Abstract
The Sofia Legionella Fluorescence Immunoassay (FIA; Quidel) is a recently introduced rapid immunochromatographic diagnostic test for Legionnaires’ disease using immunofluorescence technology designed to enhance its sensitivity. The aim of this study was to evaluate its performance for the detection of urinary antigens for Legionella pneumophila serogroup 1 in two National Reference Centers for Legionella. The sensitivity and specificity of the Sofia Legionella FIA test were determined in concentrated and nonconcentrated urine samples, before and after boiling, in comparison with the BinaxNOW® Legionella Urinary Antigen Card (UAC; Alere). Compared with BinaxNOW® Legionella UAC, the sensitivity of the Sofia Legionella test was slightly higher in nonconcentrated urine samples and was identical in concentrated urine samples. The specificity of the Sofia Legionella FIA test was highly reduced by the concentration of urine samples. In nonconcentrated samples, a lack of specificity was observed in 2.3 % of samples, all of them resolved by heat treatment. The Sofia Legionella FIA is a sensitive test for detecting Legionella urinary antigens with no previous urine concentration. However, all positive samples have to be re-tested after boiling to reach a high specificity. The reading is automatized on the Sofia analyzer, which can be connected to laboratory information systems, facilitating accurate and rapid reporting of results.
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Introduction
The accurate diagnosis of Legionella pneumonia is important for the treatment of patients by health care providers and for the control of Legionella epidemics by public health officials. Rapid urinary antigen detection kits such as lateral flow immunochromatographic assays detecting L. pneumophila serogroup 1 are the first-line diagnostic tests for Legionnaires’ disease. In Europe, the 80 % of cases were diagnosed using urinary antigen kits in 2009–2010 [1].
Various immunochromatographic membrane kits requiring no specialized laboratory equipment are commercially available. These tests are easy to perform and provide results in a few minutes, but they have not demonstrated optimal sensitivity [2–4]. Two approaches have been proposed to increase the sensitivity of the commercialized kits that are available: additional readings after a longer incubation time [2, 4–8] and the concentration of urine samples by centrifugation using filter units. Several studies showed an increase in the sensitivity of the BinaxNOW® Legionella Urinary Antigen Card (UAC) by using concentrated urine samples [4, 9–11].
The Sofia Legionella Fluorescence Immunoassay (FIA; Quidel, San Diego, CA, USA) is a recently introduced rapid diagnostic test for Legionnaires’ disease (LD) that uses immunofluorescence technology coupled with an automatic reader to enhance its sensitivity. To date, no study has been published on the performance of this test compared with the BinaxNOW® Legionella UAC (Alere, Jouy-en-Josas, France), the kit that is most commonly used. The aim of this study was to evaluate the performance of Sofia Legionella FIA in comparison with BinaxNOW® Legionella UAC in concentrated and nonconcentrated urine samples at two National Reference Centers for Legionella.
Materials and methods
Clinical specimens
A total of 269 urine samples were tested in the French and Swiss National Reference Centers (centers 1 and 2 respectively: 199 in center 1 and 70 in center 2), corresponding to 179 prospective samples submitted for Legionella urinary antigen detection between January and March 2013 (150 in center 1 and 29 in center 2) and 90 repository urine samples collected from 2009 to 2013 from patients with known LD and stored at −20 °C (49 in center 1 and 41 in center 2). Previously, urine samples had been classified as positive based on a BinaxNOW® Legionella UAC result obtained from concentrated urine samples.
A case of LD was defined according to the European Legionnaires’ Disease Surveillance Network (ELDSNet) criteria [12], i.e., clinical and/or radiological evidence of pneumonia associated with positive urinary antigens and/or positive culture or PCR on respiratory samples. In our study, cases were diagnosed by positive urinary antigens using BinaxNOW® Legionella UAC on concentrated urine samples. Twenty-five cases were confirmed by culture on respiratory samples with Legionella pneumophila serogroup 1 strain and 7 of them were Mab3/1-negative.
Legionella urinary antigen detection
The BinaxNOW® Legionella UAC is a colorimetric immunochromatographic test. A sample was considered positive when both the control line and the test line were visible after 15 min of incubation at room temperature.
The Sofia Legionella FIA kit employs a lateral-flow immunofluorescence technique. The signal is only visible under UV light. The reading is performed after 10 min of incubation and interpreted using the SOFIATM analyzer, which automatically scans the test strip, collects and analyzes the fluorescence data, and then calculates and reports the result in about 1 min.
With the Binax Legionella Urinary Antigen EIA kit (Alere), a sample was considered positive when the mean absorbance value was three times the mean value of the negative control.
Method
The tests were performed according to the manufacturers’ instructions. In addition, some samples were concentrated and heat-treated before testing. Urine samples yielding discrepant results before and after heat treatment were checked by using the Binax Legionella Urinary Antigen EIA kit.
Nonconcentrated urine samples were tested using Sofia Legionella FIA and the results were compared with those of nonconcentrated (centers 1 and 2) and those of concentrated urine samples (center 1 only) tested simultaneously using BinaxNOW® AUC.
Urinary concentration was performed in center 1 by centrifugation at 4,000 g for 10 min using Amikon Ultra-4 Ultracel-10 k (Millipore Corporation, Bedford, MS, USA) [13].
In the two centers, urine samples yielding a positive result were retested after heating at 100 °C for 5 min and centrifugation for 15 min at 1,000 g to exclude false-positive results [14]. Any sample yielding a positive result after heating was considered true-positive. Any urine sample positive before heating and negative after heating was considered false-positive.
Results
Of the 269 nonconcentrated urine samples tested, the Sofia Legionella FIA test and the BinaxNOW® Legionella UAC yielded 172 and 181 negative results and 97 and 88 positive results respectively (Table 1). Of the 9 discrepant samples yielding a positive result with the Sofia Legionella FIA test, but a negative one with the BinaxNOW® Legionella UAC, 5 urine samples (4 in center 1 and 1 in center 2) yielded a negative result with the Sofia Legionella FIA test after heating. Four of them with a sufficient amount to be checked with the BinaxNOW® EIA showed a confirmed negative result. No respiratory samples were available for these 5 patients. The 4 other urine samples (2 in center 1 and 2 in center 2) remained positive with the Sofia Legionella FIA test after heating and yielded a positive result with the BinaxNOW® EIA before and after heating. For 2 of these patients, a Legionella PCR performed on respiratory samples was positive and in addition, for one of them a L. pneumophila serogroup 1-PCR was also positive. For the other one, the L. pneumophila serogroup 1-PCR was negative.
As several studies demonstrated that urine sample concentration increased the sensitivity of urinary tests, the results of the BinaxNOW® Legionella UAC on concentrated urine samples were compared with those of the Sofia Legionella FIA on nonconcentrated urine samples in center 1 (n = 199; Table 2). The two reagents detected 49 positive samples, confirming the presence of Legionella urinary antigen. Among them, 2 samples had not been detected by the BinaxNOW® Legionella UAC before concentration. Four discrepant results were observed, yielding a positive result with the Sofia Legionella FIA and a negative one with the BinaxNOW® Legionella UAC. These samples were confirmed as negative by the Sofia Legionella FIA after heating, corresponding to false-positive results as previously described in Table 1.
Although the manufacturer of the Sofia Legionella FIA does not recommend urine concentration, we evaluated its impact on 199 concentrated urine samples in center 1. Eleven urine samples were detected as being positive with use of the Sofia Legionella FIA, but negative with use of the BinaxNOW® Legionella UAC. Among these samples, 9 turned out to have negative results after heating, confirming false positivity. In 1 case there was not enough of the urine sample available for retesting after heating; thus, it was classified as invalid. The last sample remained positive after heating, but was detected as being negative by the BinaxNOW® EIA. Legionella spp. and L. pneumophila PCR performed on respiratory samples were negative for this patient.
Discussion
In this work, we evaluated the Sofia Legionella FIA, a recently introduced rapid diagnostic test for the detection of L. pneumophila serogroup 1 urinary antigens, which uses immunofluorescence technology to enhance its sensitivity.
Although the manufacturers do not recommend urine concentration, the centrifugal ultrafiltration method for rapid concentration of L. pneumophila urinary antigen was evaluated. The Legionella urinary antigen is stable at 100 °C for 30 min [15]; therefore, all positive samples were retested as described previously after boiling to suppress nonspecific reactions and confirm positive results [9, 14, 16, 17].
Our results demonstrate that the Sofia Legionella FIA test exhibits higher sensitivity than the BinaxNOW® Legionella UAC, since it allowed the detection of all the LD-positive patients with nonconcentrated urine samples. This study also confirms that the sensitivity of BinaxNOW® Legionella UAC is enhanced by the concentration of urine without any decrease in specificity. As a matter of fact, 2 nonconcentrated urine samples that tested negative with the BinaxNOW® Legionella UAC yielded a positive result after concentration. Thus, these two tests demonstrated similar performance in terms of sensitivity only if urine samples are concentrated for the BinaxNOW® Legionella UAC.
The major limitation of the Sofia Legionella FIA is the lower specificity compared with the BinaxNOW® Legionella UAC that was detected in both concentrated and nonconcentrated urine samples. As a matter of fact, Quidel recommends using the Sofia Legionella FIA in nonconcentrated urine samples. Our results confirm this important point, since the number of false-positive results increases from 4 before to 11 after concentration. Moreover, 1 out of the 11 false-positive results in concentrated urine samples remained positive after heating.
These results of sensitivity and specificity were further confirmed in 214 new prospective urine samples tested between March and June 2014 in center 1 with Sofia Legionella FIA performed on nonconcentrated samples and the BinaxNOW® Legionella UAC on concentrated samples. Three positive urine samples were equally detected by the two kits and boiling urine samples eliminated 6 false-positive results with use of the Sofia Legionella FIA.
Finally, without heat treatment, a lack of specificity was observed for 2.3 % of all samples tested in this study (11 out of 483). Because of the consequences of false-positive results on the patient outcome and on outbreak investigation [17, 18], the recommendation of boiling any positive urine to differentiate a false-positive result from a true one should be recommended in the manufacturer’s instructions.
The Sofia Legionella FIA may represent a next step in the evolution of immunoassays, combining lateral flow and fluorescent antibody detection formats. The choice of fluorescent rather than colorimetric detection is at least partially responsible for the improved sensitivity demonstrated by the Sofia Legionella FIA, and this improvement has also been demonstrated for Influenza virus detection in comparison with several colorimetric lateral flow devices [19–22]. Because of its automated reading rather than subjective readings of the colorimetric bands, this assay produces objective data. Moreover, the Sofia analyzer can be connected to laboratory information systems to facilitate the accurate and rapid reporting of results.
References
Beauté J, Zucs P, de Jong B, European Legionnaires’ Disease Surveillance Network (2013) Legionnaires disease in Europe, 2009–2010. Euro Surveill 18:20417
Bruin JP, Diederen BM (2012) Evaluation of Meridian TRU Legionella®, a new rapid test for detection of Legionella pneumophila serogroup 1 antigen in urine samples. Eur J Clin Microbiol Infect Dis 32:333–334
Svarrer CW, Lück C, Elverdal PL, Uldum SA (2012) Immunochromatic kits Xpect Legionella and BinaxNOW Legionella for detection of Legionella pneumophila urinary antigen have low sensitivities for the diagnosis of Legionnaires’ disease. J Med Microbiol 61:213–217
Yzerman EP, den Boer JW, Lettinga KD, Schellekens J, Dankert J, Peeters M (2002) Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires’ disease in The Netherlands. J Clin Microbiol 40:3232–3236
Diederen BM, Bruin JP, Scopes E, Peeters MF, IJzerman EP (2009) Evaluation of the Oxoid Xpect Legionella test kit for detection of Legionella pneumophila serogroup 1 antigen in urine. J Clin Microbiol 47:2272–2274
Bruin JP, Peeters MF, Ijzerman EP, Diederen BM (2010) Evaluation of Legionella V-TesT for the detection of Legionella pneumophila antigen in urine samples. Eur J Clin Microbiol Infect Dis 29:899–900
Held J (2012) Increasing the sensitivity of the BinaxNOW Legionella urinary antigen immunochromatographic test by additional readings at later time points. J Med Microbiol 61:884–885
Diederen BM, Peeters MF (2007) Evaluation of the SAS Legionella Test, a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 antigen in urine. Clin Microbiol Infect 13:86–88
Domínguez JA, Galí N, Pedroso P, Fargas A, Padilla E, Manterola JM, Matas L (1998) 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 Clin Microbiol 36:2718–2722
Guerrero C, Toldos CM, Yagüe G, Ramírez C, Rodríguez T, Segovia M (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
Domínguez JA, Manterola JM, Blavia R, Sopena N, Belda FJ, Padilla E, Giménez M, Sabrià M, Morera J, Ausina V (1996) Detection of Legionella pneumophila serogroup 1 antigen in nonconcentrated urine and urine concentrated by selective ultrafiltration. J Clin Microbiol 34:2334–2336
http://ecdc.europa.eu (ECDC Portal > English > Activities > Surveillance > European Surveillance Networks/ECDC Disease Specific Surveillance > European Legionnaires’ Disease Surveillance Network (ELDSNet) > EU case definition). Accessed 19 February 2015
Blanco S, Prat C, Pallarés MA, Matas L, Domínguez J (2004) Centrifugal ultrafiltration method for rapid concentration of Legionella pneumophila urinary antigen. J Clin Microbiol 42:4410
Doskeland SO, Berdal BP (1980) Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions. J Clin Microbiol 11:380–384
Kohler RB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, White A (1981) Rapid radioimmunoassay diagnosis of Legionnaires’ disease: detection and partial characterization of urinary antigen. Ann Intern Med 94:601–605
Birtles RJ, Harrison TG, Samuel D, Taylor AG (1990) Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection. J Clin Pathol 43:685–690
Pontoizeau C, Dangers L, Jarlier V, Luyt CE, Guiller E, Fievet MH, Lecsö-Bornet M, Aubry A, Brossier F (2014) Ruling out false-positive urinary Legionella pneumophila serogroup 1 and Streptococcus pneumoniae antigen test results by heating urine. J Clin Microbiol 52:4347–4349
Rota MC, Fontana S, Montaño-Remacha C, Scaturro M, Caporali MG, Vullo V, Scorzolini L, Ercole A, Ricci ML (2014) Legionnaires’ disease pseudoepidemic due to falsely positive urine antigen test results. J Clin Microbiol 52:2279–2280
Rath B, Tief F, Obermeier P, Tuerk E, Karsch K, Muehlhans S, Adamou E, Duwe S, Schweiger B (2012) Early detection of influenza A and B infection in infants and children using conventional and fluorescence-based rapid testing. J Clin Virol 55:329–333
Lewandrowski K, Tamerius J, Menegus M, Olivo PD, Lollar R, Lee-Lewandrowski E (2013) Detection of influenza A and B viruses with the Sofia analyzer: a novel, rapid immunofluorescence-based in vitro diagnostic device. Am J Clin Pathol 139:684–689
Leonardi GP, Wilson AM, Zuretti AR (2013) Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses. J Virol Methods 189:379–382
Lee CK, Cho CH, Woo MK, Nyeck AE, Lim CS, Kim WJ (2012) Evaluation of Sofia fluorescent immunoassay analyzer for influenza A/B virus. J Clin Virol 55:239–243
Acknowledgements
The authors kindly thank the two technicians of our laboratory, Karine Droitcourt and Marielle Siffert, for performing antigen urinary assays.
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
Sofia Legionella FIA kits and equipment were kindly provided by Quidel Corp. Hospices Civils de Lyon received a grant from Quidel to support the evaluation of the Sofia Legionella FIA kits.
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Beraud, L., Gervasoni, K., Freydiere, A.M. et al. Comparison of Sofia Legionella FIA and BinaxNOW® Legionella urinary antigen card in two national reference centers. Eur J Clin Microbiol Infect Dis 34, 1803–1807 (2015). https://doi.org/10.1007/s10096-015-2415-9
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DOI: https://doi.org/10.1007/s10096-015-2415-9