Comparison of the Sofia and Veritor Direct Antigen Detection Assay Systems for Identification of Influenza Viruses from Patient Nasopharyngeal Specimens G. P. Leonardi, A. M. Wilson, I. Mitrache, A. R. Zuretti
Influenza antigen detection assays (Sofia fluorescent immunoassay [FIA] and Veritor) yield objective results, which are potentially useful for point-of-care testing. The assays were evaluated with reverse transcriptase PCR (RT-PCR) using 411 nasopharyngeal swab specimens. Sensitivity and specificity values (percentages) 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 the Sofia and Veritor assays, respectively.
I
nfluenza 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., antiviral treatment or hospitalization) to occur during the patient-physician encounter. POC assays must be easy to use, be 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% confidence interval [CI], 57.9% to 66.6%) clearly demonstrated the need for confirmation of negative results (5). Subjective visual result interpretation in a specific time frame 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 that provide objective results have recently become available. The Sofia influenza A ⫹ B fluorescent immunoassay (FIA) (Quidel, Inc., San Diego, CA) uses a fluorescence reader to detect influenza nucleoprotein antigens. The Veritor system (Becton Dickinson & Co., Sparks, MD) is a chromatographic assay which qualitatively detects influenza nucleoprotein antigens using an optical colorimetric device. These 2 assays were compared in a prospective study of nasopharyngeal swab specimens collected from symptomatic patients during the 2013 to 2014 influenza season. A total of 411 patient nasopharyngeal flocked-swab specimens, placed into 3 ml of transport medium (Copan, Inc., Marisa, CA), were prospectively studied. Specimens were mainly collected from patients exhibiting flu-like symptoms (i.e., fever, headache, body ache, malaise, upper respiratory infection [URI], pneumonia) in the hospital emergency department. Specimens were simultaneously tested using each assay following the manufacturer’s procedures. Briefly, 260 l of specimen and a preset volume of buffer were placed in a reaction tube containing Sofia lysis buffer. Using a premeasured pipette, a volume
April 2015 Volume 53 Number 4
of fluid was placed in a Sofia reaction cassette, incubated for 15 min, and analyzed using a Sofia fluorescent reader. The Veritor system utilized 300 l of specimen placed into a reagent tube containing lysis buffer. After the tip was placed on the reaction tube, 3 drops were placed into a Veritor test device cassette and allowed 10 min for incubation, and then the cassette was placed into a colorimetric reader for analysis. Specimens were tested within collection times ranging from 1 to 48 h, kept at 2 to 8°C in the interim period, and tested as they were received in the laboratory. Specimens were also tested for influenza using reverse transcriptase PCR (RT-PCR) (Lyra influenza A ⫹ B assay, Quidel, Inc.). Nucleic acid extraction was done using a NucliSENS easyMAG instrument (bioMérieux, Inc., Marcy l’Etoile, France), and the resulting aliquots (60 l) 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 the specimens were influenza antigen negative by the two assays but positive by RT-PCR. Sensitivity, specificity, and positive and negative predictive values were calculated for each assay. Ninetyfive percent confidence intervals were computed using a modified Wald method (7). Of the 411 specimens examined, 128 were influenza positive (100 influenza A and 28 influenza B specimens). Influenza A subtyping performed on 68 positive specimens yielded A/2009 H1N1like virus in 55/68 specimens (80.9%) and 13 specimens identified as A/H3N2-like virus. Other respiratory viruses (58 cases) were identified using cell culture, RT-PCR, and/or direct fluorescent
Received 3 December 2014 Returned for modification 19 December 2014 Accepted 8 January 2015 Accepted manuscript posted online 21 January 2015 Citation Leonardi GP, Wilson AM, Mitrache I, Zuretti AR. 2015. Comparison of the Sofia and Veritor direct antigen detection assay systems for identification of influenza viruses from patient nasopharyngeal specimens. J Clin Microbiol 53:1345–1347. doi:10.1128/JCM.03441-14. Editor: Y.-W. Tang Address correspondence to G. P. Leonardi, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03441-14
Journal of Clinical Microbiology
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Virology Laboratory, Department of Pathology, Nassau University Medical Center, East Meadow, New York, USA
Leonardi et al.
TABLE 1 Age distribution of 411 patients tested for influenzaa from 4 November 2013 through 14 March 2014 Distribution (no.) by age group (yr)b Test result
0–4
5–17
18–49
50–64
ⱖ65
Total
Total patients Influenza A positive Influenza B positive Percentage of group A positive Percentage of group B positive
80 10 04 12.5 05.0
64 14 02 21.8 03.1
140 53 10 37.9 07.1
81 17 07 21.0 08.6
46 06 05 13.0 10.7
411 100 28 24.3 06.8
Specimens were tested using direct antigen detection assays (Sofia [Quidel, Inc.] and Veritor [Becton Dickinson & Co.]) and by RT-PCR assay (Influenza A ⫹ B; Quidel). A second RT-PCR assay (ProFlu⫹; Hologic) was used in cases where specimens were negative by the two antigen detection assays but positive by RT-PCR. b Age ranges were chosen following those used by the CDC seasonal influenza weekly report (FluView), http://www.cdc.gov/flu/weekly/overview.htm. a
steps for operation, and the fluorescent reader was heavier, requiring technician data input for operation. Benefits of the Sofia fluorescent reader include its ability to produce hard-copy and memory-stored results and its potential to be interfaced with a laboratory information system. Importantly, the Sofia assay offers both read-now and walk-away modes of operation, enhancing laboratory workflow flexibility, depending on batch or one-at-atime specimen-testing needs. Molecular POC testing, which provides a high sensitivity and multiple pathogen results, is readily being developed (8, 9). The identification of multiple pathogens directly affects the judicious use of antibiotic and antiviral agents and reduces the number of hospital-acquired infections by cohorting those multiple agentinfected 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 be taken (8). Until resolution of these obstacles, antigen detection assays will remain in use in POC settings. The incorporation of instrumentation to provide 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 than others, 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
TABLE 2 Performance characteristics of 2 influenza direct antigen detection assaysa with respect to RT-PCRb assay results Performance characteristicc No. of TP No. of FN No. of TN No. of FP Sensitivity (% [95% CI]e) Specificity (% [95% CI]) PPV (%) NPV(%)
Influenza type A specimensd tested with:
Influenza type B specimens tested with:
Sofia
Veritor
Sofia
Veritor
79 21 308 3 79.0 (0.70–0.86) 99.0 (0.97–0.99) 96.3 95.1
64 36 309 2 64.0 (0.54–0.73) 99.4 (0.97–0.99) 97.0 92.3
26 2 371 12 92.9 (0.76–0.99) 96.7 (0.95–0.98) 68.4 99.5 98.4
22 6 378 5 78.6 (0.60–0.90) 98.7 (0.97–0.99) 81.5 98.4
Sofia influenza A ⫹ B FIA (Qiagen, San Diego, CA) and the Veritor influenza A ⫹ B assay (Becton Dickinson & Co., Sparks, MD). Influenza A ⫹ B RT-PCR assay (Qiagen). A second RT-PCR assay (ProFlu A ⫹ B; Hologic/Prodesse, Waukesa, WI) was used to confirm 13 samples as influenza A positive and 2 samples as influenza B positive by RT-PCR only. c TP, true positives; FN, false negatives; TN, true negatives; FP, false positives. Sensitivity was calculated as TP/TP ⫹ FN ⫻ 100%. Specificity was calculated as TN/TN ⫹ FP ⫻ 100%. PPV, positive predictive value, calculated as TP/TP ⫹ FP; NPV, negative predictive value, calculated as TN/TN ⫹ FN. CI, confidence interval. d A total of 411 nasopharyngeal specimens collected using flocked swabs and placed into 3-mL UTM (Copan, Inc.) were prospectively studied. e The 95% confidence interval was calculated using the modified Wald method (QuickCalcs, GraphPad Software, Inc.). a b
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antibody techniques. These included respiratory syncytial virus (RSV) (42 patients), rhinovirus (6 patients), human metapneumovirus (5 patients), adenovirus (3 patients), and parainfluenza type 3 (2 patients). Coinfection occurred in 5 patients. In 4 cases, RSV was identified along with influenza A using a direct fluorescent or rapid antigen detection assay. Rhinovirus, identified in MRC-5 culture, was isolated along with the influenza A virus. The patient age distribution is summarized in Table 1. The 18to 49-year age group accounted for 140 of 411 (34.0%) patients tested and demonstrated the greatest number of influenza-positive patients (63 of 140, 45.0%). The 0- to 4-year and ⱖ65-year age groups accounted for 14 and 11 influenza-positive patients, respectively. Assay performance data are summarized in Table 2. Using RTPCR as the gold standard method, sensitivity and specificity values (percentages) 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 falsepositive influenza B results compared with 5 false-positive results obtained from 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 nonsignificant results. The Veritor assay was easier to perform, having fewer procedural steps. The reader was compact, lightweight, and battery powered. In contrast, the Sofia assay required more procedural
Objective Influenza Antigen Testing at Point of Care
REFERENCES 1. Centers for Disease Control and Prevention. 2011. Key facts about seasonal influenza. Centers for Disease Control and Prevention, Atlanta, GA. www.cdc.gov/flu/keyfacts.htm.
April 2015 Volume 53 Number 4
2. Centers for Disease Control and Prevention. 2009. Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) virus—United States, 2009. MMWR Morb Mortal Wkly Rep 58:826 – 829. 3. Ginocchio C, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, Lotikar M, Kowerska M, Becker G, Korologos G, de Gerinomo M, Crawford JM. 2009. Evaluation of multiple test methods for the detection of novel influenza A (H1N1) during the New York City outbreak. J Clin Virol 45:191–195. http://dx.doi.org/10.1016/j.jcv.2009.06.005. 4. Leonardi GP, Mitrache I, Pigal A, Freedman L. 2010. Public hospitalbased experience during an outbreak of pandemic influenza A (H1N1) virus infections. J Clin Microbiol 48:1189 –1194. http://dx.doi.org/10.1128 /JCM.01657-09. 5. 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. http://dx.doi.org/10.7326/0003-4819-156-7-201204030-00403. 6. Leonardi GP, Wilson AM, Zuretti AR. 2013. Comparison of conventional lateral-flow immunoassays and a new fluorescent immunoassay to detect influenza viruses. J Virol Methods 189:379 –382. http://dx.doi.org /10.1016/j.jviromet.2013.02.008. 7. Agresti A, Coull BA. 1998. Approximate is better than “exact” for interval estimation of binomial proportions. Am Stat 52:119 –126. http://dx.doi .org/10.1080/00031305.1998.10480550. 8. Kiechle FL, Holland CA. 2009. Point-of-care testing and molecular diagnostics: miniaturization required. Clin Lab Med 29:555–560. http://dx .doi.org/10.1016/j.cll.2009.06.013. 9. Ince J, McNally A. 2009. Development of rapid automated diagnostics for infectious disease: advances and challenges. Expert Rev Med Devices 6:641– 651. http://dx.doi.org/10.1586/erd.09.46. 10. Gibbs J. 2001. Selecting the detection system: colorimetric, fluorescent, luminescent methods. ELISA technical bulletin 5. Corning Life Sciences, Corning, NY. 11. Fiore AE, Fry A, Shay D, Gubareva D, Bresee L, Uyeki TM, Centers for Disease Control and Prevention. 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.
Journal of Clinical Microbiology
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testing in both run-now and walk-away modes, adding an important extra dimension for workflow flexibility. Albeit statistically nonsignificant, the Sofia system demonstrated higher sensitivity values (percentages) for influenza A and B than the Veritor system (79.0/92.9 versus 64.0/78.6, respectively). Fluorescent detection methods may be responsible for this enhanced sensitivity. Fluorescent detection is known to increase sensitivity while also widening the dynamic assay detection range over that of colorimetric methods (10). The sensitivity of the Sofia assay is comparable to that reported by our laboratory when it was evaluated along with the QuickVue (Quidel, Inc.) and Directigen Flu A ⫹ B (Becton Dickinson & Co.) assays (6); however, the sensitivity values of each assay were below those 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 (more than 80%) and patient population (76/100 influenza A-positive patients more than 18 years old) may account for the lower sensitivity values obtained. The Sofia and Veritor instruments provide simple, rapid, objective results. Walk-away operation mode and increased sensitivity favor the Sofia assay 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.
Influenza antigen detection assays (Sofia fluorescent immunoassay [FIA] and Veritor) yield objective results, which are potentially useful for point-of-care testing. The assays were evaluated with reverse transcriptase PCR (RT-PCR) using 411 nasopharyngeal swab specimens. Sensitivity and specificity values (percentages) 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 the Sofia and Veritor assays, respectively.
I
nfluenza 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., antiviral treatment or hospitalization) to occur during the patient-physician encounter. POC assays must be easy to use, be 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% confidence interval [CI], 57.9% to 66.6%) clearly demonstrated the need for confirmation of negative results (5). Subjective visual result interpretation in a specific time frame 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 that provide objective results have recently become available. The Sofia influenza A ⫹ B fluorescent immunoassay (FIA) (Quidel, Inc., San Diego, CA) uses a fluorescence reader to detect influenza nucleoprotein antigens. The Veritor system (Becton Dickinson & Co., Sparks, MD) is a chromatographic assay which qualitatively detects influenza nucleoprotein antigens using an optical colorimetric device. These 2 assays were compared in a prospective study of nasopharyngeal swab specimens collected from symptomatic patients during the 2013 to 2014 influenza season. A total of 411 patient nasopharyngeal flocked-swab specimens, placed into 3 ml of transport medium (Copan, Inc., Marisa, CA), were prospectively studied. Specimens were mainly collected from patients exhibiting flu-like symptoms (i.e., fever, headache, body ache, malaise, upper respiratory infection [URI], pneumonia) in the hospital emergency department. Specimens were simultaneously tested using each assay following the manufacturer’s procedures. Briefly, 260 l of specimen and a preset volume of buffer were placed in a reaction tube containing Sofia lysis buffer. Using a premeasured pipette, a volume
April 2015 Volume 53 Number 4
of fluid was placed in a Sofia reaction cassette, incubated for 15 min, and analyzed using a Sofia fluorescent reader. The Veritor system utilized 300 l of specimen placed into a reagent tube containing lysis buffer. After the tip was placed on the reaction tube, 3 drops were placed into a Veritor test device cassette and allowed 10 min for incubation, and then the cassette was placed into a colorimetric reader for analysis. Specimens were tested within collection times ranging from 1 to 48 h, kept at 2 to 8°C in the interim period, and tested as they were received in the laboratory. Specimens were also tested for influenza using reverse transcriptase PCR (RT-PCR) (Lyra influenza A ⫹ B assay, Quidel, Inc.). Nucleic acid extraction was done using a NucliSENS easyMAG instrument (bioMérieux, Inc., Marcy l’Etoile, France), and the resulting aliquots (60 l) 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 the specimens were influenza antigen negative by the two assays but positive by RT-PCR. Sensitivity, specificity, and positive and negative predictive values were calculated for each assay. Ninetyfive percent confidence intervals were computed using a modified Wald method (7). Of the 411 specimens examined, 128 were influenza positive (100 influenza A and 28 influenza B specimens). Influenza A subtyping performed on 68 positive specimens yielded A/2009 H1N1like virus in 55/68 specimens (80.9%) and 13 specimens identified as A/H3N2-like virus. Other respiratory viruses (58 cases) were identified using cell culture, RT-PCR, and/or direct fluorescent
Received 3 December 2014 Returned for modification 19 December 2014 Accepted 8 January 2015 Accepted manuscript posted online 21 January 2015 Citation Leonardi GP, Wilson AM, Mitrache I, Zuretti AR. 2015. Comparison of the Sofia and Veritor direct antigen detection assay systems for identification of influenza viruses from patient nasopharyngeal specimens. J Clin Microbiol 53:1345–1347. doi:10.1128/JCM.03441-14. Editor: Y.-W. Tang Address correspondence to G. P. Leonardi, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03441-14
Journal of Clinical Microbiology
jcm.asm.org
1345
Downloaded from http://jcm.asm.org/ on April 7, 2015 by UNIV OF NEBRASKA-LINCOLN
Virology Laboratory, Department of Pathology, Nassau University Medical Center, East Meadow, New York, USA
Leonardi et al.
TABLE 1 Age distribution of 411 patients tested for influenzaa from 4 November 2013 through 14 March 2014 Distribution (no.) by age group (yr)b Test result
0–4
5–17
18–49
50–64
ⱖ65
Total
Total patients Influenza A positive Influenza B positive Percentage of group A positive Percentage of group B positive
80 10 04 12.5 05.0
64 14 02 21.8 03.1
140 53 10 37.9 07.1
81 17 07 21.0 08.6
46 06 05 13.0 10.7
411 100 28 24.3 06.8
Specimens were tested using direct antigen detection assays (Sofia [Quidel, Inc.] and Veritor [Becton Dickinson & Co.]) and by RT-PCR assay (Influenza A ⫹ B; Quidel). A second RT-PCR assay (ProFlu⫹; Hologic) was used in cases where specimens were negative by the two antigen detection assays but positive by RT-PCR. b Age ranges were chosen following those used by the CDC seasonal influenza weekly report (FluView), http://www.cdc.gov/flu/weekly/overview.htm. a
steps for operation, and the fluorescent reader was heavier, requiring technician data input for operation. Benefits of the Sofia fluorescent reader include its ability to produce hard-copy and memory-stored results and its potential to be interfaced with a laboratory information system. Importantly, the Sofia assay offers both read-now and walk-away modes of operation, enhancing laboratory workflow flexibility, depending on batch or one-at-atime specimen-testing needs. Molecular POC testing, which provides a high sensitivity and multiple pathogen results, is readily being developed (8, 9). The identification of multiple pathogens directly affects the judicious use of antibiotic and antiviral agents and reduces the number of hospital-acquired infections by cohorting those multiple agentinfected 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 be taken (8). Until resolution of these obstacles, antigen detection assays will remain in use in POC settings. The incorporation of instrumentation to provide 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 than others, 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
TABLE 2 Performance characteristics of 2 influenza direct antigen detection assaysa with respect to RT-PCRb assay results Performance characteristicc No. of TP No. of FN No. of TN No. of FP Sensitivity (% [95% CI]e) Specificity (% [95% CI]) PPV (%) NPV(%)
Influenza type A specimensd tested with:
Influenza type B specimens tested with:
Sofia
Veritor
Sofia
Veritor
79 21 308 3 79.0 (0.70–0.86) 99.0 (0.97–0.99) 96.3 95.1
64 36 309 2 64.0 (0.54–0.73) 99.4 (0.97–0.99) 97.0 92.3
26 2 371 12 92.9 (0.76–0.99) 96.7 (0.95–0.98) 68.4 99.5 98.4
22 6 378 5 78.6 (0.60–0.90) 98.7 (0.97–0.99) 81.5 98.4
Sofia influenza A ⫹ B FIA (Qiagen, San Diego, CA) and the Veritor influenza A ⫹ B assay (Becton Dickinson & Co., Sparks, MD). Influenza A ⫹ B RT-PCR assay (Qiagen). A second RT-PCR assay (ProFlu A ⫹ B; Hologic/Prodesse, Waukesa, WI) was used to confirm 13 samples as influenza A positive and 2 samples as influenza B positive by RT-PCR only. c TP, true positives; FN, false negatives; TN, true negatives; FP, false positives. Sensitivity was calculated as TP/TP ⫹ FN ⫻ 100%. Specificity was calculated as TN/TN ⫹ FP ⫻ 100%. PPV, positive predictive value, calculated as TP/TP ⫹ FP; NPV, negative predictive value, calculated as TN/TN ⫹ FN. CI, confidence interval. d A total of 411 nasopharyngeal specimens collected using flocked swabs and placed into 3-mL UTM (Copan, Inc.) were prospectively studied. e The 95% confidence interval was calculated using the modified Wald method (QuickCalcs, GraphPad Software, Inc.). a b
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antibody techniques. These included respiratory syncytial virus (RSV) (42 patients), rhinovirus (6 patients), human metapneumovirus (5 patients), adenovirus (3 patients), and parainfluenza type 3 (2 patients). Coinfection occurred in 5 patients. In 4 cases, RSV was identified along with influenza A using a direct fluorescent or rapid antigen detection assay. Rhinovirus, identified in MRC-5 culture, was isolated along with the influenza A virus. The patient age distribution is summarized in Table 1. The 18to 49-year age group accounted for 140 of 411 (34.0%) patients tested and demonstrated the greatest number of influenza-positive patients (63 of 140, 45.0%). The 0- to 4-year and ⱖ65-year age groups accounted for 14 and 11 influenza-positive patients, respectively. Assay performance data are summarized in Table 2. Using RTPCR as the gold standard method, sensitivity and specificity values (percentages) 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 falsepositive influenza B results compared with 5 false-positive results obtained from 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 nonsignificant results. The Veritor assay was easier to perform, having fewer procedural steps. The reader was compact, lightweight, and battery powered. In contrast, the Sofia assay required more procedural
Objective Influenza Antigen Testing at Point of Care
REFERENCES 1. Centers for Disease Control and Prevention. 2011. Key facts about seasonal influenza. Centers for Disease Control and Prevention, Atlanta, GA. www.cdc.gov/flu/keyfacts.htm.
April 2015 Volume 53 Number 4
2. Centers for Disease Control and Prevention. 2009. Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) virus—United States, 2009. MMWR Morb Mortal Wkly Rep 58:826 – 829. 3. Ginocchio C, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, Lotikar M, Kowerska M, Becker G, Korologos G, de Gerinomo M, Crawford JM. 2009. Evaluation of multiple test methods for the detection of novel influenza A (H1N1) during the New York City outbreak. J Clin Virol 45:191–195. http://dx.doi.org/10.1016/j.jcv.2009.06.005. 4. Leonardi GP, Mitrache I, Pigal A, Freedman L. 2010. Public hospitalbased experience during an outbreak of pandemic influenza A (H1N1) virus infections. J Clin Microbiol 48:1189 –1194. http://dx.doi.org/10.1128 /JCM.01657-09. 5. 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. http://dx.doi.org/10.7326/0003-4819-156-7-201204030-00403. 6. Leonardi GP, Wilson AM, Zuretti AR. 2013. Comparison of conventional lateral-flow immunoassays and a new fluorescent immunoassay to detect influenza viruses. J Virol Methods 189:379 –382. http://dx.doi.org /10.1016/j.jviromet.2013.02.008. 7. Agresti A, Coull BA. 1998. Approximate is better than “exact” for interval estimation of binomial proportions. Am Stat 52:119 –126. http://dx.doi .org/10.1080/00031305.1998.10480550. 8. Kiechle FL, Holland CA. 2009. Point-of-care testing and molecular diagnostics: miniaturization required. Clin Lab Med 29:555–560. http://dx .doi.org/10.1016/j.cll.2009.06.013. 9. Ince J, McNally A. 2009. Development of rapid automated diagnostics for infectious disease: advances and challenges. Expert Rev Med Devices 6:641– 651. http://dx.doi.org/10.1586/erd.09.46. 10. Gibbs J. 2001. Selecting the detection system: colorimetric, fluorescent, luminescent methods. ELISA technical bulletin 5. Corning Life Sciences, Corning, NY. 11. Fiore AE, Fry A, Shay D, Gubareva D, Bresee L, Uyeki TM, Centers for Disease Control and Prevention. 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.
Journal of Clinical Microbiology
jcm.asm.org
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Downloaded from http://jcm.asm.org/ on April 7, 2015 by UNIV OF NEBRASKA-LINCOLN
testing in both run-now and walk-away modes, adding an important extra dimension for workflow flexibility. Albeit statistically nonsignificant, the Sofia system demonstrated higher sensitivity values (percentages) for influenza A and B than the Veritor system (79.0/92.9 versus 64.0/78.6, respectively). Fluorescent detection methods may be responsible for this enhanced sensitivity. Fluorescent detection is known to increase sensitivity while also widening the dynamic assay detection range over that of colorimetric methods (10). The sensitivity of the Sofia assay is comparable to that reported by our laboratory when it was evaluated along with the QuickVue (Quidel, Inc.) and Directigen Flu A ⫹ B (Becton Dickinson & Co.) assays (6); however, the sensitivity values of each assay were below those 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 (more than 80%) and patient population (76/100 influenza A-positive patients more than 18 years old) may account for the lower sensitivity values obtained. The Sofia and Veritor instruments provide simple, rapid, objective results. Walk-away operation mode and increased sensitivity favor the Sofia assay 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.
