Journal of Medical Virology 87:35–38 (2015)

Evaluation of the Sofia Influenza A þ B Fluorescent Immunoassay for the Rapid Diagnosis of Influenza A and B Briony Hazelton,1* Gordana Nedeljkovic,1 V. Mala Ratnamohan,1 Dominic E. Dwyer,1,2,3 and Jen Kok1,2,3 1

Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia 2 Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Westmead Hospital, Westmead, New South Wales, Australia 3 Centre for Research Excellence in Critical Infections, University of Sydney, Westmead Hospital, Westmead, New South Wales, Australia

Rapid influenza diagnostic tests (RIDTs) can facilitate the appropriate prescription of antivirals for influenza, obviate the need for unnecessary testing and antibacterial agents and allow the implementation of infection control measures. However, the reported sensitivities and specificities of different RIDTs vary widely in clinical settings, as does assay ability to distinguish between influenza types and subtypes. To evaluate the performance of the Sofia Influenza A þ B fluorescent immunoassay (FIA) for the detection of influenza A and B during the 2013 Southern Hemisphere influenza season, a total of 209 consecutive respiratory tract swabs from adult patients with an influenza-like illness were tested by both Sofia Influenza A þ B and an in-house real-time, reverse transcription-polymerase chain reaction (RT-PCR) assay. Compared to RT-PCR, the sensitivity and specificity of the Sofia Influenza A þ B FIA for detection of influenza A was 72.4% and 98.3%, respectively. Too few influenza B positive samples were available during the study to accurately assess the Sofia’s performance for influenza B detection. The sensitivity of Sofia Influenza A þ B FIA for both influenza A and B detection correlated with the amount of influenza RNA present in the sample as indicated indirectly by the RT-PCR cycle threshold (Ct). In conclusion, the Sofia Influenza A þ B FIA continues to perform well as a RIDT with the circulating influenza strains of the 2013 Southern Hemisphere influenza season. J. Med. Virol. 87:35–38, 2015. # 2014 Wiley Periodicals, Inc. KEY WORDS:

influenza; rapid diagnostic test; fluorescent immunoassay; sensitivity; specificity

C 2014 WILEY PERIODICALS, INC. 

INTRODUCTION A rapid diagnosis of influenza infection can facilitate the prescription of antivirals, obviate the need for unnecessary testing and antimicrobials, and allows the implementation of infection control measures [Tayo et al., 2012]. Although nucleic acid testing (NAT) is now the “gold standard” for diagnosis, the necessity for specialized equipment and staff and its relatively long turnaround times preclude NAT from being used as a point-of-care test [Playford and Dwyer, 2002]. Other available methods including immunofluorescent antigen testing and serology are also not useful as rapid or bedside diagnostic assays. Rapid influenza diagnostic tests (RIDTs) provide a convenient method of diagnosing influenza A and B infection with results available within 10–30 min. However in clinical settings, reported sensitivities and specificities vary widely as do assay ability to detect and distinguish between influenza types and subtypes [Taylor et al., 2009; Kok et al., 2010; Babin et al., 2011; Tayo et al., 2012]. RIDT performance can also be adversely affected by the subjective nature of test result interpretation (particularly by non-laboratory operators) and by the circulation of new influenza strains [Foo et al., 2009; Kok et al., 2010; Baas et al., 2013]. The Sofia Influenza A þ B FIA (hereafter Sofia; Quidel, San Diego, CA) is a novel lateral-flow immunoassay that uses specific antibody-coated beads  Correspondence to: Briony Hazelton, Centre for Infectious Diseases and Microbiology Laboratory Services Level 3, Institute of Clinical Pathology and Medical Research Westmead Hospital, Westmead, New South Wales 2145, Australia. E-mail: [email protected] Accepted 15 April 2014

DOI 10.1002/jmv.23976 Published online 16 May 2014 in Wiley Online Library (wileyonlinelibrary.com).

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coupled to a heat and light-stable europium chelate instead of the traditional gold immunochromatography employed by most available RIDTs [Lee et al., 2012]. An automated Sofia reader is used to scan the nitrocellulose strip of test cassettes for fluorescence after 15 min incubation and provides a result within 1 min. Previous studies have reported sensitivities for the Sofia Influenza A þ B FIA ranging between 78–80% and 75–86% for influenza A and B, respectively, compared to RT-PCR [Leonardi et al., 2013; Lewandrowski et al., 2013]. Using NAT as the reference standard, this study evaluated the performance of Sofia Influenza A þ B FIA during the 2013 Southern Hemisphere influenza season in detection of circulating viruses in the family Orthomyxoviridae, genera Influenzavirus A and Influenzavirus B. MATERIALS AND METHODS Consecutive respiratory tract samples were processed from patients with an influenza-like illness defined as fever >38˚C with cough and/or another respiratory tract symptom. Accepted sample types were Dacron or flocked respiratory swabs (paired nose and throat swabs, nasopharyngeal swabs, or nose swabs) that were placed in viral or universal transport medium. All samples were transported to the laboratory at room temperature. Specimens were processed according to the manufacturer’s instructions [Quidel Corp, 2013]. For each specimen an aliquot of extraction solution was added to the reagent tube and 260 ml of the sample-containing transport medium was then introduced for 1 min. Using the fixed-volume pipettes provided, 120 ml of sample and reagent mixture was then inoculated onto the test cassette. Cassettes were left at room temperature for 15 min and then placed into the Sofia analyzer. The remaining viral transport medium had nucleic acid extraction performed using the Qiagen BioRobot EZ1 instrument (Qiagen, Valencia, CA). NAT was performed using a real-time in-house RT-PCR using primers and a Taqman probe targeting the matrix protein for all influenza A virus subtypes, hemagglutinin H1 for influenza A/H1N1 and A(H1N1)pdm09,

hemagglutinin H3 for influenza A/H3N2 and influenza B virus nucleoprotein, as described previously [Kok et al., 2010]. RESULTS A total of 209 specimens were tested by both Sofia and RT-PCR. The median age of patients was 56 years (range 16–98 years) with only samples from patients 16 years old included. Twenty-nine (13.9%) specimens were positive for influenza A and 6 (2.9%) for influenza B using RTPCR. Twenty-three (79.3%) of the influenza A positive samples were identified as the A(H1N1)pdm09 subtype and 6 (20.7%) were the seasonal A/H3N2 subtype. Compared to RT-PCR, 21 of 29 samples (72.4%) positive for influenza A were also positive by Sofia and 2 of 6 (33.3%) positive for influenza B. There was no significant difference in sensitivity for Sofia’s detection of influenza A(H1N1)pdm09 and seasonal A/H3N2 [18 of 23 (78.3%) influenza A(H1N1)pdm09 vs. 3 of 6 (50%) seasonal A/H3N2; P ¼ 0.30 by Fisher’s exact test]. For the NAT negative samples, Sofia gave a false positive influenza A result for 3 of 180 (1.7%) and a false positive influenza B result for 2 of 203 (1.0%). A summary of the performance statistics of Sofia in detecting influenza A and B is presented in Table I. The sensitivity of Sofia for both influenza A and B correlated with the amount of influenza RNA present in samples as indicated by the RT-PCR cycle threshold (Ct). For influenza A, samples positive by both Sofia and RT-PCR had a mean Ct of 27.74 on the subtyping RT-PCR assay and those negative on Sofia had a RT-PCR Ct mean of 35.85 (P < 0.05 by independent t-test). For influenza B, the two samples that were positive on both Sofia and RT-PCR had a mean Ct of 24.26 and those negative on Sofia had a RTPCR Ct mean of 32.53 (P < 0.05). DISCUSSION The sensitivity and specificity of Sofia Influenza A þ B FIA compared to RT-PCR for the detection of influenza A virus in this study were comparable to previous studies of the assay and similar or superior

TABLE I. Performance Statistics for Sofia Influenza A þ B Fluorescent Immunoassay Using an In-House RT-PCR Assay as the Reference Standard Statistic True positive False negative True negative False positive Sensitivity (95% CI) Specificity (95% CI) Positive predictive value (95% CI) Negative predictive value (95% CI) CI, confidence interval.

J. Med. Virol. DOI 10.1002/jmv

Influenza A (n ¼ 209)

72.4 98.3 87.5 95.7

21 8 177 3 (54.3–85.3) (95.2–99.4) (69.0–95.7) (91.7–97.8)

Influenza B (n ¼ 209) 2 4 201 2 33.3 (9.7–70.0) 99.0 (96.5–99.7) 50.0 (15.0–85.0) 98.0 (95.1–99.2)

Sofia Influenza A þ B FIA

to studies of other RIDTs [Chartrand et al., 2012; Leonardi et al., 2013; Lewandrowski et al., 2013]. There were insufficient samples in this study to accurately assess the performance of Sofia for detecting influenza B, although previous studies of RIDTs have noted generally poorer sensitivity for the detection of influenza B compared to influenza A. This is possibly secondary to the fact that infection with influenza B is often milder than influenza A, particularly that secondary to influenza A/H3N2. Milder infections are usually associated with lower viral loads, and therefore reduced assay sensitivity [Chartrand et al., 2012]. Previous studies have demonstrated the reduced sensitivity of RIDTs compared to NAT and/or viral culture in detecting influenza viruses [Chartrand et al., 2012; Lee et al., 2012; Leonardi et al., 2013; Lewandrowski et al., 2013]. This may be due, in part, to the subjective reading of the results for many RIDTs. An advantage of Sofia over other lateral immunochromatographic assays is the provision of objective results using an automated reader. Although the present study was conducted within a virology reference laboratory during normal working hours, trained personnel in a variety of healthcare settings could feasibly use this assay for the rapid diagnosis of influenza infection. RIDTs, including Sofia, can be used as point-of-care tests given their high specificity (usually >90%) for both influenza A and B when there is widespread circulation of influenza virus [Chartrand et al., 2012; Lewandrowski et al., 2013]. In the present study, the sensitivity of Sofia was directly proportional to the amount of virus present in the sample tested, as demonstrated by the increased sensitivity of Sofia when the Ct value of the corresponding sample’s RT-PCR was low. The observed sensitivity of Sofia in this evaluation was most likely reduced by the exclusion of children from the cohort, who are known to shed virus more abundantly and for longer periods when compared to adults [Fiore et al., 2011]. Sensitivity estimates in this study may have also been adversely affected by the sample types used. Nasopharyngeal swabs have been previously demonstrated to yield good RIDT sensitivity, albeit less than respiratory aspirates [Covalciuc et al., 1999; Andresen and Kesson, 2010]. As clinical details for the samples collected were not available, we were not able to determine the duration of illness prior to sample collection. Peak viral shedding generally occurs on the day of symptom onset, and thus the observed sensitivity of Sofia in this evaluation may have also been adversely affected if samples were collected significantly later [Carrat et al., 2008]. In New South Wales during 2013, influenza A and A(H1N1)pdm09 were the predominant influenza virus type and subtype, respectively [World Health Organization, 2013]. Phylogenetic analysis of influenza A(H1N1)pdm09 viruses circulating when this study was conducted showed that they were antigeni-

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cally similar to influenza A/California/7/2009, one of the predominant influenza strains in circulation when the Sofia A þ B FIA was initially developed and validated [Quidel Corp, 2013; World Health Organization, 2013]. Hence, the Sofia performed well as a RIDT during the 2013 Southern Hemisphere influenza season. Nevertheless, as with other RIDTs, negative Sofia results during periods of peak influenza activity should be followed up with NAT to definitively exclude influenza infection [Harper et al., 2009]. Re-evaluation of assay performance should also be considered with the emergence and circulation of new influenza strains. ACKNOWLEDGEMENTS The authors would like to thank Quidel for provision of the Sofia Influenza A þ B FIA test kits used in this study. REFERENCES Andresen DN, Kesson AM. 2010. High sensitivity of a rapid immunochromatographic test for detection of Influenza A virus 2009 H1N1 in nasopharyngeal aspirates from young children. J Clin Microbiol 48:2658–2659. Baas C, Barr IG, Fouchier RA, Kelso A, Hurt AC. 2013. A comparison of rapid point-of-care tests for the detection of avian influenza A(H7N9) virus, 2013. Euro Surveill 18: pii: 20487. Babin SM, Hsieh Y-H, Rothman RE, Gaydos CA. 2011. A metaanalysis of point-of-care laboratory tests in the diagnosis of novel 2009 swine-lineage pandemic influenza A (H1N1). Diagn Microbiol Infect Dis 69:410–418. Carrat F, Vergu E, Ferguson NM, Lemaitre M, Cauchemez S, Leach S, Valleron A-J. 2008. Time lines of infection and disease in human influenza: A review of volunteer challenge studies. Am J Epidemiol 167:775–785. Chartrand C, Leeflang MM, Minion J, Brewer T, Pai M. 2012. Accuracy of rapid influenza diagnostic tests: A meta-analysis. Ann Intern Med 156:500–511. Covalciuc KA, Webb KH, Carlson CA. 1999. Comparison of four clinical specimen types for detection of Influenza A and B viruses by optical immunoassay (FLU OIA test) and cell culture methods. J Clin Microbiol 37:3971–3974. Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, Uyeki TM, Centers for Disease Control and Prevention (CDC). 2011. Antiviral agents for the treatment and chemoprophylaxis of influenza —Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 60:1–24. Foo H, Blyth CC, van Hal S, McPhie K, Ratnamohan M, Fennell M, Ba Alawi F, Rawlinson W, Adamson S, Armstrong P, Dwyer DE. 2009. Laboratory test performance in young adults during influenza outbreaks at World Youth Day 2008. J Clin Virol 46: 384–386. Harper SA, Bradley JS, Englund JA, File TM, Gravenstein S, Hayden FG, McGeer AJ, Neuzil KM, Pavia AT, Tapper ML, Uyeki TM, Zimmerman RK. 2009. Seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: Clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 48: 1003–1032. Kok J, Blyth CC, Foo H, Patterson J, Taylor J, McPhie K, Ratnamohan VM, Iredell JR, Dwyer DE. 2010. Comparison of a rapid antigen test with nucleic acid testing during co-circulation of pandemic influenza A/H1N1 2009 and seasonal influenza A/ H3N2. J Clin Microbiol 48:290–291. 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. 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.

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J. Med. Virol. DOI 10.1002/jmv

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Evaluation of the Sofia Influenza A + B fluorescent immunoassay for the rapid diagnosis of influenza A and B.

Rapid influenza diagnostic tests (RIDTs) can facilitate the appropriate prescription of antivirals for influenza, obviate the need for unnecessary tes...
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