Sophie S. Arbefeville, MD, 1,2* Ann R. Fickle, SV(ASCP), 2 Patricia Ferrieri, MD 1,2 ABSTRACT

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1 Sensitivity of the Quidel Sofia Fluorescent Immunoassay Compared With 2 Nucleic Acid Assays and Viral Culture to Detect Pandemic Influenza A(H1N1)pdm09 Sophie S. Arbefeville, MD, 1,2* Ann R. Fickle, SV(ASCP), 2 Patricia Ferrieri, MD 1,2 Lab Med Summer 2015;46: DOI: /LMRXBN8P59ITDHQZ ABSTRACT To confirm a diagnosis of influenza at the point of care, healthcare professionals may rely on rapid influenza diagnostic tests (RIDTs). RIDTs have low to moderate sensitivity compared with viral culture or real-time reverse-transcription polymerase chain reaction (rrt-pcr). With the resurgence of the influenza A (Flu A; subtype H1N1) pandemic 2009 (pdm09) strain in the years 2013 and 2014, we evaluated the accuracy of the United State Food and Drug Administration (FDA)-approved Sofia Influenza A+B Fluorescent Immunoassay to detect epidemic Flu A(H1N1)pdm09 in specimens from the upper-respiratory tract. During a 3-month period, we collected 40 specimens that tested positive via PCR and/or culture for Flu A of the H1N1 pdm09 subtype. Of the 40 specimens, 27 tested positive (67.5%) via Sofia assay for Flu A. Of the 13 specimens with a negative result via Sofia testing, 4 had coinfection, as detected by the GenMark Diagnostics esensor Respiratory Viral Panel. This sensitivity of the RIDT Sofia assay to detect Flu A(H1N1) pdm09 was comparable to previously reported sensitivities ranging from 10% to 75% for older RIDTs. Keywords: influenza A, rapid influenza diagnostic test, (H1N1)pdm09, Quidel Sofia Fluorescent Immunoassay, nucleic acid amplification testing, viral culture. At points of care, healthcare professionals rely on rapid influenza diagnostic tests (RIDTs) to aid them in the clinical management of patients, particularly for the use of antiviral agents in respiratory infections caused by influenza. 1 Antiviral treatment for illness due to influenza infection can shorten the duration of fever and other symptoms, may reduce death and the risk of complications from influenza, Abbreviations: RIDTs, rapid influenza diagnostic tests; Flu A, influenza A; pdm09, pandemic 2009; FDA, United States Food and Drug Administration; rrt-pcr, real-time reverse-transcription polymerase chain reaction; FIA, fluorescent immunoassay; Flu B, influenza B; RVP, respiratory viral panel; DFA, direct fluorescent antibody (DFA); C T, cycle threshold; CLIA, Clinical Laboratory Improvement Amendments of 1988; pos, positive; neg, negative; NA, not applicable; HRV, human rhinovirus; PIV1, parainfluenza virus 1 1 Department of Laboratory Medicine and Pathology, University of Minnesota Medical School and 2 University of Minnesota Medical Center, Fairview, Minneapolis, Minnesota *To whom correspondence should be addressed. sarbefev@umn.edu and may shorten the duration of hospitalization. Clinical benefit is greatest when antiviral treatment is administered early, especially within 48 hours of the onset of influenza illness. 2 Thus, point-of-care RIDTs that provide results in less than 15 minutes are desirable. Before the appearance of the pandemic influenza A (Flu A; subtype H1N1) pdm09 in April 2009, RIDTs were widely used for the rapid diagnosis of influenza. 3 During the 2009 pandemic, the Centers for Disease Control and Prevention in the United States released a warning that RIDTs were inadequate for detecting pandemic Flu A(H1N1)pdm09. 4,5 Our review of the literature revealed that the performance of RIDTs for the detection of Flu A(H1N1)pdm09 virus varied widely, with sensitivities ranging from 10% to 75% compared with viral culture and real-time reverse-transcription polymerase chain reaction (rrt-pcr). 6-9 Since the Flu A(H1N1)pdm09 pandemic in 2009, nextgeneration immunoassay analyzers such as the BD Veritor system for rapid detection of Flu A+B (Becton, Dickinson and Company, Franklin Lakes, NJ) and the Quidel Sofia 230 Lab Medicine Summer 2015 Volume 46, Number 3

2 Table 1. Flu A Test Results for the Simplexa Flu A/B and RSV Direct rrt-pcr Assay, Viral Culture, Sofia Analyzer Influenza A+B FIA, and GenMark Diagnostics esensor Respiratory Viral Panel a Date of Collection Simplexa Focus (C T ) Viral Culture, Flu A result Sofia Flu A/B GenMark Dx Coinfection b Age January 3, 2014 Flu A (24.2) Pos Pos/Neg H1N NA 70 y January 3, 2014 Flu A (23.9) Pos Pos/Neg H1N NA 34 y January 2, 2014 Flu A (35.5) Neg Neg/Neg H1N NA 42 y December 30, 2013 Flu A (30.2) Pos Neg/Neg H1N HRV 27 y January 3, 2014 Flu A (31.0) Pos Pos/Neg H1N NA 34 y December 19, 2013 Flu A (32.4) Pos Pos/Neg H1N NA 71 y December 26, 2013 Flu A (31.0) Neg Pos/Neg H1N NA 6 mo December 27, 2013 Flu A (34.0) Pos Pos/Neg H1N NA 34 y December 12, 2013 Flu A (28.1) Pos Pos/Neg H1N NA 34 y November 14, 2013 Flu A (28.5) Pos Pos/Neg H1N NA 57 y December 22, 2013 Flu A (33.2) Neg Neg/Neg H1N PIV 1 59 y December 13, 2013 Flu A (26.0) Pos Pos/Neg H1N NA 50 y November 26, 2013 Flu A (17.6) Pos Pos/Neg H1N NA 30 y November 25, 2013 Flu A (29.0) Neg Pos/Neg H1N NA 60 y December 21, 2013 Flu A (22.1) Pos Pos/Neg H1N NA 8 y January 5, 2014 Flu A (33.7) Pos Neg/Neg H1N NA 60 y January 6, 2014 Flu A (23.4) Pos Pos/Neg H1N NA 31 y January 6, 2014 Flu A (32.3) Neg Pos/Neg H1N NA 44 y January 7, 2014 Flu A (35.6) Pos Neg/Neg H1N PIV 1 49 y January 8, 2014 Flu A (33.4) Neg Pos/Neg H1N NA 59 y January 8, 2014 Flu A (26.1) Pos Pos/Neg H1N NA 4 mo January 9, 2014 Flu A (27.9) Pos Pos/Neg H1N NA 50 y February 24, 2014 Flu A (36.0) Pos Neg/Neg H1N NA 42 y February 2, 2014 Flu A (26.9) Pos Pos/Neg H1N NA 32 y February 6, 2014 Flu A (27.5) Pos Pos/Neg H1N NA 41 y February 7, 2014 Flu A (35.5) Neg Neg/Neg H1N HRV 55 y January 24, 2014 Flu A (33.2) Neg Neg/Neg H1N NA 44 y January 11, 2014 Flu A (29.9) Pos Pos/Neg H1N NA 75 y February 6, 2014 Flu A (23.7) Pos Pos/Neg H1N NA 27 y February 5, 2014 Flu A (27.7) Pos Pos/Neg H1N NA 72 y February 5, 2014 Flu A (34.5) Neg Neg/Pos H1N NA 35 y January 22, 2014 Flu A (34.2) Pos Pos/Neg H1N NA 58 y January 16, 2014 Flu A (35.5) Neg Neg/Neg H1N NA 45 y January 11, 2014 Flu A (31.8) Neg Pos/Neg H1N NA 13 y January 3, 2014 Flu A (24.2) Pos Pos/Neg H1N NA 70 y January 21, 2014 Flu A (36.3) Pos Neg/Pos H1N NA 56 y February 4, 2014 Flu A (34.1) Neg Neg/Neg H1N NA 67 y February 2, 2014 Flu A (30.3) Pos Pos/Neg H1N NA 32 y February 2, 2014 Flu A (34.3) Pos Neg/Neg H1N NA 59 y January 10, 2014 Flu A (33.4) Neg Pos/Neg H1N NA 55 y Flu A, influenza A; rrt-pcr, real-time reverse-transcription polymerase chain reaction; FIA, fluorescent immunoassay; C T, cycle threshold; pos, positive; neg, negative; NA, not applicable; HRV, human rhinovirus; PIV1, parainfluenza virus 1 a Simplexa Flu A/B and RSV Direct real-time RT-PCR assays are manufactured by Focus Diagnostics Inc, Cypress, CA; Sofia Analyzer-influenza A+B FIA, Quidel Corporation, San Diego, CA; GenMark Diagnostics esensor Respiratory Viral Panel (GenMark Diagnostics, Inc, Carlsbad, CA). b Per the GenMark Diagnostics esensor Respiratory Viral Panel. Influenza A+B Fluorescent Immunoassay (FIA) (Quidel Corporation, San Diego, CA) have become available. These RIDTs differ from previous visually-read rapid influenzadetection tests because results of the newer tests are analyzed and interpreted digitally, potentially increasing the sensitivity of the assay by detecting a smaller amount of antigen and reducing operator variability. The Sofia Influenza A+B FIA is a novel fluorescence-based lateral flow immunoassay that uses specific antibodycoated beads coupled to a heat- and light-stable europium conjugate to detect Flu A and influenza B (Flu B) viral nucleoprotein antigens; this contrasts with the other assays, which are based on chromogenic or lateral-flow immunoassays. 10,11 The Sofia Analyzer uses the europium Downloaded from Summer 2015 Volume 46, Number 3 Lab Medicine 231

3 fluorescent tag illuminated by an ultraviolet light source, to detect antigen. The fluorescence is measured by the Sofia Influenza Analyzer, which displays the test results as positive, negative, or invalid. The performance characteristics of the Sofia Influenza A + B FIA to detect the seasonal influenza virus strains have been established, and found to be nearly as sensitive as nucleic acid testing. However, its performance in detecting the Flu A(H1N1)pdm09 has not been determined With the resurgence of the Flu A(H1N1)pdm09 during the 2013 through 2014 influenza season, we evaluated the accuracy of the FDA-cleared RIDT Sofia Influenza A+B FIA to detect Flu A(H1N1)pdm09 viral nucleoproteins in upper-respiratory-tract specimens, and compared the results with 2 nucleic-acid assays and viral culture. Materials and Methods During the peak of the influenza season, from mid- November 2013 through the beginning of February 2014, nasopharyngeal swabs in universal transport media that tested positive for Flu A via the FDA-cleared Simplexa Flu A/B and RSV Direct real-time RT-PCR assay (Focus Diagnostics Inc., Cypress, CA) or demonstrated Flu A in viral culture were saved and kept frozen at -80 C. The specimens were then thawed and tested on the FDAcleared GenMark Diagnostics Respiratory Viral Panel (RVP) featuring esensor technology (GenMark Diagnostics Inc, Carlsbad, CA) to determine the subtype of the Flu A virus. If the Flu A was of the (H1N1)pdm09 subtype, the specimen was analyzed on the FDA-cleared Sofia Influenza A+B Fluorescent Immunoassay platform. The specimens were frozen and thawed only once. All supplementary tests were set up on the same day that the specimens were thawed. A viral culture or a Simplexa PCR assay was also set up, depending on which of the 2 tests had not already been performed. The viral cultures were prepared by inoculating 0.2 ml of aliquot of the specimen into an R-Mix Too respiratory shell vial (Quidel Corporation) and incubated at 35 C to 37 C, 5% CO 2, for 48 hours. We identified Flu A virus by examining the shell vials for cytopathic effect and then used a Direct Fluorescent-Antibody (DFA) method (D3 Ultra DFA Respiratory Virus Screening and ID Kit, Quidel Corporation) to detect Flu A virus in cell cultures. Table 2. Summary of Flu A Test Results Determined via 4 Assay Systems Variable Total specimens with positive Flu A results Total specimens with negative Flu A results The main purpose of this study was to evaluate the sensitivity of the Sofia Influenza A + B FIA at detecting the Flu A(H1N1)pdm09 subtype using the Simplexa Flu A/B and RSV Direct real-time RT-PCR assay and virus culture results as standards. Results During the study period of approximately 3 months, 40 nasopharyngeal swab specimens met the criteria by testing positive for Flu A and for the subtype (H1N1)pdm09. The Simplexa PCR assay identified 37 of the specimens as having positive results for Flu A; 3 specimens were identified as testing positive for Flu A by viral culture. The 3 specimens with positive results as identified by viral culture were retrospectively tested with the Simplexa PCR assay and all 3 produced a positive result for Flu A. All 40 specimens tested positive for Flu A via the GenMark Diagnostics esensor RVP and all were of the (H1N1)pdm09 subtype (Table 1). Of the 40 specimens, 27 tested positive (67.5%) by Sofia Influenza A+B FIA for Flu A (Table 2). Of the 13 specimens that tested negative via Sofia, 4 had a coinfection that was detected by the GenMark Diagnostics esensor RVP, 2 had human rhinovirus, and 2 had parainfluenza virus type 1. Simplexa Focus a Viral Culture Sofia b GenMark Dx c Flu A, influenza A. a Simplexa Flu A/B and RSV Direct real-time reverse-transcription polymerase chain reaction (RT-PCR) assay (Focus Diagnostics Inc, Cypress, CA). b Sofia Analyzer-influenza A+B FIA (fluorescent immunoassay) (Quidel Corporation, San Diego, CA). c GenMark Diagnostics esensor Respiratory Viral Panel (GenMark Diagnostics Inc, Carlsbad, CA). Cycle threshold (C T ) values ranged from 30.2 to 36.3 (mean C T, 34.4) for the 13 specimens that tested negative via Sofia on the Simplexa RT-PCR assay and from 17.6 to 34.0 (mean C T 28.1) for the 27 specimens that tested positive via Sofia (Figure 1). The difference in the cycle threshold values between the positive and negative Sofia results was statistically significant (unpaired t test, P <.001). 232 Lab Medicine Summer 2015 Volume 46, Number 3

4 C T Value via Simplexa Focus Figure 1 Two specimens tested positive for Flu B but negative for Flu A via the Sofia Influenza A+B FIA. By culture, 27 specimens (67.5%) tested positive for Flu A; of those, 21 (77.8%) were concordant with the Sofia assay results (Table 1). The age range of patients was 4 months to 75 years. All 4 pediatric specimens tested positive via the Sofia assay for Flu A (Table 1). Discussion Accurate and timely diagnosis of Flu A is crucial for clinical management, infection-control measures, and publichealth action to limit transmission of the virus. RIDTs are useful to health care professionals at the point of care. These tests are easy to perform; also, clinic staff with limited laboratory experience can perform the Clinical Laboratory Improvement Amendments of 1988 (CLIA) waived assays. Such assays can provide results in 15 minutes, which is ideal for an acute-care or a physician-office setting in which health care professionals have limited time to examine patients. Positive for Flu A via Sofia Negative for Flu A via Sofia Mean (SD) cycle threshold (C T ) values, determined via the Simplexa Focus (Focus Diagnostics Inc., Cypress, CA), of specimens that tested positive and negative for influenza (Flu) A via the Sofia Fluorescent Immunoassay (Quidel Corporation, San Diego, CA). However, the results of RIDTs are sometimes misleading; many factors can affect the performance of these tests. The accuracy of the results depends on the prevalence of Flu A in the population at the time of testing. Falsepositive Flu A test results are more likely to occur when disease prevalence is low, and false-negative Flu A test results are more likely to occur when disease prevalence is high. The viral load present in the specimen and the Flu A virus strains tested affect test results. 15,16 The introduction of more sophisticated assays with digital analyzers, such as the Sofia and BD Veritor, has improved the sensitivity of the RIDTs by detecting the presence of viral antigens at lower concentrations and their capability to detect different circulating influenza types and strains. 5 The influenza season from 2013 through 2014 is the first season with the influenza A subtype Flu A(H1N1)pdm09 as the predominant influenza virus circulating in the United States since this subtype emerged in The resurgence of (H1N1)pdm09 provided us the opportunity to evaluate the ability of 1 of the new RIDTs to detect this Flu A subtype. This study showed that the sensitivity (67.5%) of the Sofia Influenza A+B FIA in detecting Flu A(H1N1)pdm09 was comparable to previously reported sensitivities for earlier RIDTs. 6-8 The Sofia Influenza A+B FIA misidentified 2 specimens as Flu B, whereas they were identified as Flu A by the Simplexa and the GenMark Diagnostics esensor. This raises the question of possible cross-reaction of the nucleoprotein antigens for Flu A and Flu B, which suggests decreased specificity of the assay. All 4 specimens with dual infection tested negative with the Sofia instrument, raising the possibility that coinfection may affect the ability of the assay to detect Flu A(H1N1) pdm09 or Flu A in general. The sensitivity (67.5%) of the recently introduced Sofia Influenza A+B FIA was comparable to viral culture for the detection of Flu A, as described also by others. 12 Most of the viral cultures (37 of 40 total specimens) were set up with specimens that had been frozen, which may have increased the likelihood of negative culture results. In one study, the Sofia had a high rate of false-positive results for Flu A and Flu B. 13 In our study, we could not verify this finding because all tested specimens were known to have tested positive for Flu A per the Simplexa Focus PCR assay. Test sensitivity improves in specimens from patients with higher nasopharyngeal viral load, as demonstrated indirectly by the Simplexa PCR assay mean cycle threshold values of 28.1 for the positive Sofia result and 34.4 for the negative result. 15,16 The 4 pediatric specimens all tested positive for Flu A by the Sofia assay and had a high viral load, as implied by the low cycle threshold values (31.8, 31.0, 26.1, and 22.1) on the Simplexa PCR assay. Downloaded from Summer 2015 Volume 46, Number 3 Lab Medicine 233

5 These results are consistent with previous data showing that children shed more influenza virus than adults. 9 From the presented data, the performance of the Sofia Influenza A+B FIA to detect Flu A(H1N1)pdm09 was equivalent to that of other lateral-flow immunoassays RIDTs. 6-8,18 Our study was limited by the small number of specimens and the fact that the Sofia Influenza A+B FIA and the additional studies were performed on frozen specimens. These factors may have biased the results. Conclusions Based on the results of our study, it appears that the performance of the Sofia Influenza A+B FIA as a rapid influenza test to diagnose Flu A(H1N1)pdm09 at the point of care was adequate. However, healthcare professionals cannot entirely rely on Sofia Influenza A+B FIA results and should order more sensitive assays, such as real-time RT-PCR, when the clinical findings are not concordant with the Sofia results. Acknowledgments We are grateful for the excellent technical expertise of our staff in the Clinical Microbiology Laboratory. Also, the contents of this article were presented in part at the 114th General Meeting, American Society for Microbiology, May 18, 2014, Boston, Massachusetts. LM References 1. Williams LO, Kupka NJ, Schmaltz SP, Barrett S, Uyeki TM, Jernigan DB. Rapid influenza diagnostic test use and antiviral prescriptions in outpatient settings pre- and post-2009 H1N1 pandemic. J Clin Virol. 2014;60: Fry AM, Goswami D, Nahar K, et al. Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial. Lancet Infect Dis. 2014;14: Sugaya N, Shinjoh M, Mitamura K, Takahashi T. Very low pandemic influenza A (H1N1) 2009 mortality associated with early neuraminidase inhibitor treatment in Japan: analysis of 1000 hospitalized children. 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Quantitative analysis of four rapid antigen assays for detection of pandemic H1N compared with seasonal H1N1 and H3N2 influenza A viruses on nasopharyngeal aspirates from patients with influenza. J Virol Methods. 2012;186: Nutter S, Cheung M, Adler-Shohet FC, Krusel K, Vogel K, Meyers H. Evaluation of indirect fluorescent antibody assays compared to rapid influenza diagnostic tests for the detection of pandemic influenza A (H1N1) pdm09. PLoS One. 2012;7(3):e doi: /journal. pone Lee CK, Cho CH, Woo MK, Nyeck AE, Lim CS, Kim WJ. Evaluation of Sofia fluorescent immunoassay analyzer for influenza A/B virus. J Clin Virol. 2012;55: Lewandrowski K, Tamerius J, Menegus M, Olivo PD, Lollar R, Lee- Lewandrowski E. Detection of influenza A and B viruses with the Sofia analyzer: a novel, rapid immunofluorescence-based in vitro diagnostic device. Am J Clin Pathol. 2013;139: Leonardi GP, Wilson AM, Zuretti AR. Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses. J Virol Methods. 2013;189: Dunn J, Obuekwe J, Baun T, Rogers J, Patel T, Snow L. Prompt detection of influenza A and B viruses using the BD Veritor System Flu A+B, Quidel Sofia Influenza A+B FIA, and Alere BinaxNOW Influenza A&B compared to real-time reverse transcriptionpolymerase chain reaction (RT-PCR). Diagn Microbiol Infect Dis. 2014;79: Hazelton B, Nedeljkovic G, Ratnamohan VM, Dwyer DE, Kok J. Evaluation of the Sofia Influenza A + B fluorescent immunoassay for the rapid diagnosis of influenza A and B. J Med Virol. 2015;87: Peters TR, Blakeney E, Vannoy L, Poehling KA. Evaluation of the limit of detection of the BD Veritor system flu A+B test and two rapid influenza detection tests for influenza virus. Diagn Microbiol Infect Dis. 2013;75: Boyanton BL Jr, Almradi A, Mehta T, Robinson-Dunn B. Performance of the Directigen EZ Flu A+B rapid influenza diagnostic test to detect pandemic influenza A/H1N Diagn Microbiol Infect Dis. 2014;78: Centers for Disease Control and Prevention (CDC). Update: influenza activity United States, September 29, 2013 February 8, MMWR Morb Mortal Wkly Rep. 2014;63: Nitsch-Osuch A, Woźniak-Kosek A, Brydak LB. Accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time RT-PCR in children with influenza A (H1N1) pdm09 infection. Postepy Hig Med Dosw (Online). 2012;66: Lab Medicine Summer 2015 Volume 46, Number 3