Comparison of the Alere i and BD Veritor Assays for the Rapid Detection of Influenza A and B Viruses

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1 Comparison of the Alere i and BD Veritor Assays for the Rapid Detection of Influenza A and B Viruses Gregory J. Berry, 1,2 Olajumoke Oladipo, 1 Debbie Wittnebert, 3 Michael J. Loeffelholz, 1 and John R. Petersen 1 * Background: The use of point-of care testing (POCT) in patient management decisions is becoming increasingly common. Our goal was to evaluate the diagnostic performance of 2 commercially available rapid POCT devices for influenza viruses A and B: the Alere i Instrument (Alere, Scarborough) and the BD Veritor System (BD Diagnostics). Methods: Paired nasopharyngeal swabs were collected from patients (18 71 years) presenting with influenza-like symptoms at 3 outpatient clinics. A total of 65 samples were obtained. The Alere i and BD Veritor were performed according to the manufacturers' instructions. Discordant results were resolved using real-time reverse transcription PCR (RT-PCR). Results: In a head-to-head comparison involving symptomatic adult patients visiting outpatient clinics during the and influenza seasons, the Alere i and BD Veritor had 90.63% agreement in the detection of influenza A virus and a statistically significant observed κ coefficient of (P <0.0001). Discordant results between the Alere i and BD Veritor were further investigated using RT-PCR, showing that the BD Veritor missed 5 positive influenza A virus results (false negatives) and detected 1 false positive, while the Alere i results agreed with all RT-PCR results. There were no discordant results between the Alere i and BD Veritor in the detection of influenza B virus. Conclusions: Our data suggest that the Alere i has higher sensitivity and specificity than the BD Veritor in the detection of influenza A virus. Both assays showed equal performance in the detection of influenza B virus. IMPACT STATEMENT Results of this study will assist laboratorians and clinicians in choosing between 2 different methodologies (isothermal amplification vs rapid antigen tests) in the detection of influenza A and B viruses. 1 Department of Pathology, University of Texas Medical Branch, Galveston, TX; 2 Current affiliation: Northwell Health, Department of Pathology and Laboratory Medicine, Lake Success, NY; 3 Memorial Medical Center Hospital, Port Lavaca, TX. *Address correspondence to this author at: University of Texas Medical Branch, Department of Pathology, 301 University Blvd., Galveston, TX Fax ; jrpeters@utmb.edu. DOI: /jalm American Association for Clinical Chemistry 3 Nonstandard abbreviations: POCT, point-of-care testing; RT-PCR, reverse transcription PCR. May : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

2 Comparison of Rapid Influenza Assays Point-of care testing (POCT) devices are required to generate accurate and reliable test results, making the comparison of available testing devices a necessity for deciding which testing platform to adopt. Timely and accurate diagnosis of viral infections such as influenza and the use of effective medications in patient management decreases morbidity and mortality (1) and reduces unnecessary additional testing. The BD Veritor System Flu A+B (Veritor; BD Diagnostics) is a digital read-out chromatographic immunoassay for the rapid, direct qualitative detection and differentiation of influenza A and B viruses in approximately 10 min from nasopharyngeal wash, aspirate, and swab samples from symptomatic patients. Studies evaluating the BD Veritor have reported sensitivity of 72% 89.6% and specificity of 93% 100% as compared to real-time reverse transcription PCR (RT-PCR) (2 6). The Alere i Influenza A&B (Alere i; Alere, Inc.) is a rapid isothermal nucleic acid amplification assay for the differential and qualitative detection of influenza A and B viruses in <15 min. Traditionally, molecular assays, such as the Alere i, were not available in the clinical setting due to cost, increased turnaround times, and the need for highly trained personnel for testing (7). Studies evaluating the sensitivity and specificity of the Alere i have reported sensitivities ranging from 77.8% to 99.3% and specificities ranging from 85.6% to 100% compared to RT-PCR (8 11). When the Alere i was compared to the FilmArray Respiratory Panel, it showed 100% specificity and an overall sensitivity of 73.2%, which ranged from 25% to 97.4% depending on viral strain and specimen viral titers (12). Our goal was to evaluate the diagnostic performance of these 2 commercially available rapid influenza POCT devices and determine if a methodology had better performance for detection of influenza A and B viruses. METHODS The study was approved by the Institutional Review Board and performed between February 2015 and June 2016 at the University of Texas Medical Branch (Galveston, TX). Paired nasopharyngeal swabs FLOQSwabs (Copan Diagnostics, Inc.) were collected from patients (18 71 years of age) presenting with influenza-like symptoms at 3 outpatient clinics. Specimens were also collected at the Memorial Medical Center Hospital (Port Lavaca, TX) as they presented for evaluation of influenza. One of the swabs was used for influenza testing onsite with the Veritor POCT instrument within 5 10 min of collection, while the second swab was sealed and transported to the main laboratory either immediately, or kept frozen at 80 C until analysis on the Alere i at the main laboratory site. A total of 65 samples (58 from clinic patients and 7 patient control samples supplied by the Alere i manufacturer) were obtained. Both assays were performed according to the manufacturer's instructions. Discordant results between the Alere i and Veritor were resolved using the Simplexa Flu A/B and RSV Direct real-time RT-PCR assay, which has a sensitivity/specificity of 97.1%/97.9% and 100%/99.9% for influenza A and influenza B virus, respectively (manufacturer's package insert data; Focus Diagnostics). RESULTS In the detection of influenza A virus, the Alere i detected a total of 18 positive influenza A samples and the BD Veritor detected 13 of these same specimens as positive (Table 1). The BD Veritor also detected 1 specimen as positive that was not detected by the Alere i. Overall, the 2 assays showed an agreement of 90.63% with an observed κ of ( ) and a P value of < In the case of the 6 discordant results, RT-PCR was performed to determine which result was correct. 2 JALM :06 May 2017

3 Comparison of Rapid Influenza Assays FOCUSED REPORT Table 1. Comparison of the Alere i and BD Veritor in the detection of influenza A and B viruses. Alere i Influenza A Influenza B Positive Negative Positive Negative BD Veritor Positive Negative Agreement, % Observed κ, linear weighting 0.754, 95% CI P < This analysis revealed that the 5 specimens that had been detected as positive on the Alere i, but were not detected by the BD Veritor, were positive by RT-PCR. Additionally, the 1 positive detected by the BD Veritor that was not detected by the Alere i was not detected by RT-PCR, suggesting that this was a false-positive result (Table 1). In the detection of influenza B virus, both the Alere i and the BD Veritor detected 7 positive specimens. Overall, both assays performed equally well in the detection of influenza B virus (Table 1). One specimen was invalid by the Alere i and was not included in the analysis. This Alere i invalid specimen tested positive by the BD Veritor and was confirmed positive by RT-PCR. DISCUSSION The development of rapid nucleic acid amplification tests for POCT is growing, making comparisons between these new technologies and existing methods important in utilization decisions. To our knowledge, this is the first published study comparing the Alere i to the BD Veritor in the detection of influenza. Our findings are of particular relevance because they compare the level of agreement between the Alere i (a waived nucleic acid amplification test) and the BD Veritor (a lategeneration chromatographic immunoassay) in the detection of influenza A and B viruses. The assay time for both platforms is <15 min and ease of use, instrument size, and the manufacturers' claims are comparable, making either one useful in the POCT environment. While the cost of the Alere i is substantially more than that of the BD Veritor, the reimbursement rate is commensurate with this increase. Although rapid antigen tests are clinically useful, their sensitivity and specificity have been called into question in comparison to molecular techniques, particularly when attempting to detect novel influenza strains (13). Compared to other immunoassays of varied performance, the BD Veritor has been shown to be more sensitive than several other chromatographic immunoassays, including the Alere BinaxNOW (3, 14) and the Quidel Sofia (14). Hassan et al. (3) analyzed 200 specimens and showed that the BD Veritor detected lower influenza A viral loads and was significantly better at detecting seasonal influenza A H3N2 when compared to the Alere BinaxNOW, while Dunn et al. (14) analyzed 240 specimens and showed that the accuracy of the BD Veritor was significantly higher than that of the Quidel Sofia. One interesting point is that both of these studies were performed in pediatric populations. Another study comparing the Quidel Sofia to the BD Veritor in which 76% of influenza A positive results were in adults (>18 years) showed the BD Veritor having a lower sensitivity than that of the Sofia (79% vs 64%). Of interest, >80% of positive results were the influenza A/2009/H1N1 subtype in this study (5). We only May : JALM 3

4 Comparison of Rapid Influenza Assays analyzed the adult population, which may have affected the performance of the BD Veritor for the detection of influenza A. It would be of interest to do a head-to-head comparison for the 2 assays in a pediatric population. It would also be interesting to see if the Alere i performs better in one influenza A subtype over another as compared to the BD Veritor, since a previous study showed the Alere i performed well in the detection of influenza A/2009/H1N1 and H3N2 subtypes (87.2% and 92.5% sensitivities, respectively) but did not detect untypable influenza A viruses well (25% sensitivity) when compared to a PCR-based assay (12). In light of these previous studies, our data suggest that the isothermal amplification technique of the Alere i is superior to the BD Veritor and other chromatographic immunoassays in the detection of influenza A virus. When analyzing the performance of both assays in the detection of influenza B virus, they performed identically and no discordant results were obtained. From previous studies, we know that both the Alere i (n = 360 specimens) and the BD Veritor (n = 240 specimens) are highly sensitive in the detection of influenza B virus when compared to a PCR-based method (12, 14). Our results reflect the previous findings that both assays are quite sensitive in the detection of influenza B virus. The differences in sensitivity between influenza A and B viruses could be due to better targets for influenza B virus in the chromatographic immunoassay, which leads to greater yield of positive results that equal those of the isothermal amplification method, or could be the result of our low sample number (n = 64 specimens in final analysis). Also, since RT-PCR was only used in adjudication of discordant results, it is possible that both assays could be falsely positive or negative, although they agree. Further analysis will need to be done to see if greater numbers show different results in the sensitivity of influenza B virus detection between the 2 assays. Our results show that the Alere i is a more sensitive assay than the BD Veritor in the detection of influenza A virus, while the Alere i and the BD Veritor performed equally well for detection of influenza B virus. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: J.R. Petersen, Alere. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. Acknowledgments: We would like to thank Patricia Mann for assisting with specimen collection, Alere for providing the reagents used in the study, and Focus Diagnostics for providing the Simplexa Flu A/B and RSV Direct PCR kits for discordant testing. REFERENCES 1. Chartrand C, Leeflang MM, Minion J, Brewer T, Pai M. Accuracy of rapid influenza diagnostic tests: a metaanalysis. Ann Intern Med 2012;156: Nam MH, Jang JW, Lee JH, Cho CH, Lim CS, Kim WJ. Clinical performance evaluation of the BD Veritor System Flu A+B assay. J Virol Methods 2014;204: Hassan F, Nguyen A, Formanek A, Bell JJ, Selvarangan R. Comparison of the BD Veritor System for Flu A+B with the Alere BinaxNOW Influenza A&B card for detection of influenza A and B viruses in respiratory specimens from pediatric patients. J Clin Microbiol 2014;52: Mese S, Akan H, Badur S, Uyanik A, Group IRTS. Analytical performance of the BD Veritor System for rapid detection of influenza virus A and B in a primary healthcare setting. BMC Infect Dis 2016;16: Leonardi GP, Wilson AM, Mitrache I, Zuretti AR. Comparison of the Sofia and Veritor direct antigen detection assay systems for identification of influenza 4 JALM :06 May 2017

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