharyngeal flora, suggest that cross-reaction by 
other species was not the major cause of false positives. 
Culture for anaerobes and exhaustive screening for 
cross-reacting organisms, however, were not per-
formed. Contamination of specimens with legionella 
may occur either from the patient, the container or in 
the laboratory. The patient may be colonized (8,11) may 
have been treated successfully, or may be transiently 
contaminated as a result of drinking or mouth rinsing 
with colonized potable water. The specimen may be 
contaminated by airborne organisms or splashes from 
contaminated potable water; the container may have 
been previously contaminated during production and 
irradiated, resulting in nonviable but morphologically 
intact organisms. Many of the modes of contamination 
outside the laboratory would introduce viable organ-
isms to the specimen. As culture is much more sensi-
tive than DFA. cultures could be expected to be positive 
if these modes occurred. The dilution of nonviable 
organisms introduced to a specimen would require a 
large inoculum if DFA were to be rendered positive as a 
result. In the laboratory, con tamination may occur via 
the airborne route or by contamination of reagents 
which, when used in the preparation of stains, could 
allow large numbers of viable or nonviable organisms to 
be introduced. Phosphate buffered saline has been 
determined to be the source of false positives in one 
study; the incidence of false positives dropped following 
replacement of the reagents (1 2). In the present study, 
replacement of reagents did not result in a statistically 
significant change in the number of false positive re-
sults. 
The sensitivity of DFA is not sufficient for it to be 
used to 'rule out' infection with legionella. However, 
provided clinicians recognize this limitation, it can pro-
vide useful data rapidly. Sixteen of 37 sputa (43%) and 
six of nine deep specimens (67%) were detected using 
CAN J INFECT DIS VOL4 No 2 MARCH/ A PRIL 1993 
Laboratory diagnosis of legionellosls 
TABLE 2 
Results of culture and direct immunofluorescent assay 
(DFA) for sputum and deep specimens in 1987 and 1988 
Year Culture DFA Sputum Deep Total 
1987 + + 11 4 15 
+ 10 2 12 
+ 14 5 19 
348 121 469 
1988 + + 5 2 7 
+ 11 1 12 
+ 2 0 2 
390 127 517 
TABLE 3 
Summary of the performance of direct immunofluore-
scent assay relative to c ulture in 1987 and 1988 
Overall Sputa Deep 
1987 1988 1987 1988 1987 1988 
Sensitivity 55.5 37 52.4 31 .3 66.6 66.6 
Specificity 96.1 99.6 96.1 99.5 96 100 
PPV 44.1 77.8 44 71 .4 44.4 100 
NPV 97.5 97.7 97.2 97.3 98.4 99.2 
Prevalence 5.2 3.5 5.5 3.9 4.5 2.3 
NPV Predic tive value of a negative test; PPV Predic tive value of a positive 
test 
the DFA. Clinicians must consider the results in light 
of the pretest probability of disease because of the low 
positive predictive values, and culture should always be 
used to confirm DFA results. 
Sputum was a useful specimen. Thirty-two of 38 
patients with legionellosis were detected using sputum 
culture. As the DFA can provide information within 
hours of a specimen being taken, initial examination of 
sputum samples may avoid the need for more invasive 
procedures in many patients, and specific treatment 
may be initiated earlier in the illness. Clinicians should 
not assume cultures are negative for at least six days 
after specimens are set up. Where legionella is sus-
pected as a possible cause of pneumonia, treatment 
should include coverage for legionella for the full course 
or unW it is no longer suspected. The DFA result is 
useful to supplement clinical impressions to influence 
decisions made concerning antimicrobial therapy. 
The prevalence of positive specimens in the study 
was high possibly because of an increased incidence 
relative to other institutions, and careful selection of 
patients that had cultures taken. Legionella cultures 
were not performed as part of the routine sputum 
culture. The prevalence of disease influences the pre-
dictive values of a test. In centres with low prevalence, 
below 0 .5% of specimens examined, the positive predic-
tive value may fall to unacceptable levels despite the 
specificity being 99.5%. Strict control of the specimens 
examined may increase the prevalence in the speci-
mens examined, but maintenance of expertise of read-
ing the DFA may be difficult. 
103 
HALDANE eta/ 
The number of fluorescing organisms required to call 
a specimen positive was changed from at least one to at 
least five. Unfortunately. because of the test's clinical 
importance. this change was made when reagents were 
altered to minimize possible contamination. The second 
year re ults. however. did not show a statistically sig-
nificant change in test specificity and although the 
sensitivity dropped from 52.4 to 31.3% for sputum 
specimens (ll of21 in 1987 versus five of 16 in 1988. 
two tailed P=0.54). this change did not reach statistical 
significance. Use of the less stringent criterion for posi-
tivity did not result in a significant loss of specificity 
and therefore should be preferred for routine use. 
Sputum is the initial specimen of choice for the 
laboratory diagnosis of legionellosis because it is easy 
to obtain and noninvasive. Many patients may be diag-
nosed rapidly using the DFA. and the majority detected 
by culture. The DFA of sputum using the criteria of 
more than one organism per slide for a positive result 
was a useful procedure where the prevalence of infec-
tion is sufficiently high to yield satisfactory predictive 
values and maintain expertise. The sensitivity of the 
DFA from eiU1er sputa or deep specimens is insufficient 
to rely on DFA to exclude the diagnosis of legionellosis. 
REFERENCES 
I. Dournon E. Isolation of Legionellae from clinical 
specimens. In: Harrison TG. Taylor AG. eds. A Laboratory 
Manual for Legionella. Toronto: John Wiley and Sons. 
1988:13-30. 
2. Sathapatayavongs B. Kohler RB. Wheat W. White A. 
Winn WC. Rapid diagnosis of Legionnaires· disease by 
latex agglutination. Am Rev Respir Dis 1983:127:559-62. 
104 
3. Kohler RB. Zimmerman SE. Wilson E. et al. Rapid 
radioimmunoassay diagnosis of Legionnaires· disease: 
Detection and partial characterization of urinary antigen. 
Ann Intern Med 1981:94:601-5. 
4. Sathapatayavongs B. Kohler RB. Wheat W. et al. Rapid 
diagnosis of Legionnaires· disease by urinary antigen 
detection: Comparison of ELISA and radioimmunoassay. 
Am J Med 1982:72:576-82. 
5. Kohne DE. Steigenvalt AG. Brenner OJ. Nucleic acid 
probe specific for members of the genus Legionella. In: 
Thornsberry C. Balows A. Feeley JC. Jakubowski W. eds. 
Legionella Proceedings of the 2nd International 
Symposium. Washington: An1erican Society for 
Microbiology. 1984:107-8. 
6. Cherry WB. Pittman B. Harris PP. et al. Detection of 
Legionnaires· disease bacteria by direct 
immunofluorescent staining. J Clin Microbial 
1978:8:329-38. 
7. Edelstein PH. Improved semi selective medium for 
isolation of Legionella pneumophila from contaminated 
clinical and environmental specimens. J Clin Microbial 
1981:14:298-303. 
8. Bridge JA. Edelstein PH. Oropharyngeal colonisation with 
Legionella pneumophila. J Clin Microbial 
1983:18:1108-12. 
9. McKinney RM. Diagnosis and direct immunofluorescent 
antibody test. In: Katz SM. ed. Legionellosis. vol 2. Bocca 
Raton: CRC Press. 1985:23-32. 
10. Harrison TG. Taylor AG. Demonstration of Legionellae in 
clinical specimens. In: Harrison TG. Taylor AG. eds. A 
Laboratory Manual for Legionella. Toronto: John Wiley 
and Sons. 1988:103-12. 
11. Manie TJ. Bezanson GS. Haldane DJM. Burbridge S . 
Colonisation of the respiratory tract \vith Legionella 
pneumophila for 60 days before the onset of pneumonia. 
J Infect 1992:24:81-6. 
12. Ristagno RL. Saravolatz LD. A pseudoepidemic of 
Legionella infections. Chest 1985:88:466-7. 
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Direct immuno- fluorescence for the diagnosis of legionellosis

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Direct immuno- fluorescence for the diagnosis of legionellosis

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