Evaluation of Two New Immunochromatographic Assays (Rapid U Legionella
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《微生物临床杂志》
Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, P.O. Box 747, 5000 AS Tilburg, The Netherlands
ABSTRACT
We evaluated two new immunochromatographic assays for their abilities to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained by the Binax NOW urinary antigen test. The sensitivities and specificities were estimated to be 71.2% and 96.6%, respectively, for the Rapid U test; 31.5% and 98.9%, respectively, for the SD Bioline test; and 91.8% and 100%, respectively, for the Binax NOW test.
TEXT
Legionnaires' disease (LD) is a pneumonia responsible for 1 to 5% of cases of community-acquired pneumonia (CAP) requiring hospital admission (3, 15). Legionella pneumophila causes 91% of all reported cases of LD, with serogroup 1 being the most predominant serogroup, causing approximately 80% of all culture-confirmed cases (12). However, this percentage is found to be lower in some European countries (10). Since antigen detection in urine has proved to be a sensitive and rapid method for the detection of L. pneumophila serogroup 1, this technique has become one of the most-used tools for the diagnosis of LD (10). An immunochromatographic (ICT) assay (the Binax NOW legionella urinary antigen test) that has a sensitivity and a specificity similar to those of enzyme immunoassays (EIAs) has been developed (4). ICT assays have important advantages over conventional EIAs: the tests are simple to perform, they do not require special laboratory equipment, and results can be obtained within 15 min.
The aim of our study was to evaluate two newly developed ICT urine antigen tests, the Rapid U Legionella antigen test (Rapid U test; Diamondial, Sees, France) and the SD Bioline Legionella urinary antigen test (SD Bioline test; Standard Diagnostics, Inc., Kyonggi-do, Korea), for the detection of L. pneumophila serogroup 1 in urine. Neither test is yet commercially available.
We evaluated the abilities of the new antigen tests to diagnose LD in a well-described sample of patients with and without LD using frozen urine samples. Urine samples were collected between 1997 and 2005 and were stored at –70°C until processing was performed. We included 73 urine samples from 73 patients with LD (cases). A case of LD was defined as a patient with pneumonia who had radiological signs of infiltration and who showed laboratory evidence of infection with L. pneumophila. One or more of the following criteria constituted laboratory evidence of infection with L. pneumophila: isolation of L. pneumophila from a lower respiratory tract (LRT) sample, a positive PCR result with an LRT sample by a 16S rRNA-based assay (16), or seroconversion to positivity for immunoglobulin M (IgM) and/or IgG antibodies to L. pneumophila in paired acute-phase and convalescent-phase sera by a commercial enzyme-linked immunosorbent assay (ELISA; Serion ELISA; Institut Virion/Serion GmbH, Würzburg, Germany). Urine from all patients with LD-positive samples tested negative for pneumococcal antigen (PAG; Binax NOW; Binax, Portland, Maine). The laboratory results for these patients were as follows (number of patients positive/number of patients tested): serology, 53/60 (88%); isolation, 7/17 (41%); and PCR, 37/38 (97%).
Urine samples from 89 patients with respiratory tract infections other than Legionella infections were tested in a similar manner to test the specificity of the assays. The laboratory test results for these patients were as follows: Streptococcus pneumoniae (total, 50 patients; cultured from blood [blood], PAG detected in urine [PAG], and cultured from sputum [sputum], 8 patients; blood and PAG, 23 patients; blood, 11 patients; sputum and PAG, 2 patients; sputum, 4 patients; PAG, 2 patients), Haemophilus influenzae (total, 10 patients; blood, 3 patients; sputum, 7 patients), Moraxella catarrhalis (sputum, 3 patients), Staphylococcus aureus (total, 4 patients; blood and sputum, 2 patients; sputum, 2 patients), Escherichia coli (total, 2 patients; blood and sputum, 1 patient; sputum, 1 patient), Acinetobacter baumannii (blood and sputum, 1 patient), Streptococcus pyogenes (blood and sputum, 2 patients), Klebsiella pneumoniae (sputum, 1 patient), Mycobacterium tuberculosis (sputum, 3 patients), and Pneumocystis jirovecii (Giemsa and silver stain positive, 1 patient). Twelve patients who had a fourfold rise or more in (complement-fixating) antibodies against influenza A virus (n = 3), adenovirus (n = 1), Chlamydia psittaci (n = 3), Mycoplasma pneumoniae (n = 4), and parainfluenza virus (n = 1) were included.
Urine samples were investigated for the presence of L. pneumophila antigens by using the Rapid U test and the SD Bioline test. Both tests are qualitative ICT tests. We compared the sensitivities and specificities of these assays to those of a widely used ICT assay, the Binax NOW urinary antigen test (Binax NOW; Binax). All three tests were performed simultaneously, and the results were calculated according to the manufacturers' instructions. However, to ensure maximum sensitivity for the urinary antigen tests (9), all samples were reexamined after 60 min. Fisher's exact test was used to compare categorical data. A result with a P value of <0.05 was considered statistically significant.
The results obtained are shown in Table 1. The sensitivities and specificities were 71.2% (52/73) and 96.6% (3/89), respectively, for the Rapid U Legionella antigen test; 31.5% (23/73) and 98.9% (1/89), respectively, for the SD Bioline Legionella urinary antigen test; and 91.8% (67/73) and 100% (0/89), respectively, for the Binax NOW urinary antigen test. The sensitivity of the SD Bioline test decreased to 27.4% (20/73) (P = 0.71) if the tests were reexamined after 55 min. The sensitivities of the Rapid U test and Binax NOW test increased to 80.8% (59/73) (P = 0.25) and 94.5% (69/73) (P = 0.75), respectively, if the tests were reexamined after 45 min. The differences in specificities between the three tests were not statistically significant. The Binax NOW test showed sensitivity levels significantly higher (P < 0.01) than those of the Rapid U and SD Bioline tests.
The characteristics of the first urinary antigen tests were published in the late 1970s (1, 4); and since then, numerous publications that have each described different techniques have followed and together have provided evidence that confirms the value of urinary antigen detection for the diagnosis of infections caused by L. pneumophila serogroup 1 infections (4, 7, 11). In general, none of those previous studies have demonstrated great differences in the abilities of the different tests to detect L. pneumophila antigen when nonconcentrated urine samples were tested. The specificities of the assays in the current evaluation were determined by using single urine specimens collected from 89 patients with pneumonia or respiratory tract infections of known (but not Legionella) etiology. Three control patients tested positive by the Rapid U test; two of these patients had proven pneumococcal pneumonia (blood and PAG), and one patient had S. pyogenes pneumonia (blood, sputum). Two control patients tested positive by the SD Bioline test, and both had proven pneumococcal pneumonia (blood, PAG). For an infection of low prevalence, such as LD, test specificity is of paramount importance. Although the differences in the specificities between the three tests were not statistically significant, our data suggest that in a population with a 4% prevalence of LD (e.g., hospitalized patients with community-acquired pneumonia), even if it is assumed that the Rapid U and the SD Bioline Legionella antigen tests have 100% sensitivities, the predictive values of a positive result would only be about 55% and 65%, respectively. The data further indicate that the Binax NOW urinary antigen test, in concordance with the findings of previous studies, has excellent specificity, as no false-positive results were found.
Based on prospective and retrospective studies with data from solitary cases, moderate to high urinary antigen test sensitivities have been described, ranging from 56 to 99% (2, 5, 6, 8, 13). This may be explained by differences in test and patient characteristics, the serogroup with which the patient is infected, the timing of specimen collection in the course of the illness, and whether the urine is concentrated before it is tested. In a recent evaluation, Sohn et al. (14) evaluated the SD Bioline test using samples from 11 confirmed nosocomial infections with L. pneumophila and 88 respiratory tract infections caused by species other than Legionella. The 11 LD-positive urine samples included 4 samples from culture-proven cases and 7 samples from cases with a high single Legionella immunofluorescence assay titer and a positive sputum PCR result for Legionella. They reported a sensitivity and a specificity of the SD Bioline test of 81.8% (9/11) and 100% (0/88), respectively.
In this evaluation the majority of LD-positive patients were probably infected with L. pneumophila serogroup 1, making it difficult to draw any conclusions about the assay's ability to detect L. pneumophila infections caused by other serogroups or Legionella species. Another drawback of the current evaluation is that we evaluated a relatively small group of patients; this could influence both the sensitivities and the specificities of these two new commercial kits. Despite these limitations, our study provides relevant data related to the clinical sensitivity and specificity levels of these two new commercial kits for the detection of L. pneumophila serogroup 1 antigen in urine.
In conclusion, the Binax NOW urinary antigen test is superior to the Rapid U test and the SD Bioline Legionella urinary antigen test for the diagnosis of infections caused by L. pneumophila serogroup 1. The performance of the SD Bioline test is below the acceptable level for any diagnostic assay.
ACKNOWLEDGMENTS
We thank Diamondial and Standard Diagnostics, Inc., for supplying the assays evaluated in this study.
FOOTNOTES
Corresponding author. Mailing address: Laboratory of Medical Microbiology, St Elisabeth Hospital, P.O. Box 747, 5000 AS Tilburg, The Netherlands. Phone: 31 13 539 2655. Fax: 31 13 544 1264. E-mail: b.diederen@elisabeth.nl.
REFERENCES
Berdal, B. P., C. E. Farshy, and J. C. Feely. 1979. Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay. J. Clin. Microbiol. 9:575-578.
Birtles, R. J., T. G. Harrison, D. Samuel, and A. G. Taylor. 1990. Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection. J. Clin. Pathol. 43:685-690.
Breiman, F. R., and J. C. Butler. 1998. Legionnaire's disease: clinical, epidemiological, and public health perspectives. Semin. Respir. Infect. 13:84-89.
den Boer, J. W., and E. W. Yzerman. 2004. Diagnosis of Legionella infection in Legionnaires' disease. Eur. J. Clin. Microbiol. Infect. Dis. 23:871-878.
Domínguez, J. A., N. Galí, P. Pedroso, A. Fargas, E. Padilla, J. M. Manterola, and L. Matas. 1998. Comparison of the Binax Legionella urinary antigen enzyme immunoassay (EIA) with the Biotest Legionella urine antigen EIA for detection of Legionella antigen in both concentrated and nonconcentrated urine samples. J. Clin. Microbiol. 36:2718-2722.
Dominguez, J. A., N. Gali, L. Matas, P. Pedroso, A. Hernandez, E. Padilla, and V. Ausina. 1999. Evaluation of a rapid immunochromatographic assay for the detection of Legionella antigen in urine samples. Eur. J. Clin. Microbiol. Infect. Dis. 18:896-898.
Fields, B. S., R. F. Benson, and R. E. Besser. 2002. Legionella and Legionnaires' disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506-526.
Guerrero, C., C. M. Toldos, G. Yagüe, C. Ramírez, T. Rodríguez, and M. Segovia. 2004. Comparison of diagnostic sensitivities of three assays (Bartels enzyme immunoassay [EIA], Biotest EIA, and Binax NOW immunochromatographic test) for detection of Legionella pneumophila serogroup 1 antigen in urine. J. Clin. Microbiol. 42:467-468.
Helbig, J. H., S. A. Uldum, P. C. Luck, and T. G. Harrison. 2001. Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromato-graphic assay and comparison with both Binax Legionella urinary enzyme immunoassay (EIA) and Biotest Legionella urinary antigen EIA. J. Med. Microbiol. 50:509-516.
Joseph, J. A., and the European Working Group for Legionella Infections. 2004. Legionnaires' disease in Europe 2000-2002. Epidemiol. Infect. 132:417-424.
Murdoch, D. R. 2003. Diagnosis of Legionella infection. Clin. Infect. Dis. 36:64-69.
Plouffe, J. F., T. M. File, Jr., R. F. Breiman, B. A. Hackman, S. J. Salstrom, B. J. Marston, B. S. Fields, et al. 1995. Reevaluation of the definition of Legionnaires' disease: use of the urinary antigen assay. Clin. Infect. Dis. 20:1286-1291.
Ruf, B., D. Schurmann, I. Horbach, F. J. Fehrenbach, and H. D. Pohle. 1990. Prevalence and diagnosis of Legionella pneumonia: a 3-year prospective study with emphasis on application of urinary antigen detection. J. Infect. Dis. 162:1341-1348.
Sohn, J., H. Sim, H. Jeong, D. Park, H. Cheong, W. Kim, S. Kim, B. Cho, and M. Kim. 2005. Evaluation of a new rapid immunochromatographic test using peptidoglycan-associated lipoprotein for detection of Legionella antigen in urine samples from adults with pneumonia, p. 69. In Program and Abstracts of the 6th International Conference on Legionella. American Society for Microbiology, Washington, D.C.
van der Eerden, M. M., F. Vlaspolder, C. S. de Graaff, T. Groot, W. Bronsveld, H. M. Jansen, and W. G. Boersma. 2005. Value of intensive diagnostic microbiological investigation in low- and high-risk patients with community-acquired pneumonia. Eur. J. Clin. Microbiol. Infect. Dis. 24:241-249.
van der Zee, A., H. Verbakel, C. de Jong, R. Pot, A. Bergmans, M. F. Peeters, P. Schneeberger, and J. Schellekens. 2002. Novel PCR-probe assay for detection of and discrimination between Legionella pneumophila and other Legionella species in clinical samples. J. Clin. Microbiol. 40:1124-1125.(Bram M. W. Diederen and Marcel F. Peeter)
ABSTRACT
We evaluated two new immunochromatographic assays for their abilities to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained by the Binax NOW urinary antigen test. The sensitivities and specificities were estimated to be 71.2% and 96.6%, respectively, for the Rapid U test; 31.5% and 98.9%, respectively, for the SD Bioline test; and 91.8% and 100%, respectively, for the Binax NOW test.
TEXT
Legionnaires' disease (LD) is a pneumonia responsible for 1 to 5% of cases of community-acquired pneumonia (CAP) requiring hospital admission (3, 15). Legionella pneumophila causes 91% of all reported cases of LD, with serogroup 1 being the most predominant serogroup, causing approximately 80% of all culture-confirmed cases (12). However, this percentage is found to be lower in some European countries (10). Since antigen detection in urine has proved to be a sensitive and rapid method for the detection of L. pneumophila serogroup 1, this technique has become one of the most-used tools for the diagnosis of LD (10). An immunochromatographic (ICT) assay (the Binax NOW legionella urinary antigen test) that has a sensitivity and a specificity similar to those of enzyme immunoassays (EIAs) has been developed (4). ICT assays have important advantages over conventional EIAs: the tests are simple to perform, they do not require special laboratory equipment, and results can be obtained within 15 min.
The aim of our study was to evaluate two newly developed ICT urine antigen tests, the Rapid U Legionella antigen test (Rapid U test; Diamondial, Sees, France) and the SD Bioline Legionella urinary antigen test (SD Bioline test; Standard Diagnostics, Inc., Kyonggi-do, Korea), for the detection of L. pneumophila serogroup 1 in urine. Neither test is yet commercially available.
We evaluated the abilities of the new antigen tests to diagnose LD in a well-described sample of patients with and without LD using frozen urine samples. Urine samples were collected between 1997 and 2005 and were stored at –70°C until processing was performed. We included 73 urine samples from 73 patients with LD (cases). A case of LD was defined as a patient with pneumonia who had radiological signs of infiltration and who showed laboratory evidence of infection with L. pneumophila. One or more of the following criteria constituted laboratory evidence of infection with L. pneumophila: isolation of L. pneumophila from a lower respiratory tract (LRT) sample, a positive PCR result with an LRT sample by a 16S rRNA-based assay (16), or seroconversion to positivity for immunoglobulin M (IgM) and/or IgG antibodies to L. pneumophila in paired acute-phase and convalescent-phase sera by a commercial enzyme-linked immunosorbent assay (ELISA; Serion ELISA; Institut Virion/Serion GmbH, Würzburg, Germany). Urine from all patients with LD-positive samples tested negative for pneumococcal antigen (PAG; Binax NOW; Binax, Portland, Maine). The laboratory results for these patients were as follows (number of patients positive/number of patients tested): serology, 53/60 (88%); isolation, 7/17 (41%); and PCR, 37/38 (97%).
Urine samples from 89 patients with respiratory tract infections other than Legionella infections were tested in a similar manner to test the specificity of the assays. The laboratory test results for these patients were as follows: Streptococcus pneumoniae (total, 50 patients; cultured from blood [blood], PAG detected in urine [PAG], and cultured from sputum [sputum], 8 patients; blood and PAG, 23 patients; blood, 11 patients; sputum and PAG, 2 patients; sputum, 4 patients; PAG, 2 patients), Haemophilus influenzae (total, 10 patients; blood, 3 patients; sputum, 7 patients), Moraxella catarrhalis (sputum, 3 patients), Staphylococcus aureus (total, 4 patients; blood and sputum, 2 patients; sputum, 2 patients), Escherichia coli (total, 2 patients; blood and sputum, 1 patient; sputum, 1 patient), Acinetobacter baumannii (blood and sputum, 1 patient), Streptococcus pyogenes (blood and sputum, 2 patients), Klebsiella pneumoniae (sputum, 1 patient), Mycobacterium tuberculosis (sputum, 3 patients), and Pneumocystis jirovecii (Giemsa and silver stain positive, 1 patient). Twelve patients who had a fourfold rise or more in (complement-fixating) antibodies against influenza A virus (n = 3), adenovirus (n = 1), Chlamydia psittaci (n = 3), Mycoplasma pneumoniae (n = 4), and parainfluenza virus (n = 1) were included.
Urine samples were investigated for the presence of L. pneumophila antigens by using the Rapid U test and the SD Bioline test. Both tests are qualitative ICT tests. We compared the sensitivities and specificities of these assays to those of a widely used ICT assay, the Binax NOW urinary antigen test (Binax NOW; Binax). All three tests were performed simultaneously, and the results were calculated according to the manufacturers' instructions. However, to ensure maximum sensitivity for the urinary antigen tests (9), all samples were reexamined after 60 min. Fisher's exact test was used to compare categorical data. A result with a P value of <0.05 was considered statistically significant.
The results obtained are shown in Table 1. The sensitivities and specificities were 71.2% (52/73) and 96.6% (3/89), respectively, for the Rapid U Legionella antigen test; 31.5% (23/73) and 98.9% (1/89), respectively, for the SD Bioline Legionella urinary antigen test; and 91.8% (67/73) and 100% (0/89), respectively, for the Binax NOW urinary antigen test. The sensitivity of the SD Bioline test decreased to 27.4% (20/73) (P = 0.71) if the tests were reexamined after 55 min. The sensitivities of the Rapid U test and Binax NOW test increased to 80.8% (59/73) (P = 0.25) and 94.5% (69/73) (P = 0.75), respectively, if the tests were reexamined after 45 min. The differences in specificities between the three tests were not statistically significant. The Binax NOW test showed sensitivity levels significantly higher (P < 0.01) than those of the Rapid U and SD Bioline tests.
The characteristics of the first urinary antigen tests were published in the late 1970s (1, 4); and since then, numerous publications that have each described different techniques have followed and together have provided evidence that confirms the value of urinary antigen detection for the diagnosis of infections caused by L. pneumophila serogroup 1 infections (4, 7, 11). In general, none of those previous studies have demonstrated great differences in the abilities of the different tests to detect L. pneumophila antigen when nonconcentrated urine samples were tested. The specificities of the assays in the current evaluation were determined by using single urine specimens collected from 89 patients with pneumonia or respiratory tract infections of known (but not Legionella) etiology. Three control patients tested positive by the Rapid U test; two of these patients had proven pneumococcal pneumonia (blood and PAG), and one patient had S. pyogenes pneumonia (blood, sputum). Two control patients tested positive by the SD Bioline test, and both had proven pneumococcal pneumonia (blood, PAG). For an infection of low prevalence, such as LD, test specificity is of paramount importance. Although the differences in the specificities between the three tests were not statistically significant, our data suggest that in a population with a 4% prevalence of LD (e.g., hospitalized patients with community-acquired pneumonia), even if it is assumed that the Rapid U and the SD Bioline Legionella antigen tests have 100% sensitivities, the predictive values of a positive result would only be about 55% and 65%, respectively. The data further indicate that the Binax NOW urinary antigen test, in concordance with the findings of previous studies, has excellent specificity, as no false-positive results were found.
Based on prospective and retrospective studies with data from solitary cases, moderate to high urinary antigen test sensitivities have been described, ranging from 56 to 99% (2, 5, 6, 8, 13). This may be explained by differences in test and patient characteristics, the serogroup with which the patient is infected, the timing of specimen collection in the course of the illness, and whether the urine is concentrated before it is tested. In a recent evaluation, Sohn et al. (14) evaluated the SD Bioline test using samples from 11 confirmed nosocomial infections with L. pneumophila and 88 respiratory tract infections caused by species other than Legionella. The 11 LD-positive urine samples included 4 samples from culture-proven cases and 7 samples from cases with a high single Legionella immunofluorescence assay titer and a positive sputum PCR result for Legionella. They reported a sensitivity and a specificity of the SD Bioline test of 81.8% (9/11) and 100% (0/88), respectively.
In this evaluation the majority of LD-positive patients were probably infected with L. pneumophila serogroup 1, making it difficult to draw any conclusions about the assay's ability to detect L. pneumophila infections caused by other serogroups or Legionella species. Another drawback of the current evaluation is that we evaluated a relatively small group of patients; this could influence both the sensitivities and the specificities of these two new commercial kits. Despite these limitations, our study provides relevant data related to the clinical sensitivity and specificity levels of these two new commercial kits for the detection of L. pneumophila serogroup 1 antigen in urine.
In conclusion, the Binax NOW urinary antigen test is superior to the Rapid U test and the SD Bioline Legionella urinary antigen test for the diagnosis of infections caused by L. pneumophila serogroup 1. The performance of the SD Bioline test is below the acceptable level for any diagnostic assay.
ACKNOWLEDGMENTS
We thank Diamondial and Standard Diagnostics, Inc., for supplying the assays evaluated in this study.
FOOTNOTES
Corresponding author. Mailing address: Laboratory of Medical Microbiology, St Elisabeth Hospital, P.O. Box 747, 5000 AS Tilburg, The Netherlands. Phone: 31 13 539 2655. Fax: 31 13 544 1264. E-mail: b.diederen@elisabeth.nl.
REFERENCES
Berdal, B. P., C. E. Farshy, and J. C. Feely. 1979. Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay. J. Clin. Microbiol. 9:575-578.
Birtles, R. J., T. G. Harrison, D. Samuel, and A. G. Taylor. 1990. Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection. J. Clin. Pathol. 43:685-690.
Breiman, F. R., and J. C. Butler. 1998. Legionnaire's disease: clinical, epidemiological, and public health perspectives. Semin. Respir. Infect. 13:84-89.
den Boer, J. W., and E. W. Yzerman. 2004. Diagnosis of Legionella infection in Legionnaires' disease. Eur. J. Clin. Microbiol. Infect. Dis. 23:871-878.
Domínguez, J. A., N. Galí, P. Pedroso, A. Fargas, E. Padilla, J. M. Manterola, and L. Matas. 1998. Comparison of the Binax Legionella urinary antigen enzyme immunoassay (EIA) with the Biotest Legionella urine antigen EIA for detection of Legionella antigen in both concentrated and nonconcentrated urine samples. J. Clin. Microbiol. 36:2718-2722.
Dominguez, J. A., N. Gali, L. Matas, P. Pedroso, A. Hernandez, E. Padilla, and V. Ausina. 1999. Evaluation of a rapid immunochromatographic assay for the detection of Legionella antigen in urine samples. Eur. J. Clin. Microbiol. Infect. Dis. 18:896-898.
Fields, B. S., R. F. Benson, and R. E. Besser. 2002. Legionella and Legionnaires' disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506-526.
Guerrero, C., C. M. Toldos, G. Yagüe, C. Ramírez, T. Rodríguez, and M. Segovia. 2004. Comparison of diagnostic sensitivities of three assays (Bartels enzyme immunoassay [EIA], Biotest EIA, and Binax NOW immunochromatographic test) for detection of Legionella pneumophila serogroup 1 antigen in urine. J. Clin. Microbiol. 42:467-468.
Helbig, J. H., S. A. Uldum, P. C. Luck, and T. G. Harrison. 2001. Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromato-graphic assay and comparison with both Binax Legionella urinary enzyme immunoassay (EIA) and Biotest Legionella urinary antigen EIA. J. Med. Microbiol. 50:509-516.
Joseph, J. A., and the European Working Group for Legionella Infections. 2004. Legionnaires' disease in Europe 2000-2002. Epidemiol. Infect. 132:417-424.
Murdoch, D. R. 2003. Diagnosis of Legionella infection. Clin. Infect. Dis. 36:64-69.
Plouffe, J. F., T. M. File, Jr., R. F. Breiman, B. A. Hackman, S. J. Salstrom, B. J. Marston, B. S. Fields, et al. 1995. Reevaluation of the definition of Legionnaires' disease: use of the urinary antigen assay. Clin. Infect. Dis. 20:1286-1291.
Ruf, B., D. Schurmann, I. Horbach, F. J. Fehrenbach, and H. D. Pohle. 1990. Prevalence and diagnosis of Legionella pneumonia: a 3-year prospective study with emphasis on application of urinary antigen detection. J. Infect. Dis. 162:1341-1348.
Sohn, J., H. Sim, H. Jeong, D. Park, H. Cheong, W. Kim, S. Kim, B. Cho, and M. Kim. 2005. Evaluation of a new rapid immunochromatographic test using peptidoglycan-associated lipoprotein for detection of Legionella antigen in urine samples from adults with pneumonia, p. 69. In Program and Abstracts of the 6th International Conference on Legionella. American Society for Microbiology, Washington, D.C.
van der Eerden, M. M., F. Vlaspolder, C. S. de Graaff, T. Groot, W. Bronsveld, H. M. Jansen, and W. G. Boersma. 2005. Value of intensive diagnostic microbiological investigation in low- and high-risk patients with community-acquired pneumonia. Eur. J. Clin. Microbiol. Infect. Dis. 24:241-249.
van der Zee, A., H. Verbakel, C. de Jong, R. Pot, A. Bergmans, M. F. Peeters, P. Schneeberger, and J. Schellekens. 2002. Novel PCR-probe assay for detection of and discrimination between Legionella pneumophila and other Legionella species in clinical samples. J. Clin. Microbiol. 40:1124-1125.(Bram M. W. Diederen and Marcel F. Peeter)