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COMPARISON OF THE SOFIA AND VERITOR DIRECT ANTIGEN DETECTION ASSAY 1
SYSTEMS TO IDENTIFY INFLUENZA VIRUSES FROM PATIENT NASOPHARYNGEAL 2
SPECIMENS. 3
G.P. Leonardi#, Ph.D., A.M. Wilson, Ph.D., I. Mitrache and A.R. Zuretti, M.D. 4
Virology Laboratory, Box 47, Department of Pathology, Nassau University Medical Center. 5
2201 Hempstead Turnpike, East Meadow, NY 11554. 6
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Running title: objective influenza antigen testing at point-of-care. 10
#Department of Pathology & Labs, Box 47 11
Nassau University Medical Center 12
2201 Hempstead Turnpike 13
East Meadow, NY 11554 14
Phone: 516-572-8711 15
Fax: 516-572-6409 16
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JCM Accepted Manuscript Posted Online 21 January 2015J. Clin. Microbiol. doi:10.1128/JCM.03441-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract. 19
Influenza antigen detection assays (Sofia FIA and Veritor) yield objective results, which are 20
potentially useful for point-of-care testing. Assays were evaluated with RT-PCR using 411 21
nasopharyngeal swab specimens. Sensitivity and specificity (%) of 79.0/99.0 and 64.0/99.4 for 22
influenza/A and 92.9/96.7 and 78.6/98.7 for influenza/B were obtained for Sofia and Veritor, 23
respectively. 24
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Influenza viruses are important causes of acute respiratory illness resulting in approximately 28
200,000 hospitalizations and 36,000 deaths in the United States annually.1 Point-of- care 29
(POC) testing alleviates patient surges by allowing treatment decisions (e.g. antirviral treatment 30
or hospitalization) to occur during the patient-physician encounter. POC assays must be easy to 31
use, inexpensive, provide rapid results and possess adequate sensitivity and specificity. 32
POC influenza antigen detection assays provide adequate specificity however low sensitivity 33
values require retesting of negative results. During the influenza A/ 2009 H1N1 pandemic, 34
antigen detection assays demonstrated sensitivity values ranging from 18.0% to 77.0% .2-4
In a 35
meta-analysis of 159 influenza-antigen detection assays, heterogeneous sensitivity values (95% 36
CI of 57.9% to 66.6%) clearly demonstrated the need for confirmation of negative results.5 37
Subjective visual result interpretation at a specific timeframe has also diminished the value of 38
antigen detection assays for POC testing.6 Emergency department nursing staffs, already 39
overburdened with other responsibilities, are reticent to take on this additional responsibility 40
which requires technical experience and places specific time constraints for result interpretation. 41
Antigen detection assays providing objective results have recently become available. The 42
Sofia Influenza A + B FIA assay (Quidel Inc, San Diego, CA) uses a fluorescence reader to 43
detect influenza-neucleoprotein antigens. The Veritor system (Becton Dickinson & Co., Sparks, 44
MD) is a chromatographic assay which qualitatively detects influenza neucleoprotein antigens 45
using a optical colorimetric device. 46
These 2 assays were compared in a prospective study of nasopharyngeal swab specimens 47
collected from symptomatic patients during the 2013-2014 influenza season. 48
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A total of 411 patient nasopharyngeal flocked-swab specimens, placed into 3mL of transport 49
media (Copan Inc., Marisa, CA) were prospectively studied. Specimens were mainly received 50
from the hospital emergency department from patients exhibiting flu-like symptoms (i.e., fever, 51
headache, body ache, malaise, URI, pneumonia). 52
Specimens were simultaneously tested using both assays following the manufacturer’s 53
procedures. Briefly, 260uL of specimen and a preset volume of buffer were placed in a Sofia 54
lysis-containing reaction tube. Using a premeasured pipette, a volume of fluid was placed in a 55
Sofia reaction cassette, incubated for 15 minutes, and analyzed using a Sofia fluorescent reader. 56
The Veritor system utilized 300uL of specimen placed into a reagent tube containing lysis buffer. 57
After tip placement on the reaction tube, 3 drops were placed into a Veritor test device cassette, 58
allowed 10 minutes incubation and the cassette was placed into a colorimetric reader for 59
analysis. Specimens were tested within collection times ranging from 1 to 48 hours, kept at 2-60
8oC in the interim period, and tested as received in the laboratory. 61
Specimens were also tested for influenza using RT-PCR (Lyra INF A + B; Quidel, Inc). 62
Nucleic acid extraction was done using a NucliSENSE easyMAG instrument (bioMerieux, Inc; 63
Marcy l’Etoile, France) and the resulting aliquots (60uL) were amplified using a smart cycler II 64
(Cepheid Inc., Sunnyvale, CA). A second RT-PCR assay (Pro Flu +, Hologic; San Diego, CA) 65
was used for confirmation when specimens were influenza antigen-negative by both assays but 66
were positive by RT-PCR. Sensitivity, specificity, positive and negative predictive values were 67
calculated for each assay. 68
Confidence intervals (95%) were computed using a modified method of Wald.7 69
Of the 411 specimens examined, 128 were influenza-positive (100 influenza A and 28 70
influenza B specimens). Influenza A sub-typing performed on 68 positive specimens yielded 71
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A/2009 H1N1-like virus in 55/68 specimens (80.9%) and 13 specimens identified as the A/ 72
H3N2-like virus. Other respiratory viruses (58 cases) were identified using cell culture, rt-PCR 73
and/or direct fluorescent antibody techniques. These included respiratory syncytial virus (42 74
patients), rhinovirus (6 patients), human metapneumovirus (5 patients), adenovirus (3 patients) 75
and parainfluenza type 3 (2 patients). Co- infection occurred in 5 patients. In 4 cases, RSV was 76
identified along with influenza A using direct fluorescent or rapid antigen detection assay. 77
Rhinovirus, identified in MRC-5 culture was isolated along with influenza A virus. 78
Patient age distribution is summarized in Table 1. The 18-49 year age group accounted for 79
140 of 411 (34.0%) of patients tested and demonstrated the greatest number of influenza-positive 80
patients (63 of 140; 45.0%). The 0-4 and the 65+ age groups accounted for 14 and 11 influenza-81
positive patients, respectively. 82
Assay performance data is summarized in Table 2. Using rt-PCR as the gold standard 83
method, sensitivity and specificity values (%) of 79.0/99.0 and 64.0/99.4 for influenza A and 84
92.9/96.7 and 78.6/98.7 for influenza B were obtained using the Sofia and Veritor assays, 85
respectively. The Sofia assay produced 12 false-positive influenza B results as compared with 5 86
false-positive results obtained for the Veritor system. A total of 13 influenza A-positive and 2 87
influenza B-positive specimens were identified solely by RT-PCR. In these cases, a second RT-88
PCR assay confirmed specimen positivity. Statistical analysis (95% confidence interval) of 89
sensitivity and specificity between these assays yielded non-significant results. 90
The Veritor assay easier to perform, having fewer procedural steps. The reader was 91
compact, lightweight and battery powered. In contrast, Sofia required more procedural steps for 92
operation and the fluorescent reader was heavier, requiring technician data input for operation. 93
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Benefits of the Sofia fluorescent reader include the ability to produce both hard-copy and 94
memory-stored results, and the potential to be interfaced with a laboratory information system. 95
Importantly, Sofia offers both a read-now and walk-away modes of operation, enhancing 96
laboratory workflow flexibility, depending on batch or one-at-a-time specimen testing needs. 97
Molecular POC testing which provide high sensitivity and multiple pathogen results are 98
readily being developed.8-9
Identification of multiple pathogens directly affects the judicious 99
use of antibiotic and antiviral agents and reduces hospital-acquired infection by cohorting those 100
multiple agent-infected patients. 101
A number of obstacles hinder molecular POC testing.8,9
Molecular assays are more costly. 102
They need portability, miniaturization and disposable, premeasured components. Responsibility 103
for assay maintenance, quality control, reagent storage and environmental monitoring of non-104
laboratory sites must also occur.8 Until resolution of these obstacles, antigen detection assays 105
will remain in POC. 106
The incorporation of instrumentation to providing clear, objective results marks a vast 107
improvement over assays requiring subjective result interpretation. The Veritor instrument is 108
lightweight, battery operated and portable. Although larger and more complicated to use, the 109
Sofia reader permits results to be printed, stored in the instrument or potentially interfaced to a 110
hospital information system. The Sofia instrument also allows testing in both run-now and walk-111
away modes, adding an important, extra dimension for workflow flexibility. Albeit statistically 112
non-significant, Sofia presently demonstrated higher sensitivity values (%) for influenza A / B 113
(79.0/92.9 versus 64.0/78.6) than the Veritor system. Fluorescent detection methods may be 114
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responsible for this enhanced sensitivity. Fluorescent detection is known to increase sensitivity 115
while also widening the dynamic assay detection range over colorimetric methods.10
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Sofia sensitivity is comparable to that reported by our laboratory when the former was 117
evaluated along with the Quickvue (Quidel Inc) and Directigen Flu A+B (Becton Dickinson & 118
Co.) assays,6 however, both assay’s sensitivity values were below that reported by the 119
manufacturer. Variation in patient age and the predominant influenza A subtype can influence 120
assay sensitivity.5,11
The present investigation’s influenza A/2009/H1N1 subtype (over 80%) 121
and patient population (76/100 positive-influenza A patients over 18 years old) could account 122
for the lower sensitivity values obtained. 123
Both Sofia and Veritor instrumentation provide simple, rapid, objective results. Walk-away 124
operation mode and increased sensitivity favor Sofia over the Veritor assay for POC use. 125
However, sensitivity values approaching 80% may still be insufficient to be reliably accepted, 126
thus underscoring the need for follow up testing when negative influenza results are obtained. 127
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Table 1. Age distribution of 411 patients tested for influenza1 from 11/4/2013 through 3/14/14. 131
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AGE GROUP DISTRIBUTION (YRS)2 . TOTALS: 134
ITEM 0-4 5-17 18-49 50-64 65+ 135
TOTAL PATIENTS: 80 64 140 81 46 411 136
INFLUENZA A POSITIVE: 10 14 53 17 06 100 137
INFLUENZA B POSITIVE: 04 02 10 07 05 28 138
% OF GROUP A-POSITIVE: 12.5 21.8 37.9 21.0 13.0 24.3 139
% OF GROUP B-POSITIVE: 05.0 03.1 07.1 08.6 10.7 06.8 140
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1 Specimens were tested using direct antigen detection assay (Sofia, Quidel Inc.; Veritor, Becton 142
Dickinson) and by RT-PCR assay (INF A + B, Quidel). A second RT-PCR assay (Pro FLU +, Hologic) 143
was used in cases where specimens were negative by both antigen detection assays but positive 144
by RT-PCR. 145
2 Age ranges were chosen, following that used by the CDC seasonal influenza weekly report 146
(FLUVIEW), http://www.cdc.gov/flu/weekly/overview.htm. 147
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TABLE 2. Performance characteristics of 2 influenza direct antigen detection assaysb with respect to RT- 149
PCRc assay results. 150
ITEM INFLUENZA TYPE A SPECIMENSa INFLUENZA TYPE B SPECIMENS 151
SOFIA VERITOR SOFIA VERITOR 152
TPc : 79 64 26 22 153
FN: 21 36 2 6 154
TN: 308 309 371 378 155
FP: 3 2 12 5 156
SENSITIVITY (%) 79.0 64.0 92.9 78.6 157
CI (95%)d .70-.86 .54-.73 .76-.99 .60-.90 158
SPECIFICITY (%) 99.0 99.4 96.7 98.7 159
CI (95%) 97-.99 .97-.99 .95-98 .97-.99 160
PPV (%) 96.3 97.0 68.4 81.5 161
NPV(%) 95.1 92.3 99.5 98.4 162
a A total of 411 nasopharyngeal specimens collected using flocked swabs and placed into 3mL UTM 163
(Copan, Inc) were prospectively studied 164
b Sofia influenza A + B FIA (Qiagen, San Diego, CA ) and the Veritor Flu A + B assay (Becton Dickininson 165
and Co., Sparks, MD). 166
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c INF A + B RT-PCR assay (Qiagen; San Diego, CA ). A second RT-PCR assay (ProFlu A + B; 167
Hologic/Prodesse; Waukesa, WI) was used to confirm 13 samples influenza A- positive and 2 samples 168
influenza B-positive by rt-PCR only. 169
d Confidence Interval (95%) calculated using the modified method of Wald (QuickCalcs; GraphPad 170
Software, Inc). 171
TP = true positive; FN = false negative; TN = true negative; FP = false positive. Sensitivity was calculated 172
as TP/TP + FN x 100%. Specificity was calculated as TN/TN + FP x 100%. PPV = positive predictive value, 173
calculated as TP/TP + FP; NPV = negative predictive value, calculated as TN/TN + FN. CI = 95% confidence 174
interval. 175
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