COMPARISON OF THE SOFIA AND VERITOR DIRECT ANTIGEN DETECTION ASSAY SYSTEMS TO IDENTIFY INFLUENZA VIRUSES FROM PATIENT NASOPHARYNGEAL

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1 JCM Accepted Manuscript Posted Online 21 January 2015 J. Clin. Microbiol. doi: /jcm Copyright 2015, American Society for Microbiology. All Rights Reserved COMPARISON OF THE SOFIA AND VERITOR DIRECT ANTIGEN DETECTION ASSAY SYSTEMS TO IDENTIFY INFLUENZA VIRUSES FROM PATIENT NASOPHARYNGEAL SPECIMENS. 4 G.P. Leonardi #, Ph.D., A.M. Wilson, Ph.D., I. Mitrache and A.R. Zuretti, M.D Virology Laboratory, Box 47, Department of Pathology, Nassau University Medical Center Hempstead Turnpike, East Meadow, NY Running title: objective influenza antigen testing at point-of-care. #Department of Pathology & Labs, Box 47 Nassau University Medical Center 2201 Hempstead Turnpike East Meadow, NY Phone: Fax: leonardi@numc.edu 18 1

2 19 Abstract Influenza antigen detection assays (Sofia FIA and Veritor) yield objective results, which are potentially useful for point-of-care testing. Assays were evaluated with RT-PCR using 411 nasopharyngeal swab specimens. Sensitivity and specificity (%) of 79.0/99.0 and 64.0/99.4 for influenza/a and 92.9/96.7 and 78.6/98.7 for influenza/b were obtained for Sofia and Veritor, respectively. Downloaded from on June 19, 2018 by guest 2

3 Influenza viruses are important causes of acute respiratory illness resulting in approximately 200,000 hospitalizations and 36,000 deaths in the United States annually. 1 Point-of- care (POC) testing alleviates patient surges by allowing treatment decisions (e.g. antirviral treatment or hospitalization) to occur during the patient-physician encounter. POC assays must be easy to use, inexpensive, provide rapid results and possess adequate sensitivity and specificity. POC influenza antigen detection assays provide adequate specificity however low sensitivity values require retesting of negative results. During the influenza A/ 2009 H1N1 pandemic, antigen detection assays demonstrated sensitivity values ranging from 18.0% to 77.0%. 2-4 In a meta-analysis of 159 influenza-antigen detection assays, heterogeneous sensitivity values (95% CI of 57.9% to 66.6%) clearly demonstrated the need for confirmation of negative results. 5 Subjective visual result interpretation at a specific timeframe has also diminished the value of antigen detection assays for POC testing. 6 Emergency department nursing staffs, already overburdened with other responsibilities, are reticent to take on this additional responsibility which requires technical experience and places specific time constraints for result interpretation. Antigen detection assays providing objective results have recently become available. The Sofia Influenza A + B FIA assay (Quidel Inc, San Diego, CA) uses a fluorescence reader to detect influenza-neucleoprotein antigens. The Veritor system (Becton Dickinson & Co., Sparks, MD) is a chromatographic assay which qualitatively detects influenza neucleoprotein antigens using a optical colorimetric device These 2 assays were compared in a prospective study of nasopharyngeal swab specimens collected from symptomatic patients during the influenza season. 3

4 A total of 411 patient nasopharyngeal flocked-swab specimens, placed into 3mL of transport media (Copan Inc., Marisa, CA) were prospectively studied. Specimens were mainly received from the hospital emergency department from patients exhibiting flu-like symptoms (i.e., fever, headache, body ache, malaise, URI, pneumonia). Specimens were simultaneously tested using both assays following the manufacturer s procedures. Briefly, 260uL of specimen and a preset volume of buffer were placed in a Sofia lysis-containing reaction tube. Using a premeasured pipette, a volume of fluid was placed in a Sofia reaction cassette, incubated for 15 minutes, and analyzed using a Sofia fluorescent reader. The Veritor system utilized 300uL of specimen placed into a reagent tube containing lysis buffer. After tip placement on the reaction tube, 3 drops were placed into a Veritor test device cassette, allowed 10 minutes incubation and the cassette was placed into a colorimetric reader for analysis. Specimens were tested within collection times ranging from 1 to 48 hours, kept at 2-8 o C in the interim period, and tested as received in the laboratory. Specimens were also tested for influenza using RT-PCR (Lyra INF A + B; Quidel, Inc). Nucleic acid extraction was done using a NucliSENSE easymag instrument (biomerieux, Inc; Marcy l Etoile, France) and the resulting aliquots (60uL) were amplified using a smart cycler II (Cepheid Inc., Sunnyvale, CA). A second RT-PCR assay (Pro Flu +, Hologic; San Diego, CA) was used for confirmation when specimens were influenza antigen-negative by both assays but were positive by RT-PCR. Sensitivity, specificity, positive and negative predictive values were calculated for each assay. Confidence intervals (95%) were computed using a modified method of Wald Of the 411 specimens examined, 128 were influenza-positive (100 influenza A and 28 influenza B specimens). Influenza A sub-typing performed on 68 positive specimens yielded 4

5 A/2009 H1N1-like virus in 55/68 specimens (80.9%) and 13 specimens identified as the A/ H3N2-like virus. Other respiratory viruses (58 cases) were identified using cell culture, rt-pcr and/or direct fluorescent antibody techniques. These included respiratory syncytial virus (42 patients), rhinovirus (6 patients), human metapneumovirus (5 patients), adenovirus (3 patients) and parainfluenza type 3 (2 patients). Co- infection occurred in 5 patients. In 4 cases, RSV was identified along with influenza A using direct fluorescent or rapid antigen detection assay. Rhinovirus, identified in MRC-5 culture was isolated along with influenza A virus. Patient age distribution is summarized in Table 1. The year age group accounted for 140 of 411 (34.0%) of patients tested and demonstrated the greatest number of influenza-positive patients (63 of 140; 45.0%). The 0-4 and the 65+ age groups accounted for 14 and 11 influenzapositive patients, respectively. Assay performance data is summarized in Table 2. Using rt-pcr as the gold standard method, sensitivity and specificity values (%) of 79.0/99.0 and 64.0/99.4 for influenza A and 92.9/96.7 and 78.6/98.7 for influenza B were obtained using the Sofia and Veritor assays, respectively. The Sofia assay produced 12 false-positive influenza B results as compared with 5 false-positive results obtained for the Veritor system. A total of 13 influenza A-positive and 2 influenza B-positive specimens were identified solely by RT-PCR. In these cases, a second RT- PCR assay confirmed specimen positivity. Statistical analysis (95% confidence interval) of sensitivity and specificity between these assays yielded non-significant results The Veritor assay easier to perform, having fewer procedural steps. The reader was compact, lightweight and battery powered. In contrast, Sofia required more procedural steps for operation and the fluorescent reader was heavier, requiring technician data input for operation. 5

6 Benefits of the Sofia fluorescent reader include the ability to produce both hard-copy and memory-stored results, and the potential to be interfaced with a laboratory information system. Importantly, Sofia offers both a read-now and walk-away modes of operation, enhancing laboratory workflow flexibility, depending on batch or one-at-a-time specimen testing needs Molecular POC testing which provide high sensitivity and multiple pathogen results are readily being developed. 8-9 Identification of multiple pathogens directly affects the judicious use of antibiotic and antiviral agents and reduces hospital-acquired infection by cohorting those multiple agent-infected patients. A number of obstacles hinder molecular POC testing. 8,9 Molecular assays are more costly. They need portability, miniaturization and disposable, premeasured components. Responsibility for assay maintenance, quality control, reagent storage and environmental monitoring of nonlaboratory sites must also occur. 8 Until resolution of these obstacles, antigen detection assays will remain in POC. The incorporation of instrumentation to providing clear, objective results marks a vast improvement over assays requiring subjective result interpretation. The Veritor instrument is lightweight, battery operated and portable. Although larger and more complicated to use, the Sofia reader permits results to be printed, stored in the instrument or potentially interfaced to a hospital information system. The Sofia instrument also allows testing in both run-now and walk- 112 away modes, adding an important, extra dimension for workflow flexibility. Albeit statistically non-significant, Sofia presently demonstrated higher sensitivity values (%) for influenza A / B (79.0/92.9 versus 64.0/78.6) than the Veritor system. Fluorescent detection methods may be 6

7 responsible for this enhanced sensitivity. Fluorescent detection is known to increase sensitivity while also widening the dynamic assay detection range over colorimetric methods Sofia sensitivity is comparable to that reported by our laboratory when the former was evaluated along with the Quickvue (Quidel Inc) and Directigen Flu A+B (Becton Dickinson & Co.) assays, 6 however, both assay s sensitivity values were below that reported by the manufacturer. Variation in patient age and the predominant influenza A subtype can influence assay sensitivity. 5,11 The present investigation s influenza A/2009/H1N1 subtype (over 80%) and patient population (76/100 positive-influenza A patients over 18 years old) could account for the lower sensitivity values obtained. Both Sofia and Veritor instrumentation provide simple, rapid, objective results. Walk-away operation mode and increased sensitivity favor Sofia over the Veritor assay for POC use. However, sensitivity values approaching 80% may still be insufficient to be reliably accepted, thus underscoring the need for follow up testing when negative influenza results are obtained. 7

8 Table 1. Age distribution of 411 patients tested for influenza 1 from 11/4/2013 through 3/14/ AGE GROUP DISTRIBUTION (YRS) 2. TOTALS: ITEM TOTAL PATIENTS: INFLUENZA A POSITIVE: INFLUENZA B POSITIVE: % OF GROUP A-POSITIVE: % OF GROUP B-POSITIVE: Specimens were tested using direct antigen detection assay (Sofia, Quidel Inc.; Veritor, Becton Dickinson) and by RT-PCR assay (INF A + B, Quidel). A second RT-PCR assay (Pro FLU +, Hologic) was used in cases where specimens were negative by both antigen detection assays but positive by RT-PCR. 2 Age ranges were chosen, following that used by the CDC seasonal influenza weekly report (FLUVIEW),

9 TABLE 2. Performance characteristics of 2 influenza direct antigen detection assays b with respect to RT- PCR c assay results. ITEM INFLUENZA TYPE A SPECIMENS a INFLUENZA TYPE B SPECIMENS SOFIA VERITOR SOFIA VERITOR TP c : FN: TN: FP: SENSITIVITY (%) CI (95%) d SPECIFICITY (%) CI (95%) PPV (%) NPV(%) a A total of 411 nasopharyngeal specimens collected using flocked swabs and placed into 3mL UTM (Copan, Inc) were prospectively studied 165 b Sofia influenza A + B FIA (Qiagen, San Diego, CA ) and the Veritor Flu A + B assay (Becton Dickininson 166 and Co., Sparks, MD). 9

10 c INF A + B RT-PCR assay (Qiagen; San Diego, CA ). A second RT-PCR assay (ProFlu A + B; Hologic/Prodesse; Waukesa, WI) was used to confirm 13 samples influenza A- positive and 2 samples influenza B-positive by rt-pcr only. 170 d Confidence Interval (95%) calculated using the modified method of Wald (QuickCalcs; GraphPad Software, Inc). TP = true positive; FN = false negative; TN = true negative; FP = false positive. Sensitivity was calculated as TP/TP + FN x 100%. Specificity was calculated as TN/TN + FP x 100%. PPV = positive predictive value, calculated as TP/TP + FP; NPV = negative predictive value, calculated as TN/TN + FN. CI = 95% confidence interval. Downloaded from on June 19, 2018 by guest 10

11 177 REFERENCES: Centers for Disease Control and Prevention Key Facts about Seasonal Influenza Centers for Disease Control and Prevention Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) virus-united States, MMWR Morb. Mortal. Wkly. Rep. 58: Ginocchio C, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, Lotikar M, Kowaska M, Becker G, Korologos G, de Gerinomo M, Crawford, JM Evaluation of multiple test methods for the detection of novel influenza A (H1N1) during the New York City outbreak. J. Clin. Virol. 45: Leonardi GP, Mitrache I, Pigal A, Freedman L Public hospital-based experience during an outbreak of pandemic influenza A (H1N1) virus infections. J. Clin. Microbiol. 48: Chartrand C, Leeflang MG, Minion J, Brewer T, Madhukar P Accuracy of rapid influenza diagnostic tests. A meta-analysis. Ann. Int. Med. 156: Leonardi GP, Wilson AM, Zuretti AR Comparison of conventional lateral-flow immunoassays and a new fluorescent immunoassay to detect influenza viruses. J. Virol. Meth. 189(2): Agresti A, Coull BA Approximate is better than exact for interval estimation of binomial proportions. The American Statistician, 52:

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