Direct Immunofluorescence in the Diagnosis of Legionnaires' Disease

Direct Immunofluorescence in the Diagnosis of Legionnaires' Disease" L. D. Saravolatz, M .D.;t G. Bussell, B.S.; and D. Cvitkovich, M.D. !

Over a 2¥.z-year period, 61 cHnicai specimens from 41 patients with pneumonia of uncertain etiology were evaluated for the presence of Legionella pneumophila (serogroups 1 to 4) by immunolluorescent antibody techniques. In 13 of 19 patients with Legionnaires' disease, the diagnosis was established by lIuorescent antibody (FA) staining of lung biopsies, pleuralllnids, or respiratory tract secretions. In tbe 19 patients with Legionnaires' disease, the diagnosis W88 confirmed by isolation of L pneumophila by in vitTo culture tecbniques in five or by measurement of serum antibody titers in 17. Although the FA staining tecbnique W88 of Umited sensitivity (68 per-

cent), it w. bighIy speclfic: DO patients with non-Legionnaires' pneumonia bad a faIse-positive lIuorescent stain. In addition, the FA staining of lung tissue W. positive only wben performed daring the first nine days of antimicrobial thenpy and wben an acute bronchopneumonia noted bistologicaDy. In cases of a nonspecific interstitial pneumonitis, FA stain was always negative, and the diagnosis could be confirmed only by serum antibody me&'lurements. Tests for serogroups 1 to 4 with a polyvalent conjugate sbowed that L pneum&fJhila serogrOup 1 W88 the predominant strain detected in pneumonia of uncertain etiology 10 the Detroit area.

Legionnaires' disease is suspected on the basis of a characteristic clinical presentation and the failure to identify another etiologic agent. Most cases are confirmed by demonstrating antibody rises from acute and convalescent sera. However, because antibody may not develop until two to six weeks after the onset of symptoms, other tests are needed to establish the diagnosis. 1 The preferred method for establishing the diagnosis is to recover L pneumophila from clinical specimens. While use of guinea pig inoculation and subsequent passage into embryonated eggs has been advocated by the Center for Disease Control (CDC), in Atlanta, this procedure is not readily available in a community hospital," Nevertheless, the organism can be recovered on artificial medium available in a microbiology laboratory that has the capability of culturing Neisseria gonorrhoeae or Hemophilus influenzae. However, because it may take several days for cultures of L pneumophila to become positive, the isolation of the etiologic agent is still unsatisfactory in providing a rapid diagnosis. Recovery of the organism from living patients has thus far been uncommon. This report describes such recovery from five patients in whom isolates from lung tissue, pleural fluids, or bronchial washings

were obtained. Although direct fluorescent antibody (FA) staining has been used to demonstrate L pneumophila in clinical specimens, it has been used primarily in autopsy specimens for retrospective diagnosis," Recently, the procedure has been advocated as a rapid diagnostic test when used on sputum specimens. However, in one retrospective study, only five of 26 patients had positive FA staining of sputum specimens that were stored for various periods," Since the sensitivity and specificity of this procedure on clinical specimens are not known, we report the results of 61 clinical specimens evaluated prospectively from 41 patients with pneumonia of undetermined etiology that occurred in a community hospital over a 2~­ year period. In addition, the newly recognized serogroups 2 to 4 were included in the evaluation of lung biopsy specimens to determine the predominant strain of L pneumcphila responsible for pneumonia of uncertain etiology.

·From the Infectious Disease, Microbiology, and Pulmonary Divisions, Henry Ford Hospital, Detroit. tClinical Assistant Professor of Medicine, University of Michigan Medical School, Ann Arbor. tCurrently in Muskegon, Mich. Presented in part at the American Thoracic Society 74th Annual Meeting, Las Vegas, May 16, 1979. Reprint requests: Dr. Sat'avolatz, Department of Infectious Disease, Henry Fcwd Hospital, Detroit 48202

566 SARAVOLATZ, RUSSELL, CrnKOVICH

w.

MATERIALS AND METHODS

Specimens

Sixty-one clinical specimens from 41 patients were submitted for examination by FA technique over a 2~year period from May 1977 to Nov 1979. The specimens were divided into two patients groups : group 1 with 19 legionnaires' disease patients and group 2 with 22 non-Legionnaires' disease cases. Cases were defined as Legionnaires' disease if L pneumophUa was recovered from clinical specimens (five patients ), or if the patient demonstrated a fourfold rise in antibody titer between acute and convalescent sera (17 patients) . Two of the five patients in whom the diagnosis was confirmed by isolation of L pneumophila died during the first

CHEST, 79: 5, MAY, 1981

the polyvalent conjugate, they were stained with each of the monovalent components. For controls, smears from homologous and heterologous bacteria (Klebsiella pneumonia) were run at the same time as each specimen was processed. In addition, a control with fluorescein-conjugated antigammaglobulin (Baltimore Biological Laboratories), from an unimmunized animal was used on each clinical specimen. Slides were examined with a Zeiss fluorescence microscope equipped with HBD 200 lamp, BG 12 exciter filter, and K510 barrier filter. Smears were considered positive if five or more bacillary forms of morphology similar to L pneumophila demonstrated a typical apple-green fluorescence (Fig 1). They were considered negative if no fluorescent organisms could be detected after a ten-minute examination with the 100x oil immersion objective. FIGURE 1. Direct fluorescent antibody stain of isolate from case 1 cultivated on MHIH agar (original magnification X 400).

Culture Techniques Lung biopsy, pleural fluid, transtracheal aspirates, and bronchial washing specimens submitted for FA staining were also cultured for L pneumophila. These specimens were inoculated onto Mueller-Hinton agar (MHIH) and g-c base (Difco Laboratories, Detroit), supplemented with 2 percent hemoglobin (Bioquest, Inc) and 1 percent IsoVitaleX (Baltimore Biological Laboratories), adjusted to pH=7.0 and incubated under 5 percent CO? at 3·5°C for 20 days. In addition, charcoal yeast extract agar containing Difco charcoal agar, 0.04 percent L-cysteine hydrochloride, and 0.025 percent ferric pyrophosphate adjusted to a pH=6.9 to 7.0 was used for culture during the last six months of this study. This medium was added because of the superior recovery reported to be associated with it. Culture plates were examined for growth every 24 hours, and direct fluorescence as described previously was performed when growth was visible.

week of their illness. In these cases, no rise in antibody could be demonstrated. In addition, the demographic features, clinical presentation, antimicrobial therapy, and histopathologic findings of biopsy specimens were reviewed.

Fluorescent Antibody Techniques Clinical specimens submitted for FA staining included lung biopsies, pleural fluid, and respiratory tract secretions (sputum, transtracheal aspirates, and bronchial washings). Lung tissue submitted was obtained from an open lung biopsy and measured at least 2 cm X 2 ern X 2 cm. Specimens were processed within 24 hours after the laboratory received them. A touch imprint was performed on all lung biopsy specimens, and smears were made from pleural fluid and respiratory tract secretions. All specimens were heat fixed and then fixed in 10 percent formalin solution for 15 minutes. They were then stained with a conjugate prepared from hyperimmune rabbit antiserum from the Knoxville No.1 strain serogroup 1 ( courtesy of the CDC) by the method of Cherry et al. 5 The conjugate was employed at its diagnostic use of 1:80. Rhodamine-labeled bovine albumin (Baltimore Biological Laboratories, Cockeysville, Md) was used as a counterstain to increase visualization of L pneumophila. Lung biopsy specimens that did not stain with the serogroup 1 conjugate were subsequently stained with a polyvalent conjugate containing serogroup 1 (Knoxville No.1), serogroup 2 (Togus I), serogroup 3 (Bloomington 2), and serogroup 4 (Los Angeles 1), courtesy of the CDC. When specimens were positive with

Serology Serum specimens were obtained during the first week of illness and convalescent sera during the second and fourth weeks. If no rise in serum antibody was detected in the second specimen, a third specimen was obtained at six weeks after the illness began. A fourfold rise in titer to :2': 64 was required for a serologic diagnosls.s These tests were confirmed by the Michigan Department of Public Health Laboratories, Lansing. Paired sera was stored at _70 0 C and tested simultaneously for antibody to all four serogroups of L pneumophila with the same antigen that was used in preparing the polyvalent conjugate for FA testing.

Table I-Results of Direct Immunofluorescent Staining of 61 Clinical Specimens Group 1 Legionnaires' Disease Patients

Group 2 Non-Legionnaires' Disease Cases

----..A

.A-

% of

% of

No. Done

No. of Patients

No. of Specimens Positive

14

14

9 (5)*

64

64

0

15

15

4

4

3 (1)

75

75

0

6

6

Respiratory tract secretions

15

4

9t (1)

60

70

0

7

6

Total

33

19

21 (7)

64

68

0

28

22

Lung biopsies Pleural fluid

Specimens Positive

Patients Positive

No. Positive

No. Done

No. of Patients

*Number in parentheses is the number of positive cultures from the clinical specimen indicated. [Includes seven sputum, one transtracheal, and one bronchial washing.

CHEST, 79: 5, MAY, 1981

DIAGNOSIS OF LEGIONNAIRES' DISEASE 567

Table 2-Lu,.. B;o".r Rea"" of Group 1 Palie....

FACase Results Culture

2 3 -I

5 6

7

8 9

Histology

Duration of Antimicrobial Therapy'[

+

+

Acute bronchopneumonia with necrotic parenchyma

7

+ +

+ +

Acute bronchopneumonia

5

+ + + + + +

+

+

Acute bronchopneumonia with necrotic parenchyma

4

Acute bronchopneumonia

9

Acute bronchopneumonia

3

Acute bronchopneumonia

7

Acute bronchopneumonia

6

Acute bronchopneumonia

8

Acute bronchopneumonia

10

Interstitial pneumonia

12

11

Interstitial pneumonia

9

12

Interstitial pneumonia

10

13

Interstitial pneumonia

10

14

ND

Congestive ehangesf

3

-FA - Direct fluorescent antibody staining of lung biopsy specimens. tDuration of therapy before lung biopsy was performed. tInadequate specimen obtained from lung biopsy.

REsuLTS Clinical characteristics of both groups of patients were similar. Since our emphasis is on the diagnostic aspects of Legionnaires' disease, we will summarize the clinical details briefly. All patients were immunosuppressed, febrile, and had a pulmonary infiltrate. A lung biopsy was performed if no cause could be found to account for the pulmonary infiltrate. In each case, the primary physician believed that the clinical presentation might be compatible with a diagnosis of Legionnaires' disease. Respiratory secretions were evaluated only during the second year of this study. The FA staining and culture results are summarized in Table 1. Cultures of L pneumophila were confirmed by the CDC. Four isolates were from serogroup 1, and one isolate was from serogroup 3. Table 2 presents the lung histology and duration of antimicrobial therapy before the lung biopsies were performed. Since respiratory tract secretions generally contained few organisms, a full ten minutes was required before a slide was interpreted as negative. Lung tissue imprints of patients with Legionnaires' disease contained extremely large numbers of organisms, with more than 25 organisms per field during a brief slide examination . 568 SARAYOLATZ, RUSSELL, CYITIOYlCH

In all cases in which lung biopsies were performed during the first nine days of illness, the histology showed an acute bronchopneumonia, and the FA procedure was positive. In four cases in which the lung biopsy was performed after nine days of antimicrobial therapy, the direct FA test was negative, and the histology showed a nonspecific interstitial process. The one exception was case 14 (Table 2) , in which an inadequate lung biopsy specimen was obtained at the time of surgery. In all cases in which FA staining was negative with the serogroup conjugate, the lung biopsies were stained with a polyvalent (serogroup 1 to 4) conjugate. These specimens were all negative except case 9 (Table 2), which stained with 4 + intensity and was found to be a serogroup 3 case when stained with monovalent components of the polyvalent conjugate. Respiratory tract secretions evaluated by direct immunofluorescence from group 2 patients contained H influenzae, Proteus mirabilis, Pseudomonas aeruginosa, Enterobacter aerogenes, and K pneumonia. None of these specimens reacted with the Legionnaires' disease conjugate. The etiology of the pneumonia in group 2 patients was established by lung biopsy in nine of 15 patients (Table 3). In six cases, a nonspecific interstitial pneumonia was present. These patients had no antibody response to L pneumonia, and a definite cause'for the pneumonia was not established. . DISCUSSION

The diagnosis of Legionnaires' disease is generally made from specific tests. Though recovery of L pneumonia has been uncommon from living patients," the organism was recovered in five of the 19 cases (26 percent) in this series. Recovery was from lung tissue, pleural fluid, and bronchial washings. In addition, others 7•8 have reported recovery from transtracheal aspirates and even from the blood. Use of media such as the Feeley-Gorman agar and the charcoal yeast extract media have been advocated because they are believed to be superior to the Table 3--Lu,.. Bio".r Ruul.. of Group 2 Patien..

Nonspecific interstitial pneumonia

6

Anaerobic pneumonia (Clostridia sporogems, Peptostreplococcu.8 (2 cases)

3

Underlying malignancy

2

Pneumocysti& carinii

M ycobaclerium tuberculosis Pseudomonas aeruginosa Protoiheca wickerhamii

Total

1

15

CHEST, 79: 5, MAY, 1981

MHIH agar," Nonetheless, in our series the techniques employed by Dumoff 10 yielded five isolates. In the last isolation the organism was recovered on both charcoal yeast extract agar and the MHIH agar. Difficulty in recovering L pneumophila may be due to its fastidious growth requirements and its slow-growing nature. In addition, the exposure of the organism in vivo to antimicrobial agents that are inhibitory in vitro may also reduce the yield of cultures. FA staining may be used as a rapid diagnostic test on respiratory tract secretions such as sputum, transtracheal aspirates, or even bronchial washings. The test is easy to perform and can be done quickly to avoid more invasive tests. Since few organisms are seen in respiratory tract secretions, a negative result should not be interpreted as excluding the diagnosis of Legionnaires' disease . Pleural fluid and lung tissue imprints may be positive even when respiratory tract secretions are negative. The control group described in this article has the obvious limitation that no test has been established that can exclude the diagnosis of Legionnaires' disease with absolute certainty. However, the failure to demonstrate an antibody rise plus the isolation of another etiologic agent in two-thirds of these patients is the strongest evidence against a diagnostic of Legionnaires' disease that can be made at the present time. While the direct FA test has a high degree of specificity (100 percent in this limited series of 22 non-Legionnaires' patients), some bacteria may be antigenically related to L pneumophila. Cherry noted one strain of Pseudomonas fluorescens that cross-reacted with the Legionnaires' conjugate, but it was only one bacterial strain among 374 cultures evaluated.S Other bacteria, such as P alcaligenes, have been identified as reacting weakly.' Bacteria demonstrating high-intensity fluorescence with the Legionnaires' conjugate have been uncommon. In addition, Cherry noted that the sera of immunized rabbits from which the conjugate is prepared may contain natural antibodies to streptococci and staphylococci.s This could cause some confusion and false-positive tests if the morphology of these organisms are altered by antimicrobial therapy. The sensitivity of FA staining procedure is not as high as one would desire, since it was positive only in 68 percent (13/19) of the known cases of Legionnaires' disease and in only 64 percent (21/33) of the clinical specimens from patients with Legionnaires' disease. The predictive accuracy of a positive test was 100 percent. One limitation that may reduce the sensitivity of the direct FA staining of lung biopsy specimens may be the duration of antimicrobial

CHEST, 79: 5, MAY, 1981

therapy that the patient receives before specimens are collected. H this test is performed after prolonged antitnicrobial therapy (ie longer than nine days) , organisms no longer appear to be viable or detectable by direct FA staining in lung tissue. The release of proteolytic enzymes by the polymorphonuclear leukocytes and alveolar macrophages seen on the histologic secretions are probably responsible for removing this bacterium. The limitations of direct FA staining of clinical specimens may be reduced by using newly recognized serogroups (2 to 4) to prepare the conjugate. To date, there are four known serogroups. However, even when we included these antigenically dissimilar strains in FA techniques, we detected only one additional case. This did not significantly improve the sensitivity of the test. This result may reflect merely the prevalence of serogroup 1 as the predominant pathogen in our geographic area or that there are yet-to-be-discovered strains of L pneumophila. Though the Dieterle silver impregnation stain has been advocated as a sensitive stain for L pneumaphila, the test does not appear to be very specific." Since other gram-negative bacteria may stain with the silver stain, this technique was not performed in all the lung specimens reviewed in this article, and the sensitivity of the Dieterle silver stain and the FA stain cannot be compared. The histopathology of Legionnaires' disease is that of an acute fibrinopurulent bronchopneumonia in which the alveoli are filled with neutrophils and macrophages. In all cases in which histology revealed this finding, FA staining was positive. In cases in which an interstitial process alone was identified, the bacteria were not found on FA staining. This could suggest several possibilities. One possibility is that L pneumophila coexisted with a viral pathogen. Another is that L pneumophila is respon sible for a hypersensitivity pneumonitis by releasing a toxin affecting lung tissue. L pneumophila does have endotoxin activity, and an exotoxin has been suggested by Baine." Still another consideration is that L pneumophila has been responsible for nonspecific inflammation in compromised hosts for years. Such inflammation has frequently been found in compromised hosts with pneumonia in anywhere from 13 to 34 percent of lung biopsies. 12 •13 In this study, over a 2*-year period, nonspecific interstitial pneumonia was present in 35 percent (10/29) of lung biopsies, and in four of these cases patients demonstrated a rise in antibody to L pneumophila. In summary, the direct FA stain of specimens from the lung, pleural fluid, and respiratory tract secretions is very specific but has a limited sensitivity.

DIAGNOSIS OF LEGIONNAIRES' DISEASE 569

Although sensitivity may be increased if new serogroups are added, it has been minimal with the addition of serogroups 2 to 4 in our geographic area. Also, the direct FA staining procedure has little value in nonspecific interstitial pneumonia, although Legionnaires' disease may have a role as an etiologic agent in these cases. Viral pneumonia may predispose to infection with L pneumophUa and account for the histologic findings noted here. Finally, relatively simple culture techniques, if handled properly, may be used to recover L pneumophUa in fresh clinical specimens of lung tissue, pleural fluid, and respiratory tract secretions. With these techniques, a more rapid, specific diagnosis may be established earlier in the course of illness so that specific antimicrobial therapy can be selected. ACKNOWLEDGMENT: The authors thank T. KiDip, M.D., J. Ohordniek, M.D., E. Metzger, M.D., T. Neblett, Ph.D., J. Eisses, Ph.D., L. Ross, P. Cornett, Ph.D .; the physicians of Henry Ford Hospital; W. Cherry, Ph.D., of the Center for Disease Control, Atlanta; B. B. Wentworth, Ph.D., of the Michigan Department of Public Health; P. McFarland, and C. Saunders.

3 4 5

6 7

8 9 10 11

1 Kirby BD, Snyder KM, Meyer RD, Finegold SM. Legionnaires' disease: clinical features of 24 cases. Ann Intern Med 1978; 89:297-309 2 McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR. Laboratory investigation team. Legionnaires' disease: isolation of a bacterium and dem-

510 SAlAYOLATZ, RUSSnt., CYlTKOYlCH

12 13

onstration of its role in other respiratory disease. N Engl J Med 1977; 297:1197-03 Cherry WB, Pittman B, Harris PP, et al. Detection of Legionnaires' disease bacteria by direct immunofluorescent staining. J Clin Microbiol 1978; 8 :329-38 Broome CV, Cherry WB, Winn WC, MacPherson BR. Rapid diagnosis of Legionnaires' disease by direct immunofluorescent staining. Ann Intern Med 1979; 90 :1-4 Cherry WB, McKinney RM. Detection in clinical specimens by direct immunofluorescence. In Legionnaires'-the disease, the bacterium, and the methodology (as manual) . Atlanta: CDC, 1978; 129-46 Tsai TF, Fraser DW. The diagnosis of Legionnaires' disease (editorial). Ann Intern Med 1978; 89:413-14 Lattimer CL, McCrone C, Caison J. Diagnosis of Legionnaires' disease from transtracheal aspirate by direct fluorescent antibody staining and isolation of the bacterium. N Engl J Med 1978; 299:1172-73 Edlestein PH, Meyer RD, Finegold SM. Isolation of Legionella pneumophila from blood . Lancet 1979; 1:75051 Feeley JC, Corman CW, Weaver RE, Mackel DC, Smith HW . Primary isolation media for Legionnaires' disease bacterium. J Clin Microbiol1978; 8:320-25 Dumoff M. Direct in vitro isolation of the Legionnaires' disease bacterium in two fatal cases. Ann Intern Med 1979; 90:694-96 Baine WB, Rasheed JK, Mackel DC, Bopp CA, Wells JC, Kaufman AF. Exotoxin activity associated with the Legionnaires' disease bacterium. J Clin Microbiol 1979; 9:453-56 Creenman RL, Goodall PT, King D. Lung biopsy in immunocompromised hosts. Am J Med 1975; 59:488-96 Feldman NT, Pennington IE, Ehrie MC. Transbronchial lung biopsy in the compromised host. JAMA 1977; 238:1377-79

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