JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1998, p Vol. 36, No. 9. Copyright 1998, American Society for Microbiology. All Rights Reserved.

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1998, p Vol. 36, No /98/$ Copyright 1998, American Society for Microbiology. All Rights Reserved. Comparison of the Binax Legionella Urinary Antigen Enzyme Immunoassay (EIA) with the Biotest Legionella Urin Antigen EIA for Detection of Legionella Antigen in both Concentrated and Nonconcentrated Urine Samples J. A. DOMÍNGUEZ,* N. GALÍ, P. PEDROSO, A. FARGAS, E. PADILLA, J. M. MANTEROLA, AND L. MATAS Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Facultat de Medicina de la Universitat Autònoma de Barcelona, Barcelona, Spain Received 19 February 1998/Returned for modification 31 March 1998/Accepted 15 June 1998 We evaluated a newly commercial enzyme immunoassay (EIA) (Biotest Legionella Urin Antigen EIA; Biotest AG, Dreieich, Germany) for detection of antigens of all Legionella pneumophila serogroups with a relatively wide spectrum of cross-reactivity as well as antigens of other Legionella spp. by comparing its sensitivity and specificity with those of an EIA for detection of L. pneumophila serogroup 1 antigen (Legionella urinary antigen EIA; Binax, Portland, Maine). Both tests were performed with both concentrated and nonconcentrated urine samples. We also evaluated the capabilities of both EIAs to detect extracted soluble antigens of American Type Culture Collection (ATCC) Legionella strains (L. pneumophila serogroups 1 to 14, L. bozemanii, and L. longbeachae). The sensitivity of the Biotest EIA was 66.66% in nonconcentrated urine and 86.66% in concentrated urine. The sensitivity of the Binax EIA was 63.76% and 88.88% in nonconcentrated and concentrated urine, respectively. The specificity was 100% in nonconcentrated and concentrated urine for both assays. The Binax EIA and Biotest EIA detected extracted soluble antigens of L. pneumophila serogroups 1 to 14 and L. bozemanii ATCC strains. The cross-reactions observed with the Binax EIA were probably due to common epitopes directly related to lipopolysaccharide. Further studies are required to determine the usefulness of the Binax EIA for detection of urinary antigens from Legionella species and serogroups other than L. pneumophila serogroup 1. The Biotest EIA proved to be as rapid, sensitive, and specific as the Binax EIA for the diagnosis of legionellosis. Concentration of antigen present in urine increased the sensitivities of both techniques with no reduction in specificity. Since the initial description of Legionnaires disease in 1976, Legionella pneumophila has been increasingly recognized as a pathogen causing both community-acquired and nosocomial pneumonia (23). Legionella pneumonia can be difficult to diagnose because the signs and symptoms are nonspecific and do not distinguish Legionella infection from other common causes of pneumonia (11). Diagnosis by culture of respiratory-tract secretions requires 3 to 5 days of incubation (10). Many laboratories do not culture Legionella spp. or are unable to do so (7). Determination of antibody titers can provide only a retrospective diagnosis, since seroconversion usually occurs 2 to 9 weeks after the onset of infection (12). Direct fluorescent-antibody staining of respiratory secretions is a rapid test, but it has a low level of sensitivity and cross-reactions with other bacteria in the clinical laboratory. The number of etiologic diagnoses could increase, providing therapeutic benefit, if a rapid, sensitive, and specific technique for detecting the soluble antigen of Legionella in urine were used. The detection of the soluble antigen of L. pneumophila serogroup 1 in urine by an enzyme immunoassay (EIA) from Binax (Portland, Maine) has proven rapid, sensitive, and specific (8, 9, 14, 18), and the use of concentrated urine improves the yield significantly (8, 9). Although L. pneumophila serogroup 1 is responsible for more than 80% of Legionnaires * Corresponding author. Mailing address: Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Ctra. Canyet s/n, Badalona, Barcelona, Spain. Phone: , ext Fax: jadoming@ns.hugtip.scs.es. disease cases (11), other species or serogroups can cause infection in humans (23, 27). The value of urinary antigen detection assays to diagnose Legionnaires disease would be enhanced if Legionella infections other than L. pneumophila serogroup 1 infections could also be detected. The aim of the present study was to evaluate a newly available kit for performing an EIA to detect all antigens of L. pneumophila serogroups with a relatively wide spectrum of cross-reactivity, as well as antigens of other Legionella species (Biotest AG, Dreieich, Germany) by comparing its sensitivity and specificity with those of the EIA to detect L. pneumophila serogroup 1 antigen (the Binax EIA). Both tests were performed with both concentrated and nonconcentrated urine samples in order to assess whether concentration improves the yield in the Biotest EIA as it does in the Binax EIA. MATERIALS AND METHODS Patients and samples. Urine samples were obtained from four groups of patients. First, we studied 69 nonconcentrated and 45 concentrated urine samples from 65 patients (48 men and 17 women) with pneumonia caused by L. pneumophila. The mean age of the patients in this group was 61 years (age range, 28 to 86 years). Pneumonia was community acquired for 31 patients and nosocomial for 34 patients. Legionnaires disease was diagnosed by isolating L. pneumophila from respiratory specimens for 19 patients, by seroconversion for 41 patients, and by culture plus seroconversion for 5 patients. L. pneumophila was isolated on selective buffered charcoal-yeast extract- medium (13), and identification was based on the usual criteria (31). Detection of specific antibodies was carried out by indirect immunofluorescence (13) for at least two serum samples, one obtained during the initial phase of disease and one obtained during convalescence (3 to 9 weeks); the criterion for seroconversion was a fourfold increase in antibody titer (13). The second group consisted of 51 nonconcentrated and 41 concentrated urine samples from 46 patients (27 men and 19 women) with Legionnaires disease. 2718

2 VOL. 36, 1998 COMPARISON OF BINAX EIA AND BIOTEST EIA 2719 Species TABLE 1. Reference strains used in this study Serogroup Strain designation ATCC strain no. L. pneumophila 1 Philadelphia Togus Bloomington Los Angeles Dallas Chicago Chicago Concord IN-23-GI-C Leiden PA-H CO-H A MN-H L. bozemanii 1 WIGA L. longbeachae 1 Long Beach Patients were diagnosed by detection of soluble antigen from L. pneumophila serogroup 1 by the Legionella urinary antigen EIA (Binax), but culture results were negative and there was no seroconversion. Pneumonia was community acquired for 19 patients and nosocomial for 27 patients. The mean age of the patients in this group was 59 years (age range, 16 to 90 years). The third group contained 129 nonconcentrated and 88 concentrated urine samples from 90 patients with pneumonia of other etiologies (31 from Streptococcus pneumoniae, 23 from Chlamydia pneumoniae, 13 from Mycoplasma pneumoniae, 6 from Coxiella burnetii, 1 from Legionella longbeachae, 7 from Pseudomonas aeruginosa, 6 from Haemophilus influenzae, 2 from Staphylococcus aureus, and 1 from Aspergillus fumigatus). The fourth group consisted of 78 nonconcentrated and 78 concentrated urine samples from 78 patients with no clinical or radiological signs of pneumonia, 26 of whom were healthy and 52 of whom had urinary-tract infections (22 with Escherichia coli, 4 with Proteus mirabilis, 2 with Klebsiella pneumoniae, 4 with Klebsiella oxytoca, 1 with Citrobacter freundii, 1 with Enterobacter aerogenes, 3 with Staphylococcus saprophyticus, 3 with P. aeruginosa, 1 with Providencia stuartii, 1 with Enterococcus faecalis, 2 with Morganella morganii, 4 with Candida albicans, and 4 with mixed infections: 1 with K. oxytoca and P. aeruginosa, 1 with P. mirabilis and E. faecalis, 1 with E. coli and K. pneumoniae, and 1 with E. coli and E. faecalis). The urine specimens of the 52 patients with urinary-tract infections each yielded 10 6 bacteria or fungi/ml. Sample treatment. The samples were boiled for 5 min and centrifuged at 1,000 g for 15 min to prevent nonspecific reactions (6, 8, 9, 25, 28). The antigen present in the urine was concentrated 25-fold by selective ultrafiltration (8, 9) (Urifil-10 Concentrator; Millipore Corporation, Bedford, Mass.). Samples were tested by the Binax EIA immediately or after being stored from 1 to 12 months. The Biotest EIA results were compared to the prior results of the Binax EIA. Soluble-antigen preparation. Soluble antigens of the American Type Culture Collection (ATCC) (Manassas, Va.) Legionella strains listed in Table 1 were prepared by a method adapted from that of Kohler et al. (22). Each Legionella strain was grown on buffered charcoal-yeast extract- agar at 35 C for 48 h in 3% CO 2 and harvested in 3 ml of 0.05 M phosphate-buffered saline (ph 7.4). The cell suspensions were centrifuged, and 0.5 ml of wet-packed cells was autoclaved at 100 C for 1 h and then left at 4 C for 10 days in order to extract soluble antigens. The suspensions were centrifuged at 1,200 g for 10 min, and the supernatants were collected. Binax Legionella urinary antigen EIA kit. The Binax EIA method is a direct sandwich assay that uses polyclonal rabbit immunoglobulin G specific for L. pneumophila serogroup 1 as the capture and detection antibody. The test was performed according to the manufacturer s instructions. Samples were considered positive when the absorbance units were triple those recorded for the negative control. Biotest Legionella Urin Antigen EIA. The Biotest assay is a direct sandwich assay that uses polyclonal rabbit antibodies which react with antigens of all L. pneumophila serogroups as well as with antigens of other Legionella species as the capture and detection antibodies. The test was performed by following the manufacturer s instructions. The cutoff value was calculated as the mean absorbance of the negative controls plus Urine samples with an extinction equal to or greater than the cutoff were considered positive. Testing of urine samples with absorbance values in the region of the cutoff plus U had to be repeated for confirmation, and when absorbance values were again in this range or higher, the samples were considered positive. Statistical analysis. Data were analyzed by the SAS statistics program (24). Concentrated and nonconcentrated urine samples were compared by a Student t test. Results by the Binax EIA and the Biotest EIA were compared, and specificity was evaluated, by means of McNemar s test. RESULTS Detection of Legionella antigens in patients urine. By using the Binax EIA, soluble antigen was detected in 44 nonconcentrated samples and in 40 concentrated samples from group 1 patients. By using the Biotest EIA, Legionella antigen was detected in 46 nonconcentrated samples and in 39 concentrated samples from group 1 patients. The sensitivity of the Binax EIA was 63.76% with nonconcentrated urine and 88.88% with concentrated samples. The sensitivities of the Biotest EIA with nonconcentrated and concentrated urine samples were and 86.66%, respectively (Table 2). Urine samples from patients with no clinical symptoms or signs of pneumonia or with pneumonia of other etiologies were all negative, whether they were concentrated or not. The specificity for both EIAs was 100% with both nonconcentrated and concentrated urine samples. Grouping the sensitivity results by the means of diagnosis (positive culture, seroconversion, and seroconversion plus positive culture) reveals that concentrating the urine provides an increase in sensitivity. The sensitivities for nonconcentrated urine in these three groups were 80, 52.38, and 85.71% by the Binax EIA and 90, 52.38, and 85.71% by the Biotest EIA, respectively, and the sensitivities for concentrated urine were 92.85, 85.71, and 100% by the Binax EIA and 92.85, 82.14, and 100% by the Biotest EIA, respectively (Table 2). There were no significant differences between the Binax EIA and the Biotest EIA. We did not find any differences between the Biotest and Binax EIAs when the samples were tested immediately by the Binax EIA or when samples were evaluated after being stored. For the samples from group 2 patients diagnosed by detection of soluble antigen, but with negative culture results and without seroconversion, the Binax EIA detected soluble antigen in 25 of the nonconcentrated samples (49.01%) and in all 41 concentrated samples (100%). The Biotest EIA detected soluble antigen in 28 of the nonconcentrated urine samples (54.90%) and in 36 of the concentrated urine samples (87.80%). We found no significant differences in sensitivity between the Binax EIA and the Biotest EIA when we evaluated both tests according to whether the legionellosis case was a community-acquired or a nosocomial pneumonia case (Table 3). The absorbances of samples from patients with diagnoses of legionellosis (groups 1 and 2) obtained by the Biotest EIA TABLE 2. L. pneumophila serogroup 1 patients, by diagnosis, and sensitivities of the Binax EIA and the Biotest EIA with concentrated and nonconcentrated urine samples Test and sample a No. of patients (sensitivity of test) b by means of diagnosis Total Positive culture Seroconversion Positive culture plus seroconversion Binax EIA NCU 44 (63.76) 16 (80) 22 (52.38) 6 (85.71) CU 40 (88.88) 13 (92.85) 24 (85.71) 3 (100) Biotest EIA NCU 46 (66.66) 18 (90) 22 (52.38) 6 (85.71) CU 39 (86.66) 13 (92.85) 23 (82.14) 3 (100) a NCU, nonconcentrated urine; CU, concentrated urine. b Measured in percentages.

3 2720 DOMÍNGUEZ ET AL. J. CLIN. MICROBIOL. Test and sample a TABLE 3. Binax EIA and Biotest EIA sensitivities with concentrated and nonconcentrated urine samples from patients with confirmed community-acquired and nosocomial Legionnaires disease Sensitivity b (no. of positive samples/total no.) for patients with: Community-acquired legionellosis Nosocomial legionellosis Binax EIA NCU (21/32) (23/37) CU (18/21) (22/24) Biotest EIA NCU (22/32) (24/37) CU (18/21) (21/24) a NCU, nonconcentrated urine; CU, concentrated urine. b Measured in percentages. were significantly higher (P ) for concentrated urine samples than for nonconcentrated samples in both assays. Concentrating urine by selective ultrafiltration increased the number of detections of legionellosis, whether by the Binax EIA or the Biotest EIA, in patients with Legionnaires disease. There were no significant differences between the Binax EIA and the Biotest EIA (Tables 2 and 3). In this study, nonconcentrated urine samples from patients with Legionnaires disease, regardless of the ratio, could give positive results by the Binax EIA when concentrated. This is always true for nonconcentrated urine samples with ratios between 2.0 and 3.0. By the Biotest EIA, nonconcentrated urine samples from patients with legionellosis with negative results, independently of the absorbance value, could give positive results when concentrated. When nonconcentrated urine samples from groups 1 and 2 were used, there were 11 cases of discrepant results between the Binax EIA and the Biotest EIA. For three patients (one diagnosed by seroconversion and two diagnosed by antigenuria), the Binax EIA was positive and the Biotest EIA was negative. For eight patients (two diagnosed by culture, one by seroconversion, and five by antigenuria), the Binax EIA was negative (in six cases, ratios were between 2.0 and 3.0) and the Biotest EIA was positive. Using concentrated urine samples, we found eight cases in which results obtained by the Binax EIA failed to agree with those obtained by the Biotest EIA; in seven of these (two diagnosed by seroconversion and five by antigenuria), the Binax EIA was positive and the Biotest EIA was negative. For one patient diagnosed by seroconversion, the Binax EIA was negative (ratio, 2.03) and the Biotest EIA was positive. When concentrated urine from patients diagnosed by culture was used, we found equivalent results in both assays. In five cases (three nonconcentrated and two concentrated urine samples), results from the Biotest EIA were in the range of the cutoff plus All samples were retested. For four cases, results were again in this range, and for one case, results were higher. All the samples were considered positive by the Biotest EIA, and the Binax EIA was negative only in one of these cases (ratio, 2.9). Detection of Legionella soluble antigens from culture extracts. The Binax EIA and Biotest EIA were tested for their capabilities to detect soluble antigens of Legionella. For the 14 serogroups from L. pneumophila and for the Legionella bozemanii ATCC strain, both tests were positive; for the L. longbeachae ATCC strain, both tests were negative. DISCUSSION The EIA for detection of L. pneumophila serogroup 1 antigens has been shown to be a sensitive and specific test when applied to urine specimens (4 6, 19, 21, 23, 25, 29). Several authors (9, 14, 18) have confirmed the usefulness of the EIA kit available from Binax for detecting antigenuria in Legionnaires disease. Our previous study (9) indicated that the Binax EIA was rapid, sensitive, and specific and was comparable in sensitivity and specificity to the Binax radioimmunoassay (RIA). Hackman et al. (14) also compared the Binax EIA with the Binax RIA. The sensitivities were equal (77%), and the EIA was as useful as the RIA for detecting L. pneumophila serogroup 1. Kazandjian et al. (18) evaluated the Binax EIA in cases of suspected or proven legionellosis, detecting antigen in samples from 37% of patients with suspected legionellosis and from 83% of those with proven L. pneumophila serogroup 1 infection. The recently available EIA for detecting antigen from all L. pneumophila serogroups from Biotest was evaluated recently by the manufacturer (26). Biotest developed the EIA to detect Legionella antigen in urine, and it found antigen in nonconcentrated urine from 102 patients with a specificity of 99.8% and a sensitivity of 100%. Tang et al. (28) also developed an EIA that detected soluble antigens of L. pneumophila serogroups 1, 3, 4, 6, and 8, Legionella micdadei, and L. longbeachae serogroup 1 in 25 of 35 patients with legionellosis with 100% specificity. In our study we found no significant differences in sensitivity between the Binax EIA and the Biotest EIA, although nonconcentrated urine gave a slightly higher level of sensitivity for the Biotest EIA, and the sensitivity of the Binax EIA was slightly higher than that of the Biotest EIA when concentrated urine was used. The sensitivities of the Binax EIA and the Biotest EIA for detecting antigenuria, whether in nonconcentrated or concentrated urine, were higher for patients diagnosed by positive culture than for those diagnosed by seroconversion alone. Culture-positive patients may excrete more antigen than do culture-negative patients (20). Our study indicated that boiling samples and concentrating the antigen is useful with both EIAs. Concentrating the antigen present in urine by selective ultrafiltration increases sensitivity with no decrease in specificity. Urine concentration by this method is thus a simple procedure which contributes substantially toward increasing the utility of techniques for diagnosing Legionnaires disease. Given that soluble antigen is stable at high temperatures (22), boiling urine for 5 min and centrifuging the sample at 1,000 g is recommended to prevent nonspecific reactions (8, 9, 23, 25, 28). Antigenuria is detectable from the onset of disease, and the results have been unaffected by prior administration of antibiotics in several studies (11, 21, 22). Other authors have reported that soluble antigen can be detected in urine many days after the start of symptoms: from 10 to 15 days (1, 23, 25), 60 days (6), and 42 days or longer (21). In the present study, we were able to detect antigen from the onset of symptoms up to 50 days later by both the Binax EIA and the Biotest EIA. Birtles et al. (6) obtained a sensitivity of 77% when testing urine samples collected from the start of symptoms up to 28 days afterwards and a sensitivity of 86% when only urine from the first 7 days of illness was used. The coefficient by the Binax EIA for samples from patients not suffering from Legionnaires disease never produced ratios over 2.0, and the results by the Biotest EIA never were in the range of the cutoff plus 0.200, whether urine was concentrated or nonconcentrated.

4 VOL. 36, 1998 COMPARISON OF BINAX EIA AND BIOTEST EIA 2721 We observed no cross-reactions with other microorganisms in urine samples from patients with urinary-tract infections and high bacterial counts, unlike other authors (1, 22, 28). Although cross-reactions among species of the genus Legionella have been described (5, 20, 23), Kazandjian et al. (18), using the Binax EIA, observed no cross-reactions of L. pneumophila serogroup 1 with other species or serogroups (L. longbeachae, and L. pneumophila serogroups 2, 3, 4, and 10). Our results for the single patient with pneumonia due to L. longbeachae were negative by both the Binax EIA and the Biotest EIA. The major advantage of the Biotest EIA over the Binax EIA is that the first detects the antigens from all L. pneumophila serogroups with a wide spectrum of cross-reactivity as well as antigens from other Legionella species, but with regard to the detection of Legionella soluble antigens from culture extracts, both EIAs were able to detect antigen from all strains tested, except for L. longbeachae. Further studies involving urine samples from patients with legionellosis due to Legionella species or serogroups other than L. pneumophila serogroup 1 are required in order to determine the real usefulness of the Biotest EIA, given that Legionella soluble antigens from culture extracts are not an adequate substitute. Kohler et al. (20) reported cross-reactive urinary antigens among patients with legionellosis caused by L. pneumophila serogroups 1 and 4 and the Leiden 1 strain. Kohler and Sathapatayavongs (19) have reported on the detection of antigen in the urine of patients infected with L. pneumophila serogroup 4 and Legionella dumoffii with an L. pneumophila serogroup 1 direct system. Bibb et al. (5) found that extracts of cells of the reference strains of L. pneumophila serogroups 2 through 8 cross-react with serogroup 1 antibodies in their direct enzyme-linked immunosorbent assay (ELISA). Recently Benson et al. (3) evaluated the usefulness of the Binax RIA for detection of serogroups and species of Legionella other than L. pneumophila serogroup 1. They tested 34 urine samples from non-l. pneumophila serogroup 1 patients diagnosed by a broad-spectrum ELISA (28) and found that 14 of the 34 samples were positive with the Binax RIA. The antigen detected by both the Binax EIA and the Biotest EIA is now believed to be a lipopolysaccharide (LPS) (30). Although the reference strain for each serogroup of L. pneumophila possesses at least one LPS-specific epitope not found on any other reference strain and therefore designated the serogroup-specific epitope (15), when antiserum prepared against serogroup 5 was used to probe the LPS from L. pneumophila serogroups 1 to 14, the antibodies recognized a common epitope harbored by all L. pneumophila serogroups (16, 17). Barthe et al. (2) also detected an epitope that was common to serogroups 1 to 8 of L. pneumophila and was attached to the LPS. The EIA manufactured by Binax was initially developed by Kohler and coworkers. Given that the Binax EIA still produces polyclonal antibody using whole cells from L. pneumophila serogroup 1, as in Kohler s assay (22), it is more probable that the immune sera obtained contain antibodies against common epitopes harbored by all L. pneumophila serogroups. This fact could explain why the Binax EIA detects antigens from L. pneumophila serogroups other than serogroup 1. However, antigens of other species of Legionella and other serogroups of L. pneumophila may or not be excreted in urine. An antigen may be excluded from passage across the walls of the glomerular capillaries by virtue of its molecular weight or charge and thus may not be present in urine. Further studies are required in which multiple urine samples from patients with legionellosis due to species or serogroups other than L. pneumophila serogroup 1 are tested in order to determine the usefulness of the Binax EIA. The fact that the Biotest EIA is able to detect all L. pneumophila serogroups does not imply a decrease in sensitivity in the detection of L. pneumophila serogroup 1. In cases of outbreaks of nosocomial pneumonia due to L. pneumophila serogroup 1, we found no significant differences between the Binax EIA and the Biotest EIA. Furthermore, the Biotest EIA could be especially useful in geographic areas where a minority of proven Legionella infections are caused by L. pneumophila serogroup 1. The Biotest EIA is rapid, sensitive, and specific, comparable in sensitivity and specificity to the Binax EIA. The drawback of the Biotest EIA lies in its inability to identify the particular Legionella species and/or serogroup that is causing the infection. The Binax EIA contains 12 strips with eight separable wells each. When a unique sample is tested, it uses only four wells (one positive control, one negative control, one blank, and the sample). However, in the Biotest EIA the eight wells from a strip are inseparable. Thus, when a unique sample is tested, the Biotest EIA has to be performed with one strip using five wells (for one positive control, two negative controls, one blank, and the sample), wasting the three left, since they cannot be stored for further determinations. Consideration should be given to designing Biotest EIA strips with separate wells so that it will not be necessary to wait until there are enough samples to use all the wells of one strip, since the major interest of these soluble-antigen detection techniques is the rapid determination of the urine antigen. Both the Binax EIA and the Biotest EIA are useful tools for assisting physicians in the diagnosis and treatment of patients with Legionnaires disease. ACKNOWLEDGMENT We thank Carmen Pelaz from Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain, for providing Legionella ATCC strains. REFERENCES 1. Aguero-Rosenfeld, M. E., and P. H. Edelstein Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans. J. Clin. Microbiol. 26: Barthe, C., J. R. Joly, D. Ramsay, M. Boissinot, and N. 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