Vol. 29, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1991, p. 2220-2223
0095-1137/91/102220-04$02.00/0 Copyright © 1991, American Society for Microbiology
Evaluation of the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test DIANE S. LELAND'* AND RICHARD B. KOHLER',2 Departments of Pathology' and Medicine,2 Indiana University Medical Center, Indianapolis, Indiana 46202 Received 21 November 1990/Accepted 1 July 1991
The purpose of this study was to evaluate the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) for detection of Legionella antigen in urine. A total of 481 frozen urine samples previously tested by an in-house solid-phase radioimmunoassay (RIA) was thawed and retested by using L-CLONE. Included in this sample were 140 RIA-positive samples from culture-positive or serologically confirmed cases of legionellosis and 341 RIA-negative samples from patients with nonLegionella respiratory disease or bacteriuria. The original RIA test result was accepted as the true value. L-CLONE correctly identified 76 of 140 (54%) known positive samples. False-negative results could not be attributed to a low Legionella antigen concentration or to a LegioneUla antigen subgroup. L-CLONE correctly identified 252 of 341 (74%) known negative samples. False-positive results were experienced in all groups of negative samples, regardless of the patients' underlying diseases. A total of 141 fresh urine samples was tested; all were Legionella antigen negative by RIA. L-CLONE provided 86% specificity. The sensitivity of the L-CLONE in testing fresh urine samples could not be evaluated because of the lack of Legionella antigen RIA-positive samples.
Legionella pneumophila serogroup 1 causes the majority of cases of legionellosis (9). The infection may result in death in 25% of untreated, nonimmunosuppressed patients and in 80% of untreated immunosuppressed patients (4). Because the antibiotics that are used to treat pneumonias empirically are not optimal for the treatment of Legionnaires disease, it is essential that the infection be accurately and rapidly diagnosed early in the course of the disease so that appropriate specific antimicrobial therapy can be initiated. Although Legionella species can be isolated on buffered charcoal-yeast extract agar or can be identified through direct fluorescent-antibody staining, these methods may be either too slow or inaccurate, and both require specimens collected by invasive techniques such as transtracheal aspiration or bronchoalveolar lavage (3, 13, 14). Serum antibody to Legionella species can be identified by an indirect fluorescent-antibody test, but seroconversion may not occur until 4 to 6 weeks after the onset of disease (12). Approximately 80% of patients with Legionnaires disease (93% of those proven by culture to be infected) excrete soluble Legionella antigens in their urine in sufficient quantity to be detected by radioimmunoassay (RIA) (7). Urinary antigen has been detected within 1 to 3 days of the onset of symptoms just as often as it has at 4 to 7, 8 to 14, and 14 or more days after the onset of symptoms (8). Legionella antigenuria can be detected by RIA, enzyme immunoassay (EIA), and latex agglutination (10, 11). Until recently, these assays were not available commercially and were performed in reference and research laboratories only. The L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) has recently become available commercially. It is a reverse passive agglutination system consisting of a buffered suspension of black latex particles coated with murine monoclonal antibody; the antibody is directed against a soluble lipopolysaccharide monovalent hapten *
found in the urine of patients infected with L. pneumophila serogroup 1. If the lipopolysaccharide antigen is present in urine, it binds through its single reactive site to the sensitized latex. No lattice is formed, so the binding acts to competitively inhibit any agglutination. If LPS antigen is absent from the urine, the sensitized latex remains unbound and agglutinates to produce clumping that is visible against the white background of the slide (1). The manufacturer of the L-CLONE kit reports that it is both sensitive and specific in the identification of Legionella serogroup 1 antigen. The purpose of this study was to evaluate the L-CLONE kit by using it to test a large group of stored urine samples that included Legionella antigen-positive and -negative samples. Previously, these samples were tested for Legionella urinary antigen by RIA, and the patients from whom the specimens were collected were evaluated and classified by disease state. The L-CLONE kit was also used to test fresh urine samples, and the L-CLONE results were compared with the RIA results for these samples. The reproducibility of the test and the technical skill required for test performance were also evaluated. MATERIALS AND METHODS
Urine samples. Four hundred eighty-one samples were previously tested by RIA for Legionella antigen, and a portion of each sample was stored frozen at -20°C. Of 140 RIA-positive samples, 84 were from culture-positive cases and 56 were from serologically confirmed cases of serogroup 1 L. pneumophila pneumonia. A group of 109 of the RIApositive samples was tested by quantitative RIA to determine the Legionella antigen titer and by a monoclonal antibody EIA (6) to determine the Legionella urine antigen subgroup. Three hundred forty-one RIA-negative samples were from patients with non-Legionella respiratory disease or bacteriuria, as follows: 7 were from patients with empyema associated with Streptococcus pneumoniae, Klebsiella pneumoniae, anaerobes, or other bacteria; 15 were from patients with mycobacterial infections; 75 were from pa-
Corresponding author. 2220
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1991
tients with bacterial pneumonia caused by common respiratory pathogens; 20 were from patients with other pulmonary diseases including sarcoidosis, Hodgkins disease, and various fungal pneumonias; and 224 had >105 CFU of common bacterial urinary pathogens per ml. A total of 141 fresh urine samples received for routine bacterial cultures in the microbiology laboratory was tested for Legionella urine antigen by both RIA and L-CLONE. L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test. Test kits were purchased from Access Medical Systems. Kits were from three different lots, K022701, K110805, and KF02027, with expiration dates of February 1990, November 1990, February 1991, respectively. All testing was performed according to the instructions of the manufacturer. Reactants were mixed on a slide and rotated at 100 rpm under a moisture cover for 15 min. Agglutination was graded from negative through 4+, depending on the size of the clumps and the appearance of background liquid as described and pictured in the package insert of the manufacturer (1). Samples which yielded 2+ or stronger agglutination were identified as negative for Legionella antigen. An aliquot from each sample yielding weak or no agglutination (105 CFU/ml) Total
20 15 75 7 224 341
16 (80) 8 (53) 46 (61) 5 (71) 177 (79) 252a
76a
a The sensitivity of the L-CLONE test for specimens positive by RIA was 54%. The specificity of the L-CLONE test for specimens negative by RIA was 74%. Repeat RIA testing of all samples with discordant L-CLONE and RIA results showed that, following freezer storage, 3% of RIA-positive samples may have become RIA negative and that 1% of RIA-negative samples may have become RIA positive. When L-CLONE sensitivity and specificity calculations were adjusted to reflect RIA changes, the sensitivity was 56% and the specificity was 75%.
were selected at random from those specimens that contained sufficient urine to permit additional testing. Thirtyone samples were chosen from the 89 false-positive samples, and 29 samples were chosen from the 64 false-negative samples. Repeat RIA testing was performed on all of these samples as part of the retesting protocol used for all samples that yielded discordant results in initial testing. The technologist was asked to comment concerning the technical skill required for test performance and the clarity of the test endpoint. Calculations. The RIA result was accepted as the true result in all comparisons. Comparisons were made by using the chi-square test; no continuity correction was used. Significance was determined at P < 0.05.
RESULTS A total of 481 stored urine samples were tested by using the L-CLONE kit. L-CLONE correctly identified 76 of 140 (54%) urine samples that had positive RIA results when they were tested previously (Table 1). The RIA was repeated for the 64 samples in which L-CLONE was negative and RIA was positive, and 2 (3%) of these samples were RIA negative. The maximum L-CLONE sensitivity was 56% after sensitivity calculations were adjusted to compensate for a possible 3% change in RIA results in previously RIApositive samples. Although the sensitivity of L-CLONE was somewhat higher in urine samples with high antigen titers, there was no significant difference in L-CLONE sensitivity between urine samples with low (1:1 to 1:8) and high (.1:256) titers, between culture and serologically confirmed Legionella cases, or among antigens of subgroup A or B or an unknown subgroup (Table 2). The L-CLONE specificity was 74% when 341 known negative urine samples were tested (Table 1). In repeat testing by RIA of all 89 samples that had results that were L-CLONE positive and RIA negative, one sample (1%) was positive by RIA. The maximum L-CLONE specificity was 75% after specificity calculations were adjusted to compensate for a possible 1% change in the RIA results. There was
2222
LELAND AND KOHLER
J. CLIN. MICROBIOL.
TABLE 2. Sensitivity of L-CLONE in detecting various amounts and subgroups of Legionella urine antigen Legionella urine antigen titer and subgroup
No. of urine
specimens
No. (%) positive by L-CLONE
Antigen titer by RIA 1:1-1:8 1:16-1:32 1:64-1:128 .1:256
25 23
15 (50) 15 (48) 14 (56) 15 (65)
Antigen subgroup by EIAa Type A Type B Unknown
63 23 23
33 (52) 10 (43) 12 (52)
30 31
a Subgroups were determined by using monoclonal antibodies and an EIA method. Monoclonal antibody A was from the University of Missouri School of Medicine, and monoclonal antibody B was from the Indiana University School of Medicine. Production of these antibodies has been described in detail previously (6).
no significant difference in the rates of false positivity among the various groups of patients. Within the groups with respiratory diseases, there was no apparent difference in specificity, regardless of the infecting agent. Among the 224 samples from patients with bacteriuria, there was no apparent difference in L-CLONE specificity for any single organism or between gram-positive and gram-negative organisms or mixtures of the two. In testing of 141 fresh urine samples, all were negative by RIA; 121 of 141 (86% specificity) were negative by L-CLONE. L-CLONE reagents from several lots were used to test fresh urine samples. The test specificity was 83% for 40 samples tested with lot K022701 and 87% for 101 samples tested with lot K110805 or KF02027 (data not shown). This 86% overall specificity for fresh samples was greater than the 75% specificity obtained when 341 known RIA-negative stored samples were tested. A known Legionella antigenpositive urine sample was added to four additional fresh samples; all four were positive by RIA but negative by L-CLONE. Reproducibility data for L-CLONE testing of 60 urine samples that initially yielded false-positive or false-negative L-CLONE results were as follows. All samples were retested by RIA, and repeat results were identical to initial results in all samples except one (see below). All samples were tested twice with L-CLONE lot K022701 and once with L-CLONE lot K110805. Of 29 urine samples that were falsely identified as positive by L-CLONE (results for 2 of the initial 31 samples were discarded, 1 because of fungal contamination of the sample and 1 because the repeat RIA yielded a positive result), 15 were consistently false positive in three L-CLONE determinations; but 4 samples had two positive results with lot K022701 and one negative result with lot K110805, 8 samples had one positive result and one negative result with lot K022701 and one negative result with lot K110805, and 2 samples had one positive and one negative result with lot K022701 and one positive result with lot K110805. Of 29 samples identified by L-CLONE as falsely negative, 20 were consistently negative, 5 samples had one positive and one negative result with lot K022701 and one negative result with lot K110805, and 4 samples had one positive and one negative result with lot K022701 and 1 positive result with lot K110805. L-CLONE reagents were provided in convenient-to-use dropper bottles, and the testing procedure required little
technical expertise. It was not difficult to differentiate positive from negative agglutination patterns. The 1.0-ml quantity of control samples provided in the kit was probably inadequate for actual clinical use, because 0.5 to 1.0 ml of control samples must be boiled on each day that tests are performed and would likely be consumed long before the other reagents in the 40-test kit would. In replicate testing of control material by using latex from two different lots of L-CLONE, the technologist noted that the strength of agglutination was inconsistent between the lots, ranging from 1+ to 3+ on the same sample. DISCUSSION When we used L-CLONE to evaluate 481 stored urine samples that were previously tested by RIA, the L-CLONE result agreed with the RIA result for 76 of 140 (54%) of the RIA-positive samples and for 252 of 341 (74%) of the RIA-negative samples. Although the antigen in the samples may have changed during storage or there may have been errors in the initial testing, repeat RIA testing of all samples with discordant results showed this to be true in only 2% of the samples (2 of 64 samples initially RIA positive and 1 of 89 samples initially RIA negative). The lack of L-CLONE sensitivity was consistent with all Legionella urine antigen subgroups and quantities and in culturally and serologically confirmed Legionella infections. The L-CLONE specificity did not vary significantly in testing of urine samples from patients with mycobacteriosis, bacterial pneumonia, empyema, other pulmonary disease, or bacteriuria. The L-CLONE sensitivity of 54% and specificity of 74% we obtained when we tested stored urine samples differed substantially from the 95% sensitivity (37 of 39 samples) and 87% specificity (219 of 248 samples) reported by the manufacturer in the package insert for the L-CLONE test kit (1). Durda and Winn (2) tested the L-CLONE kit previously when it was marketed by Meridian Diagnostics, Inc. They found 92% sensitivity (34 of 37 samples) and 46% (6 of 13 samples) specificity. When we tested 141 fresh urine samples, all of which were Legionella antigen negative by RIA, L-CLONE specificity was 86%. Our specificity results differed from those provided in the L-CLONE package insert (1) when we used the kit to test stored urine but not when we tested fresh urine. Although the differences in test specificity between fresh and stored negative control urine samples may be partially due to the fact that the 341 stored urine samples were likely collected from a more heterogeneous control population than the 141 fresh urine samples were, our use of stored frozen urine may have adversely affected the assay's performance. This is difficult to evaluate. Because the test mechanism, which is described by the manufacturer as relying on "a careful balance in the stoichiometry of the reagents" (1), is insufficiently explained and defined, it is difficult to pinpoint specific interferences. When we tested stored frozen urine specimens from patients with serogroup 1 L. pneumophila infections, we encountered fewer true-positive results than the package insert (1) predicted for the testing of fresh urine specimens. However, because we also encountered fewer true-negative results in frozen urine samples than the manufacturer predicted for the testing of fresh samples, it appears that the effect on the kit's performance of freezing and/or storing urine is not simply a matter of nonspecifically increasing the tendency of the kit's latex particles to agglutinate. Alternatively, given the small number of samples tested to
VOL. 29, 1991
arrive at the package insert (1) sensitivity estimate (39 samples), perhaps the set of samples tested was not representative of the overall population of urine samples likely to come from patients with serogroup 1 L. pneumophila pneumonia. The details regarding how samples from patients with serogroup 1 L. pneumophila pneumonia were collected or stored for testing by L-CLONE have not been published, nor are they provided in the package insert. If, in fact, they were stored urine samples, then our estimate, which was obtained by testing stored urine samples, may be more accurate given the larger sizes of our test groups. We attempted to compensate for the lack of availability of fresh urine samples from patients with serogroup 1 L. pneumophila pneumonia by adding a small quantity of a high-titer antigen-positive stored urine sample to several fresh normal urine samples. The L-CLONE kit failed to detect antigen in these samples, although all of them were highly positive in the reference RIA. Given the low prevalence of legionellosis and the requirements of the kit for fresh urine samples, it seems unlikely that the sensitivity claims made in the package insert (1) can be validated independently in any reasonable time period. Even if the specificity of the L-CLONE kit is 87%, as claimed in the package insert (1), one would expect that 13 of every 100 urine samples tested from patients without serogroup 1 L. pneumophila pneumonia would yield positive results. For a low-prevalence disease like legionellosis, this means that most positive results would be false positives. If, for example, the prevalence of serogroup 1 L. pneumophila pneumonia among the test population were 5%, one might expect, from each 100 patients tested, approximately 13 false-positive and 5 true-positive results. Thus, 13 of 18 (72%) of all positive results would be false positive. There is a need for a rapid and accurate Legionella urine antigen test. L-CLONE is rapid and requires little technical expertise. However, in our opinion, the sensitivity and specificity of L-CLONE were too low and intra- and interlot inconsistencies were too common to allow the test to be seriously considered for clinical use. REFERENCES 1. Access Medical Systems, Inc. 1988. Package insert for L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test. Access Medical Systems, Inc., Branford, Conn.
LEGIONELLA URINE ANTIGEN LATEX TEST
2223
2. Durda, J. P., and W. C. Winn. 1988. Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, C-51, p. 340. 3. Edelstein, P. H. 1985. Legionella, p. 373-381. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 4. Kirby, B. D., K. M. Snyder, R. D. Meyer, and S. M. Finegold. 1980. Legionnaires' disease: report of sixty-five nosocomially acquired cases and review of the literature. Medicine 59:188205. 5. Kohler, R. B. 1986. Antigen detectiun for the rapid diagnosis of Mycoplasma and Legionella pneumonia. Diagn. Microbiol. Infect. Dis. 4:47S-59S. 6. Kohler, R. B., C. Wilde III, W. Johnnson, J. Joly, L. J. Wheat, R. Baker, and M. Misfeldt. 1988. Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays. J. Clin. Microbiol. 26:2059-2063. 7. Kohler, R. B., W. C. Winn, Jr., J. C. Girod, and L. J. Wheat. 1982. Rapid diagnosis of pneumonia due to Legionella pneumophila serogroup 1. J. Infect. Dis. 146:444. 8. Kohler, R. B., W. C. Winn, Jr., and L. J. Wheat. 1984. Onset and duration of urinary antigen excretion in Legionnaires' disease. J. Clin. Microbiol. 20:605-607. 9. Reingold, A. L., B. M. Thomason, B. J. Brake, L. Thacker, H. W. Wilkinson, and J. N. Kuritsky. 1984. Legionella pneumonia in the United States: the distribution of serogroups and species causing human illness. J. Infect. Dis. 148:819. 10. Sathapatayavongs, B., R. B. Kohler, L. J. Wheat, A. White, and W. C. Winn, Jr. 1983. Rapid diagnosis of Legionnaires' disease by latex agglutination. Am. Rev. Respir. Dis. 127:559-562. 11. Sathapatayavongs, B., R. B. Kohler, L. J. Wheat, A. White, W. C. Winn, Jr., J. C. Girod, and P. H. Edelstein. 1982. Rapid diagnosis of Legionnaires' disease by urinary antigen detection. Comparison of ELISA and radioimmunoassay. Am. J. Med.
72:576-582. 12. Wilidnson, H. W. 1986. Serodiagnosis of Legionella pneumophila disease, p. 395-398. In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical laboratory immunology, 3rd ed.
American Society for Microbiology, Washington, D.C. 13. Wilkinson, H. W. 1988. Legionellosis, p. 320-332. In A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Turano (ed.), Laboratory diagnosis of infectious diseases, principles and practice, Vol. 1. Springer-Verlag, New York. 14. Zuravleff, J. J., V. L. Yu, J. W. Shonnard, B. K. Davis, and J. D. Rihs. 1983. Diagnosis of Legionnaires disease: an update of laboratory methods with new emphasis on isolation by culture. J. Am. Med. Assoc. 250:1981-1985.
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1991, p. 2220-2223
0095-1137/91/102220-04$02.00/0 Copyright © 1991, American Society for Microbiology
Evaluation of the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test DIANE S. LELAND'* AND RICHARD B. KOHLER',2 Departments of Pathology' and Medicine,2 Indiana University Medical Center, Indianapolis, Indiana 46202 Received 21 November 1990/Accepted 1 July 1991
The purpose of this study was to evaluate the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) for detection of Legionella antigen in urine. A total of 481 frozen urine samples previously tested by an in-house solid-phase radioimmunoassay (RIA) was thawed and retested by using L-CLONE. Included in this sample were 140 RIA-positive samples from culture-positive or serologically confirmed cases of legionellosis and 341 RIA-negative samples from patients with nonLegionella respiratory disease or bacteriuria. The original RIA test result was accepted as the true value. L-CLONE correctly identified 76 of 140 (54%) known positive samples. False-negative results could not be attributed to a low Legionella antigen concentration or to a LegioneUla antigen subgroup. L-CLONE correctly identified 252 of 341 (74%) known negative samples. False-positive results were experienced in all groups of negative samples, regardless of the patients' underlying diseases. A total of 141 fresh urine samples was tested; all were Legionella antigen negative by RIA. L-CLONE provided 86% specificity. The sensitivity of the L-CLONE in testing fresh urine samples could not be evaluated because of the lack of Legionella antigen RIA-positive samples.
Legionella pneumophila serogroup 1 causes the majority of cases of legionellosis (9). The infection may result in death in 25% of untreated, nonimmunosuppressed patients and in 80% of untreated immunosuppressed patients (4). Because the antibiotics that are used to treat pneumonias empirically are not optimal for the treatment of Legionnaires disease, it is essential that the infection be accurately and rapidly diagnosed early in the course of the disease so that appropriate specific antimicrobial therapy can be initiated. Although Legionella species can be isolated on buffered charcoal-yeast extract agar or can be identified through direct fluorescent-antibody staining, these methods may be either too slow or inaccurate, and both require specimens collected by invasive techniques such as transtracheal aspiration or bronchoalveolar lavage (3, 13, 14). Serum antibody to Legionella species can be identified by an indirect fluorescent-antibody test, but seroconversion may not occur until 4 to 6 weeks after the onset of disease (12). Approximately 80% of patients with Legionnaires disease (93% of those proven by culture to be infected) excrete soluble Legionella antigens in their urine in sufficient quantity to be detected by radioimmunoassay (RIA) (7). Urinary antigen has been detected within 1 to 3 days of the onset of symptoms just as often as it has at 4 to 7, 8 to 14, and 14 or more days after the onset of symptoms (8). Legionella antigenuria can be detected by RIA, enzyme immunoassay (EIA), and latex agglutination (10, 11). Until recently, these assays were not available commercially and were performed in reference and research laboratories only. The L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) has recently become available commercially. It is a reverse passive agglutination system consisting of a buffered suspension of black latex particles coated with murine monoclonal antibody; the antibody is directed against a soluble lipopolysaccharide monovalent hapten *
found in the urine of patients infected with L. pneumophila serogroup 1. If the lipopolysaccharide antigen is present in urine, it binds through its single reactive site to the sensitized latex. No lattice is formed, so the binding acts to competitively inhibit any agglutination. If LPS antigen is absent from the urine, the sensitized latex remains unbound and agglutinates to produce clumping that is visible against the white background of the slide (1). The manufacturer of the L-CLONE kit reports that it is both sensitive and specific in the identification of Legionella serogroup 1 antigen. The purpose of this study was to evaluate the L-CLONE kit by using it to test a large group of stored urine samples that included Legionella antigen-positive and -negative samples. Previously, these samples were tested for Legionella urinary antigen by RIA, and the patients from whom the specimens were collected were evaluated and classified by disease state. The L-CLONE kit was also used to test fresh urine samples, and the L-CLONE results were compared with the RIA results for these samples. The reproducibility of the test and the technical skill required for test performance were also evaluated. MATERIALS AND METHODS
Urine samples. Four hundred eighty-one samples were previously tested by RIA for Legionella antigen, and a portion of each sample was stored frozen at -20°C. Of 140 RIA-positive samples, 84 were from culture-positive cases and 56 were from serologically confirmed cases of serogroup 1 L. pneumophila pneumonia. A group of 109 of the RIApositive samples was tested by quantitative RIA to determine the Legionella antigen titer and by a monoclonal antibody EIA (6) to determine the Legionella urine antigen subgroup. Three hundred forty-one RIA-negative samples were from patients with non-Legionella respiratory disease or bacteriuria, as follows: 7 were from patients with empyema associated with Streptococcus pneumoniae, Klebsiella pneumoniae, anaerobes, or other bacteria; 15 were from patients with mycobacterial infections; 75 were from pa-
Corresponding author. 2220
VOL. 29,
1991
tients with bacterial pneumonia caused by common respiratory pathogens; 20 were from patients with other pulmonary diseases including sarcoidosis, Hodgkins disease, and various fungal pneumonias; and 224 had >105 CFU of common bacterial urinary pathogens per ml. A total of 141 fresh urine samples received for routine bacterial cultures in the microbiology laboratory was tested for Legionella urine antigen by both RIA and L-CLONE. L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test. Test kits were purchased from Access Medical Systems. Kits were from three different lots, K022701, K110805, and KF02027, with expiration dates of February 1990, November 1990, February 1991, respectively. All testing was performed according to the instructions of the manufacturer. Reactants were mixed on a slide and rotated at 100 rpm under a moisture cover for 15 min. Agglutination was graded from negative through 4+, depending on the size of the clumps and the appearance of background liquid as described and pictured in the package insert of the manufacturer (1). Samples which yielded 2+ or stronger agglutination were identified as negative for Legionella antigen. An aliquot from each sample yielding weak or no agglutination (105 CFU/ml) Total
20 15 75 7 224 341
16 (80) 8 (53) 46 (61) 5 (71) 177 (79) 252a
76a
a The sensitivity of the L-CLONE test for specimens positive by RIA was 54%. The specificity of the L-CLONE test for specimens negative by RIA was 74%. Repeat RIA testing of all samples with discordant L-CLONE and RIA results showed that, following freezer storage, 3% of RIA-positive samples may have become RIA negative and that 1% of RIA-negative samples may have become RIA positive. When L-CLONE sensitivity and specificity calculations were adjusted to reflect RIA changes, the sensitivity was 56% and the specificity was 75%.
were selected at random from those specimens that contained sufficient urine to permit additional testing. Thirtyone samples were chosen from the 89 false-positive samples, and 29 samples were chosen from the 64 false-negative samples. Repeat RIA testing was performed on all of these samples as part of the retesting protocol used for all samples that yielded discordant results in initial testing. The technologist was asked to comment concerning the technical skill required for test performance and the clarity of the test endpoint. Calculations. The RIA result was accepted as the true result in all comparisons. Comparisons were made by using the chi-square test; no continuity correction was used. Significance was determined at P < 0.05.
RESULTS A total of 481 stored urine samples were tested by using the L-CLONE kit. L-CLONE correctly identified 76 of 140 (54%) urine samples that had positive RIA results when they were tested previously (Table 1). The RIA was repeated for the 64 samples in which L-CLONE was negative and RIA was positive, and 2 (3%) of these samples were RIA negative. The maximum L-CLONE sensitivity was 56% after sensitivity calculations were adjusted to compensate for a possible 3% change in RIA results in previously RIApositive samples. Although the sensitivity of L-CLONE was somewhat higher in urine samples with high antigen titers, there was no significant difference in L-CLONE sensitivity between urine samples with low (1:1 to 1:8) and high (.1:256) titers, between culture and serologically confirmed Legionella cases, or among antigens of subgroup A or B or an unknown subgroup (Table 2). The L-CLONE specificity was 74% when 341 known negative urine samples were tested (Table 1). In repeat testing by RIA of all 89 samples that had results that were L-CLONE positive and RIA negative, one sample (1%) was positive by RIA. The maximum L-CLONE specificity was 75% after specificity calculations were adjusted to compensate for a possible 1% change in the RIA results. There was
2222
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J. CLIN. MICROBIOL.
TABLE 2. Sensitivity of L-CLONE in detecting various amounts and subgroups of Legionella urine antigen Legionella urine antigen titer and subgroup
No. of urine
specimens
No. (%) positive by L-CLONE
Antigen titer by RIA 1:1-1:8 1:16-1:32 1:64-1:128 .1:256
25 23
15 (50) 15 (48) 14 (56) 15 (65)
Antigen subgroup by EIAa Type A Type B Unknown
63 23 23
33 (52) 10 (43) 12 (52)
30 31
a Subgroups were determined by using monoclonal antibodies and an EIA method. Monoclonal antibody A was from the University of Missouri School of Medicine, and monoclonal antibody B was from the Indiana University School of Medicine. Production of these antibodies has been described in detail previously (6).
no significant difference in the rates of false positivity among the various groups of patients. Within the groups with respiratory diseases, there was no apparent difference in specificity, regardless of the infecting agent. Among the 224 samples from patients with bacteriuria, there was no apparent difference in L-CLONE specificity for any single organism or between gram-positive and gram-negative organisms or mixtures of the two. In testing of 141 fresh urine samples, all were negative by RIA; 121 of 141 (86% specificity) were negative by L-CLONE. L-CLONE reagents from several lots were used to test fresh urine samples. The test specificity was 83% for 40 samples tested with lot K022701 and 87% for 101 samples tested with lot K110805 or KF02027 (data not shown). This 86% overall specificity for fresh samples was greater than the 75% specificity obtained when 341 known RIA-negative stored samples were tested. A known Legionella antigenpositive urine sample was added to four additional fresh samples; all four were positive by RIA but negative by L-CLONE. Reproducibility data for L-CLONE testing of 60 urine samples that initially yielded false-positive or false-negative L-CLONE results were as follows. All samples were retested by RIA, and repeat results were identical to initial results in all samples except one (see below). All samples were tested twice with L-CLONE lot K022701 and once with L-CLONE lot K110805. Of 29 urine samples that were falsely identified as positive by L-CLONE (results for 2 of the initial 31 samples were discarded, 1 because of fungal contamination of the sample and 1 because the repeat RIA yielded a positive result), 15 were consistently false positive in three L-CLONE determinations; but 4 samples had two positive results with lot K022701 and one negative result with lot K110805, 8 samples had one positive result and one negative result with lot K022701 and one negative result with lot K110805, and 2 samples had one positive and one negative result with lot K022701 and one positive result with lot K110805. Of 29 samples identified by L-CLONE as falsely negative, 20 were consistently negative, 5 samples had one positive and one negative result with lot K022701 and one negative result with lot K110805, and 4 samples had one positive and one negative result with lot K022701 and 1 positive result with lot K110805. L-CLONE reagents were provided in convenient-to-use dropper bottles, and the testing procedure required little
technical expertise. It was not difficult to differentiate positive from negative agglutination patterns. The 1.0-ml quantity of control samples provided in the kit was probably inadequate for actual clinical use, because 0.5 to 1.0 ml of control samples must be boiled on each day that tests are performed and would likely be consumed long before the other reagents in the 40-test kit would. In replicate testing of control material by using latex from two different lots of L-CLONE, the technologist noted that the strength of agglutination was inconsistent between the lots, ranging from 1+ to 3+ on the same sample. DISCUSSION When we used L-CLONE to evaluate 481 stored urine samples that were previously tested by RIA, the L-CLONE result agreed with the RIA result for 76 of 140 (54%) of the RIA-positive samples and for 252 of 341 (74%) of the RIA-negative samples. Although the antigen in the samples may have changed during storage or there may have been errors in the initial testing, repeat RIA testing of all samples with discordant results showed this to be true in only 2% of the samples (2 of 64 samples initially RIA positive and 1 of 89 samples initially RIA negative). The lack of L-CLONE sensitivity was consistent with all Legionella urine antigen subgroups and quantities and in culturally and serologically confirmed Legionella infections. The L-CLONE specificity did not vary significantly in testing of urine samples from patients with mycobacteriosis, bacterial pneumonia, empyema, other pulmonary disease, or bacteriuria. The L-CLONE sensitivity of 54% and specificity of 74% we obtained when we tested stored urine samples differed substantially from the 95% sensitivity (37 of 39 samples) and 87% specificity (219 of 248 samples) reported by the manufacturer in the package insert for the L-CLONE test kit (1). Durda and Winn (2) tested the L-CLONE kit previously when it was marketed by Meridian Diagnostics, Inc. They found 92% sensitivity (34 of 37 samples) and 46% (6 of 13 samples) specificity. When we tested 141 fresh urine samples, all of which were Legionella antigen negative by RIA, L-CLONE specificity was 86%. Our specificity results differed from those provided in the L-CLONE package insert (1) when we used the kit to test stored urine but not when we tested fresh urine. Although the differences in test specificity between fresh and stored negative control urine samples may be partially due to the fact that the 341 stored urine samples were likely collected from a more heterogeneous control population than the 141 fresh urine samples were, our use of stored frozen urine may have adversely affected the assay's performance. This is difficult to evaluate. Because the test mechanism, which is described by the manufacturer as relying on "a careful balance in the stoichiometry of the reagents" (1), is insufficiently explained and defined, it is difficult to pinpoint specific interferences. When we tested stored frozen urine specimens from patients with serogroup 1 L. pneumophila infections, we encountered fewer true-positive results than the package insert (1) predicted for the testing of fresh urine specimens. However, because we also encountered fewer true-negative results in frozen urine samples than the manufacturer predicted for the testing of fresh samples, it appears that the effect on the kit's performance of freezing and/or storing urine is not simply a matter of nonspecifically increasing the tendency of the kit's latex particles to agglutinate. Alternatively, given the small number of samples tested to
VOL. 29, 1991
arrive at the package insert (1) sensitivity estimate (39 samples), perhaps the set of samples tested was not representative of the overall population of urine samples likely to come from patients with serogroup 1 L. pneumophila pneumonia. The details regarding how samples from patients with serogroup 1 L. pneumophila pneumonia were collected or stored for testing by L-CLONE have not been published, nor are they provided in the package insert. If, in fact, they were stored urine samples, then our estimate, which was obtained by testing stored urine samples, may be more accurate given the larger sizes of our test groups. We attempted to compensate for the lack of availability of fresh urine samples from patients with serogroup 1 L. pneumophila pneumonia by adding a small quantity of a high-titer antigen-positive stored urine sample to several fresh normal urine samples. The L-CLONE kit failed to detect antigen in these samples, although all of them were highly positive in the reference RIA. Given the low prevalence of legionellosis and the requirements of the kit for fresh urine samples, it seems unlikely that the sensitivity claims made in the package insert (1) can be validated independently in any reasonable time period. Even if the specificity of the L-CLONE kit is 87%, as claimed in the package insert (1), one would expect that 13 of every 100 urine samples tested from patients without serogroup 1 L. pneumophila pneumonia would yield positive results. For a low-prevalence disease like legionellosis, this means that most positive results would be false positives. If, for example, the prevalence of serogroup 1 L. pneumophila pneumonia among the test population were 5%, one might expect, from each 100 patients tested, approximately 13 false-positive and 5 true-positive results. Thus, 13 of 18 (72%) of all positive results would be false positive. There is a need for a rapid and accurate Legionella urine antigen test. L-CLONE is rapid and requires little technical expertise. However, in our opinion, the sensitivity and specificity of L-CLONE were too low and intra- and interlot inconsistencies were too common to allow the test to be seriously considered for clinical use. REFERENCES 1. Access Medical Systems, Inc. 1988. Package insert for L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test. Access Medical Systems, Inc., Branford, Conn.
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2. Durda, J. P., and W. C. Winn. 1988. Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, C-51, p. 340. 3. Edelstein, P. H. 1985. Legionella, p. 373-381. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 4. Kirby, B. D., K. M. Snyder, R. D. Meyer, and S. M. Finegold. 1980. Legionnaires' disease: report of sixty-five nosocomially acquired cases and review of the literature. Medicine 59:188205. 5. Kohler, R. B. 1986. Antigen detectiun for the rapid diagnosis of Mycoplasma and Legionella pneumonia. Diagn. Microbiol. Infect. Dis. 4:47S-59S. 6. Kohler, R. B., C. Wilde III, W. Johnnson, J. Joly, L. J. Wheat, R. Baker, and M. Misfeldt. 1988. Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays. J. Clin. Microbiol. 26:2059-2063. 7. Kohler, R. B., W. C. Winn, Jr., J. C. Girod, and L. J. Wheat. 1982. Rapid diagnosis of pneumonia due to Legionella pneumophila serogroup 1. J. Infect. Dis. 146:444. 8. Kohler, R. B., W. C. Winn, Jr., and L. J. Wheat. 1984. Onset and duration of urinary antigen excretion in Legionnaires' disease. J. Clin. Microbiol. 20:605-607. 9. Reingold, A. L., B. M. Thomason, B. J. Brake, L. Thacker, H. W. Wilkinson, and J. N. Kuritsky. 1984. Legionella pneumonia in the United States: the distribution of serogroups and species causing human illness. J. Infect. Dis. 148:819. 10. Sathapatayavongs, B., R. B. Kohler, L. J. Wheat, A. White, and W. C. Winn, Jr. 1983. Rapid diagnosis of Legionnaires' disease by latex agglutination. Am. Rev. Respir. Dis. 127:559-562. 11. Sathapatayavongs, B., R. B. Kohler, L. J. Wheat, A. White, W. C. Winn, Jr., J. C. Girod, and P. H. Edelstein. 1982. Rapid diagnosis of Legionnaires' disease by urinary antigen detection. Comparison of ELISA and radioimmunoassay. Am. J. Med.
72:576-582. 12. Wilidnson, H. W. 1986. Serodiagnosis of Legionella pneumophila disease, p. 395-398. In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical laboratory immunology, 3rd ed.
American Society for Microbiology, Washington, D.C. 13. Wilkinson, H. W. 1988. Legionellosis, p. 320-332. In A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Turano (ed.), Laboratory diagnosis of infectious diseases, principles and practice, Vol. 1. Springer-Verlag, New York. 14. Zuravleff, J. J., V. L. Yu, J. W. Shonnard, B. K. Davis, and J. D. Rihs. 1983. Diagnosis of Legionnaires disease: an update of laboratory methods with new emphasis on isolation by culture. J. Am. Med. Assoc. 250:1981-1985.
