Infectious Diseases, Internal Medicine, College of Medicine, Korea University, Seoul,

Transcription

1 JCM Accepts, published online ahead of print on 26 November 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Clinical performance evaluation of Sofia RSV FIA rapid antigen test for the diagnosis of respiratory syncytial virus infection Jin Woo Jang, 1 Chi Hyun Cho, 1 Myung-Hyun Nam, 1 Soo Young Yoon, 1 Chang Kyu Lee, 1 Chae Seung Lim, 1# and Woo Joo Kim 2 1 Department of Laboratory Medicine, College of Medicine, Korea University; 2 Division of Infectious Diseases, Internal Medicine, College of Medicine, Korea University, Seoul, Republic of Korea Running title: Evaluation of Sofia RSV FIA rapid antigen test # Correspondent footnote: Chae Seung Lim, Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Guro 2 Dong, GuroGu, Seoul, , Republic of Korea; malarim@korea.ac.kr; Tel: ; Fax: , the authors contributed equally to this study. 1

2 Abstract A recently introduced Sofia RSV FIA was evaluated comparatively with the BinaxNow RSV Card and the SD Bioline RSV test using 348 respiratory samples. The Sofia, BinaxNow and SD Bioline kits showed sensitivities of 66%, 65%, and 64% for RSV A, and 71%, 63%, and 65% for RSV B, respectively Key Words: respiratory syncytial virus, rapid antigen test, real-time RT PCR, sensitivity, specificity 2

3 Although the reverse transcriptase polymerase chain reaction (RT-PCR) method is widely used for respiratory syncytial virus (RSV) detection due to its high sensitivity and specificity, rapid antigen detection tests (RADTs) remain practical tests for the detection of RSV in clinical practice (1, 2). The Sofia RSV FIA (Quidel Corp., San Diego, CA, USA) is a recently introduced U.S. FDA-cleared rapid RSV antigen assay that employs immunofluorescence technology and is interpreted with a portable automatic reader (Sofia). The Sofia RSV FIA eliminates subjective interpretation, thereby greatly reducing the chance of human error. The aim of this study was to evaluate the performance of the Sofia RSV FIA in comparison with conventional RSV antigen tests, BinaxNow RSV Card (Alere Scarborough, Inc., Scarborough, ME, USA) and SD Bioline RSV (Standard Diagnostics Inc., Kyonggi, Korea) test, using RSV RT-PCR as the reference standard (3). Between January and November 2013, a total of 348 respiratory specimens (nasopharyngeal aspirates (NPA) or nasopharyngeal swabs (NS)) were collected from consecutive patients suspected of having respiratory infection at the Korea University Guro Hospital, Seoul, Korea. The mean age of patients was 28.1 years (0-98 years), and the M:F ratio was 1:0.78. Patient samples were transported in a vial containing 3 ml of viral transport medium and were immediately used for viral cell culture and real-time RT-PCR. The leftover specimens were then cryo-preserved at -80 C until RSV rapid antigen testing in March All of the specimens underwent a single freeze-thaw cycle. Among the 348 patient samples, we identified 97 RSV A-positive and 101 RSV B-positive samples by RT-PCR and viral cell culture. One hundred and fifty samples were negative for RSV. This study was approved by the Institutional Research Ethics Board of the Korea University Guro Hospital (Approval No.: KUGH ). For the Sofia RSV FIA, 260 μl of nasopharyngeal specimen in VTM was added to the same volume of the provided reagent in a test tube. A total volume of 120 μl of the 3

4 obtained reaction mixture was then added to the test kit. After waiting approximately 15 min to allow the lateral flow of the reaction mixture, the test cassette was inserted into the portable fluorescence analyzer, called Sofia (Quidel Corp., San Diego, CA, USA), and the results were automatically printed as positive, negative or invalid within one minute. For the BinaxNow RSV Card, 50 μl of nasopharyngeal specimen in VTM was mixed with the same volume of reagent solution. A total volume of 100 μl of the reaction mixture was then added to the cassette. For the SD Bioline RSV test, 50 μl of nasopharyngeal specimen in VTM was mixed with the same volume of reagent solution. The test strip was inserted into a tube containing a total volume of 100 μl of the reaction mixture. For both of the kits, the test result was visually examined after approximately 15 min and interpreted as positive, negative or invalid according to the manufacturer s instructions. For a home-brewed RSV RT-PCR, viral RNAs were extracted from 140 µl of respiratory specimen by the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). A one-step real-time RT-PCR method was performed according to van Elden et al. (4), with minor modifications. Briefly, each tube contained a 25-μL reaction mix that included 2.5 μl of isolated viral RNA, 0.1 μm forward and reverse primer, and 0.1 μm probe. TaqMan amplification and detection were performed with a real-time thermocycler CFX96 (Bio-Rad, Hercules, CA, USA). Thermocycling conditions were as follows: reverse transcription at 50 for 20 min and then initial denaturation at 95 for 10 min followed by 45 cycles at 95 for 15 sec and at 60 for 60 sec. Two laboratory technicians who performed the rapid tests and RT-PCR were blinded to the results of the other tests as well as to the clinical presentations of the patients. The performance parameters, such as sensitivity, specificity, positive predictive value (PPV), negative predictive value, positive likelihood ratio and negative likelihood ratio, of 4

5 the three RADTs were calculated using the RT-PCR results as standards (3) and expressed as a 95% confidence interval. Statistical analysis was performed with Excel software (Microsoft Corporation, Redmond, WA, USA), using a McNemar test or Chi-squared test or independent t test with P < 0.05 considered statistically significant. The performance parameters for the three RADT kits are summarized in Table 1. The differences in sensitivities between the Sofia RSV FIA and BinaxNow RSV Card kit were 1.0% (95% CI, , P = 1.000) for RSV A and 8.0% (95% CI, , P = 0.043) for RSV B. For the comparison between the Sofia RSV FIA and SD Bioline RSV test, the differences in sensitivities were 2.0% (95% CI, , P = 0.724) for RSV A and 6.0% (95% CI, , P = 0.181) for RSV B. The Sofia RSV FIA showed slightly lower specificity than the other kits, but the difference (2.0%) was not statistically significant (95% CI, , P=0.13). PPV did not vary substantially for any of the three RADTs (92-100%). The mean RT-PCR threshold cycle (Ct) values (Standard deviation, SD) for RSV A or RSV B positive specimens for each of the three RADTs are presented in Table 2. Regarding samples that were positive with the Sofia RSV FIA but negative with the other kits, the average RT-PCR Ct (SD) was 25.0 (±3.8) for RSV A (n = 5) and 26.3 (±3.3) for RSV B (n = 9). The sensitivity of the Sofia RSV FIA kit was slightly higher than those of the two other RADTs, but a statistically significant difference in sensitivity was noted only for RSV B detection in comparison with the BinaxNow RSV Card kit. Therefore, the Sofia RSV FIA kit demonstrated similar sensitivity for the detection of RSV A and B compared with the BinaxNow RSV Card or SD Bioline RSV test. The sensitivity values of our study were lower than those reported in previous studies. One reason for this lower sensitivity could be the broad age distribution of enrolled patients in our study. The mean ages of our study population for RSV A and B were 17.8 and

6 years, respectively, and 25% (24/97) of the RSV A positive group was more than 5 years of age, while 32% (32/101) of the RSV B positive group was more than 5 years of age. Compared to subjects 5 years, RADT sensitivity in the group that was over 5 years old was significantly lower in all three RDATs for the detection of RSV A and B. The sensitivities between two groups differed by 20.0% (BinaxNow RSV Card) to 22.6% (SD Bioline RSV test) for RSV A (P < 0.05), and 40.3% (Sofia RSV FIA) to 50.0%( SD Bioline RSV test) for influenza B (P = 0.00). In this study, RT-PCR analysis was performed with fresh specimens, but the RADT analysis was performed with stored frozen specimens. However, a previous study showed similar sensitivities with both fresh and stored frozen specimens for RSV diagnosis in an elderly population (5). Accordingly, the use of stored specimens was assumed to not have affected the sensitivity values in our study. In conclusion, the Sofia RSV FIA kits showed comparable performance measures for RSV A and B compared to the BinaxNow RSV Card and SD Bioline RSV test. The Sofia RSV FIA can be used as a reliable point-of-care (POC) device for rapid detection of RSV in clinical practice. Acknowledgement This study was supported by a Korea University R & D Grant in Conflict of interest None. 6

7 References 1. Rabon-Stith KM, McGuiness CB, Saunders B, Edelman L, Kumar VR, Boron ML Laboratory testing trends for respiratory syncytial virus, J. Clin. Virol. 58: Popow-Kraupp T, Aberle JH Diagnosis of respiratory syncytial virus infection. Open. Microbiol. J. 5: Papenburg J, Buckeridge DL, De Serres G, Boivin G Host and viral factors affecting clinical performance of a rapid diagnostic test for respiratory syncytial virus in hospitalized children. J Pediatr 163: van Elden LJ, van Loon AM, van der Beek A, Hendriksen KA, Hoepelman AI, van Kraaij MG, Schipper P, Nijhuis M Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J. Clin. Microbiol. 41: Casiano-Colón AE, Hulbert BB, Mayer TK, Walsh EE, Falsey AR Lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults. J. Clin. Virol. 28:

8 Table 1. Performance characteristics of three rapid tests for the detection of RSV A/B compared to RT-PCR. RSV Rapid Positive predictive Negative predictive Positive likelihood Negative likelihood Sensitivity (n, 95% CI) Specificity (n, 95% CI) type tests value (n, 95% CI) value (n, 95% CI) ratio (95% CI)* ratio (95% CI) SO 66% (64/97, ) 98.0% (246/251, % (64/69, % (145/178, ( ) 0.35 ( ) 99.1) 96.9) 86.5) A BN 65% (63/97, ) 100.0% (251/251, 100.0% (63/63, % (150/184, 75.3 NA 0.35 ( ) ) 100.0) 86.5) SD 64% (62/97, ) 100.0% (251/251, 100.0% (62/62, % (150/185, 74.8 NA 0.36 ( ) ) 100.0) 86.1) SO 71% (72/101, ) 98.0% (242/247, % (72/77, % (145/174, ( ) 0.30 ( ) 99.1) 97.2) 88.1) B BN 63% (64/101, ) 100.0% (247/247, 100.0% (64/64, % (150/187, 73.9 NA 0.37 ( ) ) 100.0) 85.3) SD 65% (66/101, ) 100.0% (247/247, 100.0% (66/66, % (150/185, 74.8 NA 0.35 ( ) ) 100.0) 86.1) *, Positive likelihood ratio was not applicable when the denominator was zero. Abbreviations: CI, confidence interval; NA, not applicable; RSV, respiratory syncytial virus; RT-PCR, real-time reverse transcription polymerase chain reaction; SO, Sofia RSV FIA kit; BN, BinaxNow RSV Card kit; SD, SD BIOLINE RSV kit.

9 6 7 8 Table 2. Threshold cycle (Ct) levels of real-time PCR for RSV A/B in rapid RSV test positive and negative cases. Influenza type Rapid tests Positive Mean Ct Negative Mean Ct P No. No. (SD) (SD) value * SO 20.0 (3.1) (5.1) 33 <0.05 BN 19.7 (2.7) (4.8) 34 <0.05 A SD 19.6 (2.7) (4.8) 35 <0.05 SO only 25.0 (3.8) positive SO 22.3 (3.2) (4.2) 29 <0.05 BN 21.7 (2.7) (4.2) 37 <0.05 B SD 22.1 (3.1) (4.5) 35 <0.05 SO only 26.3 (3.3) positive *, Independent t test between Positive and Negative Mean Ct Abbreviations: SO, Sofia RSV FIA kit; BN, BinaxNow RSV Card kit; SD, SD BIOLINE RSV kit; SD, Standard Deviation.