1. E-mail: am.freydiere@chu-lyon.fr A.M. Freydiere 1, T. Goyard 1, C. Ploton 1, D. Thouvenot 2, A. Rousson 1 and F. Vandenesch 1 1 Hôpital Debrousse, Hospices Civils de Lyon, 2 Laboratoire deVirologie, Hospices Civils de Lyon, Lyon, France. Comparative Evaluation of Four Enzyme Immunoassay Tests versus Cell Culture for the Rapid Diagnosis of Respiratory Syncytial Virus Infection in a Pediatric Hospital Abstract Rapid diagnosis of Respiratory Syncytial Virus (RSV) impacts positively on patient care by reducing hospital stays, and decreasing the need for additional diagnostic procedures. This study assesses the performance of three rapid membrane enzyme immunoassay tests (MEIA) and one ELISA test for RSV detection compared with cell-culture considered as the reference method. One hundred eighty six endotracheal and nasopharyngeal aspirates from hospitalized infants were tested prospectively, 53 with the DAKORapide VRS kit (J2L ELITECH, France), 102 with the Directigen RSV kit (BECTON DICKINSON, France), 103 with the RSV TESTPACK kit (ABBOTT, France), and all the specimens were tested both with ELISA Vidas test (bioMérieux, France) and cell- culture. By using culture as the “gold standard”, the sensitivities, specificities, positive predictive values (PPV) and negative predictive values (NPV) were 75, 67.8, 66.6, and 76 % for DAKORapide VRS; 80.6, 61.3, 81.8, and 59.4 % for Directigen RSV; 86.2, 77.7, 83.3, and 81.4 % for RSV TESTPACK; 87.2, 82.1, 85.5, and 84.1 % for ELISA test. DAKORapide VRS and Directigen RSV yielded 0.2 and 3.9 % of non-interpretable results, respectively, due to difficulties in readings while the other tests did not yield any non-interpretable results. Retrospectively, 84 frozen endotracheal and nasopharyngeal aspirates were simultaneously tested with the MEIA tests. Compared to cell-culture, the sensitivities, specificities, PPV and NPV were 82.3, 60.6, 76.4, and 68.9 % for DAKORapide VRS; 78.4, 66.6, 78.4, and 65.6 % for Directigen RSV; 86.3, 63.6, 78.6, and 75 % for RSV TESTPACK. With frozen specimens DAKORapide VRS showed a highest sensitivity than with fresh specimens. Directigen RSV presented similar results with either frozen or fresh specimens. RSV TESTPACK achieved the best results among the three MEIA tests and similar results to the ELISA test. This test is rapid (20 min vs 2h 30 for ELISA) and easy-to-perform; it appears to be the most convenient test for rapid diagnosis of RSV in a microbiological laboratory. Introduction Respiratory syncytial virus (RSV) represents the single most important cause of serious lower respiratory tract infection being involved, especially bronchiolitis, tracheobronchitis, and pneumoniae, among infants and children in all parts of the world (3). It is the primary cause of hospitalization in the first year of life. The rapid identification of respiratory pathogens is of great importance for the prevention of nosocomial spread, for monitoring infected patients and for improving clinical management. Since the virology laboratory in not located within our pediatric hospital, we determined whether rapid membrane enzyme immunoassay tests (MEIA) might be used in our local bacteriological laboratory and which of the different commercialized tests yielded the best performance. This study assesses the performance of three rapid MEIA and one ELISA test (VIDAS RSV test) for RSV detection compared with cell-culture considered as the reference method by using nasopharyngeal aspirates obtained from pediatric infants with suspected viral lower respiratory tract infections. II) Comparative evaluation of the three MEIA, using frozen clinical specimens. I) Comparison of the three MEIA for RSV detection with cell culture, using fresh specimens Material and Methods Specimens. Specimens. From October 1999 to April 2000 and from October 2000 to April 2001, 186 endotracheal aspirates or nasopharyngeal aspirates were collected by physicians from infants and children (from 1 to 18 months) with apparent viral respiratory tract infections, admitted at Debrousse Hospital. 84 of the 186 specimens were then frozen at -20°C for further testing by the three MEIA in parallel. Reagents. Reagents. The three MEIA evaluated were the DAKORapide VRS kit (J2L ELITECH, France), the Directigen RSV kit (BECTON DICKINSON, France) and the RSV TESTPACK kit (ABBOTT, France). Each MEIA was performed following manufacturer’s instruction. The VIDAS RSV ELISA test (bioMérieux) is an automated qualita- tive test using the Enzyme Linked Fluorescent Assay Technique. Cell Culture. Cell Culture. Samples are inoculated onto the following cell lines: LLC-MK2, MDCK, Vero, HEp-2 and MRC-5. Cultures were performed on shell-vial with a centrifugation after inoculation and incubation at 34°C with a 5% CO2 atmosphere. Cytopathic effect was regularly checked for 10 days in HEp-2 and RSV and identified by immunofluorescence with the RSV Direct IF reagent (bioMérieux) I) Comparison of methods for the detection of the RSV with cell culture, using fresh specimens. I) Comparison of methods for the detection of the RSV with cell culture, using fresh specimens. All the clinical specimens received at the laboratory were tested, prospectively, in real clinical laboratory settings, at least by one of the three MEIA, and were sent in parallel to the clinical virology laboratory for testing by ELISA and cell culture. Among the total of 186 specimens tested, 53 were tested by the DAKORapide VRS, 102 by the Directigen RSV, 103 by the RSV TESTPACK and 186 by the VIDAS RSV ELISA. All the results were compared to the cell culture. II) Comparative evaluation of the three MEIA using frozen clinical specimens. II) Comparative evaluation of the three MEIA using frozen clinical specimens. 84 frozen specimens were retrospectively tested in parallel by the three MEIA, in the same conditions. The results were compared with the cell culture performed before freezing of the specimens. DAKORapide VRS and Directigen RSV yielded 0.2 and 3.9 % of non-interpretable results, respecti- vely, due to difficulties in readings. Cell culture method Non-interpretable result Positive Negative Concordant results (%) DAKORapide VRS (J2L ELITECH) n = 53 Directigen RSV (Becton Dickinson) n = 102 RSV TESTPACK (ABBOTT) n = 103 VIDAS RSV ELISA test (bioMérieux) n = 186 18 9 6 19 1 69.8 54 13 12 19 4 71.6 50 8 10 35 0 82.5 89 13 15 69 0 84.9 Number of specimens PositiveNegativePositiveNegativePositiveNegativePositiveNegative Table 1 a. Comparison of the three MEIA for the detection of the RSV with cell culture, using fresh specimens These findings, in accordance with other studies (7, 8, 12) are most likely due to insensitive RSV culture methods and/or problems with transport of specimens to the laboratory since RSV is a relatively labile virus and isolation is optimized if specimens are inoculated rapidly. On the contrary, in some other cases, only cell culture was positive. Some authors used freeze-thawing to increase the sensitivity of EIAs (4, 10). In this study only the DAKORapide VRS showed a higher sensitivity after freezing (82.3 % versus 75 %). Again, RSV TESTPACK had the highest sensitivity and overall similar results in specificity in comparison with the two other MEIA. The commercially-available MEIA tests for RSV detection are notable because of their convenience and ease-to perform. Moreover, the use of these rapid methods have been demonstrated to have clinical and financial benefits (1, 11). Recent studies have demonstrated that genetical methods are more sensitive than both cell culture and immunological methods (2, 5, 6), however, until real-time genetical methods becomes a reality, direct specimen testing such as immunoenzymatic methods is likely to remain the most rapid, sensitive and specific methodology for detection of RVS in clinical specimens. Amongst the three MEIA, the RSV TESTPACK had the highest sensitivity and specificity. In previous studies, the sensitivity of RSV TESTPACK compared with culture varies from 83% to 96 % and specificity from 84 to 97 % according to the authors (3, 9). The specificity of the three MEIA increased when combining, for the reference method, the results obtained by cell culture and VIDAS RSV ELISA test. Table 1 b. Sensitivity, specificity, PPV and NPV for the different immunological methods using either (i) cell culture or (ii) cell culture + VIDAS RSV ELISA test as reference method. (i) Reference method: cell culture Sensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) (ii) Reference method: cell culture + VIDAS RSV ELISA test Sensitivity (%) Specificity (%) Positive Predictive Value (%) 75 67.8 66.6 76 80.6 61.3 81.8 59.4 86.2 83.3 81.4 87.2 82.1 85.5 84.1 79.2 70.4 70.4 82.6 82.6 93.9 87.9 94.6 96.6 Negative Predictive Value (%)79.259.481.4 DAKORapide VRS (J2L ELITECH) n = 53 Directigen RSV (Becton Dickinson) n = 102 RSV TESTPACK (ABBOTT) n = 103 VIDAS RSV ELISA test (bioMérieux) n = 186 77.7 102 nd A.S.M. May 19-23 Salt Lake City UTAH C-62 Results are presented in Table 1 a and 1 b. 84 frozen specimens were tested in parallel with the three MEIA in the same conditions. Results Acknowledgements We thank the technicians of the two laboratories for their technical assistance, ABBOTT, BECTON DICKINSON, and J2L ELITECH for providing us their respective reagents, and Anne Frangin for her valuable help in producing this poster. 1 - Barenfangen J. et al. 2000. J. Clin. Microbiol. 38:2824-2828 2 - Freymuth F. et al. 1995. J. Clin. Microbiol. 33:3352-3353 3 - Ismail N. et al. 2001. Clin. Microbiol. Newsletter 23:91-94 4 - Kok T. et al. 1990. J. Clin. Microbiol. 27:1151-1154 5 - Liolios I. et al. 2001. J. Clin. Microbiol. 39:2779-2783 6 - Lipson S. M. 2002. J. Clin Microbiol. 40:733-734 7 - Olsen M. A. et al. 1993. Diagn. Microbiol. Infect. Dis.16:105-109 8 - Swierkosz E. M. et al. 1989. J. Clin. Microbiol. 27:1151-1154 9 - Todd S. et al. 1995. J. Clin. Microbiol. 33:1650-1651 10 - Waner J. L. et al. 1990. J. Clin. Microbiol. 28:480-483 11 - Woo P. C. Y. et al. 1997. J. Clin. Microbiol. 35:1579-1581 12 - Wren C. G. et al. 1990. J. Clin. Microbiol. 28:1395-1397 References Table 2. Sensitivity, Specificity, PPV and NPV for the three MEIA using 84 frozen specimens and the cell culture as reference method. 78.4 65.6 78.4 65.6 82.3 60.6 76.4 68.9 86.3 63.6 78.6 75 Reference method: cell culture before freezing Sensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) DAKORapide VRS (J2L ELITECH) n = 84 Directigen RSV (Becton Dickinson) n = 84 RSV TESTPACK (ABBOTT) n = 84 The results of this comparative study, allowed us to use the rapid RSV TESTPACK from ABBOTT to screen incoming clinical specimens to identify any positive results in 20 minutes. All the negative specimens are sent to the clinical virology laboratory for further testing using more sensitive diagnostic tests. Conclusion