Performance of Binax NOW Flu A and B and direct fluorescent assay in
comparison with a composite of viral culture or reverse transcription
polymerase chain reaction for detection of influenza
infection during the 2006 to 2007 season☆
Mahbubur Rahmana,⁎, Mary F. Vandermauseb, Burney A. Kiekeb, Edward A. Belongiab
aCenter for Interdisciplinary Research in Women's Health, The University of Texas Medical Branch, Galveston, TX 77555-0587, USA
bMarshfield Clinic Research Foundation and Marshfield Clinic, Marshfield, WI 54449, USA
Received 15 June 2007; accepted 9 October 2007
The Binax NOW Flu A and Flu B (Binax NOW), direct fluorescent assay (DFA), and viral culture were evaluated and compared with a
composite of viral culture or reverse transcription polymerase chain reaction (RT-PCR). Participants with medically attended acute respiratory
illness were identified through active surveillance during the 2006 to 2007 season, and consenting individuals (n = 932) were tested for
influenza by culture and RT-PCR. Physicians ordered a rapid antigen test (Binax NOW [n = 73] or DFA [n = 70]) according to their clinical
judgment. The Binax NOW detected 11 of 18 influenza infections (sensitivity, 61%; 95% confidence interval [CI], 36–83%), whereas DFA
detected 17 of 21 influenza infections (sensitivity 81%, 95% CI, 58–95%). Compared with culture/RT-PCR, specificity of both Binax NOW
and DFAwas 100%. During the 2006 to 2007 influenza season, DFA and Binax NOW demonstrated high specificity but failed to identify a
substantial proportion of influenza infections.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Influenza virus; Rapid test; Binax NOW; Direct fluorescent assay; Sensitivity; Specificity
Influenza infections are an important cause of mortality
and morbidity worldwide. Rapid diagnosis of influenza
infections is important because antiviral drugs are only
effective if started within 48 h after symptom onset
(Younkin et al., 1983; Reuman et al., 1989; Hayden et al.,
1999). Early diagnosis is also important because it reduces
the length of hospital stays and may reduce unnecessary
antibiotic use (Woo et al., 1997; Barenfanger et al., 2000).
However, rapid diagnosis is a challenging task. Diagnosis of
influenza infection based on only clinical features is
difficult due to its nonspecific presentation and cocircula-
tion of other respiratory viruses during influenza epidemics.
Several office-based commercial rapid tests are available for
detecting influenza viruses to assist in patient management
(Storch, 2003). These tests do not require special expertise
and can be completed within minutes, but low test
sensitivity has been a concern despite high specificity.
Binax NOW Influenza A and B test kit (Binax, Portland,
ME) is a recent addition to the list of commercial rapid tests,
but limited information is available regarding the perfor-
mance of the test in different populations. Direct fluorescent
assay (DFA) is another option at some laboratories for
timely access to influenza test results, but it requires
significant technical expertise and equipment (Landry and
Ferguson, 2003). Reverse transcription polymerase chain
reaction (RT-PCR) provides sensitive and specific detection
of influenza viruses, but this technology currently requires
special equipment and expertise, and is not widely available
in most clinical settings.
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 62 (2008) 162–166
☆Funding for this research was provided by a cooperative agreement
with the Centers for Disease Control and Prevention, Atlanta, GA (1 U01
CI000192-01). The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the funding agency.
⁎Corresponding author. Tel.: +1-409-772-2978; fax: +1-409-747-5129.
E-mail address: email@example.com (M. Rahman).
0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
During the 2006 to 2007 influenza season, we evaluated
the performance of the Binax NOWand DFA in comparison
with a composite gold standard based on viral culture and
RT-PCR. The latter tests were performed on patients with
acute respiratory illness who were recruited for a study of
influenza vaccine effectiveness.
2. Materials and methods
Participants for this investigation were derived from a
population-based study of influenza vaccine effectiveness
conducted within the Marshfield Epidemiologic Study Area,
a 14-ZIP code region surrounding Marshfield, WI (DeSte-
fano et al., 1996). Patients receiving inpatient or outpatient
care for acute respiratory illness b10 days in duration were
recruited and tested for influenza infection by viral culture
and RT-PCR. During the influenza season, 932 patients were
enrolled and tested from a source population of 20693
residents who were eligible to receive the 2006 to 2007
influenza vaccine based on recommendations from the
Advisory Committee on Immunization Practices (ACIP)
(Smith et al., 2006). These included all children 6 to
59 months old, all adults ≥50 years old, and individuals
aged 5 years through 49 years with specific high-risk
Enrollment began on January 22, 2007, and continued
for 10 weeks. For this study, we identified enrolled
patients who also had a rapid test ordered by the physician
in their routine clinical practice. Physicians ordered a rapid
test based on clinical indication, independent of the
research tests for influenza. The latter test results were
usually not available until more than 1 week later, and the
research test results were not included in the patient's
medical record. As a result, primary care physicians were
encouraged to maintain their usual testing procedures for
influenza despite concurrent testing for a vaccine effec-
Physicians did not specify the type of rapid test when
ordered, and the use of DFA or Binax NOW was determined
by the timing and location of the sample. Binax NOW was
performed in the regional clinic centers and in the main clinic
during evening and weekend hours. DFA was performed in
the main clinic during normal working hours when
experienced technicians were available.
2.2. Collection of specimens
All samples that had a Binax NOWor DFA test performed
were ordered and collected by the physician or physician's
medical assistant according to clinic protocol. In all cases,
nasopharyngeal swabs were obtained, placed in M4-RT viral
transport media (Remel, Lenexa, KS), and transported to the
clinical laboratory on ice within 2 h. The same sample swab
was used for viral culture, RT-PCR, and rapid antigen tests
(Binax NOW or DFA).
2.3. Binax NOW
The Binax NOW Influenza A and B test kit is an
immunochromatographic membrane assay that uses highly
sensitive monoclonal antibodies to detect influenza A and B
nucleoprotein antigens in 15 min. Nasopharyngeal swabs,
collected and transported in the viral transport media, were
vortexed. One hundred microliters were applied directly to
the test cartridge. Results were read in 15 min, as per
manufacturers package insert. For a negative sample, the
blue control line turns to a pink-to-purple color. No other
line appears. For an influenza A-positive sample, the blue
control line turns to a pink-to-purple color, and a 2nd
pink-to-purple line appears in the designated Flu A area.
For an influenza B-positive sample, the blue control line
turns to a pink-to-purple color, and a 2nd pink-to-purple
line appears in the designated Flu B area. A test is
considered invalid if the control line remains blue or is not
present at all.
2.4. Direct fluorescent assay
DFAs allow visual localization of viral antigen within
infected cells. Nasopharyngeal swabs, collected and trans-
ported in the viral transport media, were vortexed. The
beads contained in the viral transport aided in breaking up
mucus in the specimen and releasing cells from the
mucous and swab fibers. The swab was removed and the
specimen was centrifuged at 600×g for 5 min to obtain a
cell pellet concentrated in the bottom of the conical
transport tube. The supernatant was transferred to a
separate tube and stored at 4 to 8 °C for any subsequent
testing. The pellet was twice washed in phosphate-buffered
saline (PBS) and centrifuged again. The final centrifuged
pellet was resuspended in 400 μL of PBS and spotted onto
a 3-well microscope slide. Slides were dried, acetone fixed,
and stained using monoclonal fluorescein-labeled antibo-
dies for influenza A and B (D3Influenza A/Influenza B
DFA Reagent Kit; Diagnostic Hybrids, Athens, OH). The
slides were read by an experienced technician using a
fluorescence microscope. The sample was considered
adequate if 200 cells were observed on the slide with at
least 2 columnar epithelial cells/250× field. Specimens
were considered positive for influenza A or B if at least
1 cell showed characteristic influenza nuclear and/or
2.5. Viral culture
Virus cultures for the isolation of influenza A and B were
performed by inoculating monolayered Madin–Darby
canine kidney epithelial cells in shell vial cell cultures
(Diagnostic Hybrids) with 200 μL of the sample supernatant.
Inoculated shell vials were centrifuged for 1 h at 700×g
(2000 rpm) and incubated at 35 to 37 °C. Cultures were
examined microscopically for cytopathic effect for up to 72 h
postinoculation. Cell culture shell vials were maintained
using standard shell vial techniques. Laboratory protocols
163M. Rahman et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 162–166
for quality control and monitoring cell suitability were
followed. After detection of cytopathic effect, or if no
cytopathic effect was detected by 72 h, the cell sheet was
scraped and/or dislodged from the surface of the coverslip at
the bottom of the shell vial. Slides were prepared using the
cell scraping, dried, acetone fixed, and stained with
fluorescein-labeled virus-specific monoclonal antibody
reagents (D3Influenza A/Influenza B DFA Reagent Kit,
Diagnostic Hybrids) intended for the qualitative detection
and identification of influenza A and influenza B. Slides
were read using a fluorescent microscope.
2.6. Reverse transcription polymerase chain reaction
RT-PCR was performed on nucleic acid extracts from
specimens using the LightCycler® Real-Time PCR System
(Roche Diagnostics, Basel, Switzerland). Total nucleic acid
isolation and purification were performed using automated
magnetic bead technology. RT-PCR primers, probes, and
protocols for influenza Aand B were provided by Centers for
Disease Control and Prevention. The primers and probes
have been previously proven to be highly conserved and
make an effective target for detection. The primer/probe sets
were designed for universal detection of type A and type B
influenza viruses. The human RNase P gene (RNP) primer
and probe set serves as an internal positive control for human
RNA. Standard laboratory safeguards were incorporated into
the protocol to decrease the risk of carryover amplicon
contamination including the use of uracil glycosylase, 3
separate unidirectional workflow areas, and laboratory
surface decontamination products to eliminate residual
For this study, positive viral cultures for influenza A
or B or positive results in RT-PCR were considered as
2.7. Statistical analysis
Sensitivity, specificity, positive predictive value, and
negative predictive value of the Binax NOW and DFA tests
were calculated in comparison with a composite gold
standard of viral culture or RT-PCR. Corresponding 95%
confidence intervals (CIs) were computed using exact
methods (Collett, 1991). Univariate comparisons were
performed using the χ2test, Fisher's exact test, or Wilcoxon
rank sum test as appropriate. All analyses were performed
using SAS 9.1 (SAS Institute, Cary, NC) or STATA 9 (Stata,
College Station, TX).
3.1. Demographics and clinical characteristics
Physicians ordered an influenza rapid test in 143 (15%) of
study participants with acute respiratory illness b10 days in
duration. The clinical laboratory performed Binax NOW in
73 (51%) and DFA in 70 (49%) of these patients. Patients
who received a rapid influenza test were older and were more
likely to have a high-risk medical condition relative to
patients with acute respiratory illness who did not receive a
rapid test. Patients who had a rapid test also had a higher
prevalence of influenza infection and were more likely to
present for care during the first 2 days after symptom onset
(Table 1). Gender and vaccination status during the 2006 to
2007 season were similarly distributed for those who did and
Baseline characteristics of the study participants with and without Binax
(n = 143)
(n = 789)
≥ 65 years
With high-risk medical
during 2006 to 2007
Duration of symptoms
at 1st clinical encounter
74 (52)455 (58)0.189
aP value for the χ2test.
bHigh-risk conditions were based on ACIP criteria for persons at
increased risk of influenza complications due to chronic disease (specific
diagnosis codes available on request).
Performance of Binax NOW and DFA compared with a composite gold
standard (viral culture or RT-PCR) for detection of influenza virus
Age groupNo of specimensSeSpPPV NPV
TPFP FN TN Total
6 months to 17 years
6 months to 17 years
61 100 100
83 100 100
50 100 100
81 100 100
69 100 100
100 100 100
TP = true positive; FP = false positive; FN = false negative; TN = true
negative; Se = sensitivity; Sp = specificity; PPV = positive predictive value;
NPV = negative predictive value.
aAll influenza A infections.
bFive influenza A and 2 influenza B infections.
cOne influenza B infection.
164M. Rahman et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 162–166
who did not have a rapid test. No significant demographic
differences were found between those who had the Binax
NOW and DFA.
3.2. Accuracy of Binax NOW
Of the 73 specimens, Binax NOW identified 11 of 18
culture/RT-PCR–positive influenza infections. The sensitiv-
ity and specificity of Binax NOW was 61% (95% CI, 36–
83%) and 100% (95% CI, 94–100%), respectively, with a
positive predictive value of 100% (95% CI, 72–100%) and a
negative predictive value of 89% (95% CI, 78–95%).
Although the sensitivity of Binax NOW was higher in
children and adolescents compared with the adult popula-
tion, the difference was not statistically significant (P =
0.316) (Table 2). Performance of Binax NOW did not differ
when either viral culture or RT-PCR was used as the gold
standard (Table 3).
3.3. Accuracy of DFA
DFA identified 17 of 21 culture/RT-PCR–positive
influenza infections, yielding a sensitivity of 81% (95%
CI, 58–95%). DFA was negative in all 49 patients with a
negative culture/RT-PCR (specificity, 100%; 95% CI, 93–
100%). The positive and negative predictive values were
100% (95% CI, 80–100%) and 92% (95% CI, 82–98%),
respectively. DFA had a higher sensitivity in the adult
population compared with children and adolescents, but
the difference was not statistically significant (P = 0.131)
(Table 2). Accuracy of the test did not change substantially
when either viral culture or RT-PCR test was used as the
gold standard (Table 3).
The sensitivity of DFAwas not significantly different than
that of Binax NOW (P = 0.17).
3.4. Detection of influenza infection by viral culture
Based on a composite gold standard (culture confirmed
or PCR positive), viral culture detected 93 (sensitivity,
91%; 95% CI, 84–96%) and RT-PCR detected 98 of 102
influenza infections (sensitivity, 96%; 95% CI, 90–99%).
In total, 89 specimens were positive for influenza virus
both with RT-PCR and viral culture, 9 with RT-PCR only,
and 4 with viral culture only. Specificity of both viral
culture and RT-PCR was 100%. Although based on a
limited number of observations, both viral culture and RT-
PCR demonstrated 100% sensitivity for detecting influenza
During the 2006 to 2007 season, Binax NOW detected
approximately two thirds of influenza infections. The
sensitivity estimate of the assay generated in this study
(61%) was within the range reported in the literature (56–
76%) (Cruz et al., 2006; Smit et al., 2007; Landry et al.,
2004; Weinberg and Walker, 2005; Fader 2005; Hurt et al.,
2007). Although we did not observe a statistically
significant difference in test sensitivity in children versus
adults, the relatively high sensitivity in children is
consistent with previously published results (Landry
et al., 2004; Weinberg and Walker, 2005; Fader, 2005;
Hurt et al., 2007). A decrease in sensitivity in adult
patients has also been observed with other enzyme
immunoassay rapid tests for detecting influenza infections
(Yamazaki et al., 2000; Chan et al., 2002; Reina et al.,
2002; Cazacu et al., 2003; Hamilton et al., 2002; Ruest
et al., 2003; Cazacu et al., 2004; Landry et al., 2004;
Alexander et al., 2005). Higher viral excretion by children
has been suggested as a possible mechanism for higher
sensitivity in children (Landry et al., 2000).
The sensitivities and the specificities of DFA reported
range from 59% to 100% and 90% to 100%, respectively
(Daisy et al., 1979; Reina et al., 1996; Landry et al., 2000;
Chan et al., 2002; Quach et al., 2002; Habib-Bein et al.,
2003; Landry and Ferguson, 2003; Shetty et al., 2003;
Rahman et al., 2007), which are also consistent with our
findings. We observed that DFA had greater sensitivity
in adults relative to children, although the difference
was not statistically significant. However, power was
limited to assess age-related differences because only 8
influenza infections were detected in adults who were
tested by DFA.
The strength of our study includes its population-based
study design and the use of 2 different gold standard tests.
The limitations of our study were a small sample size and
the lack of standardized criteria for use of the rapid test
because clinicians ordered rapid influenza tests based on
their clinical judgment. Few influenza B infections
occurred in the study population, and it was not possible
to assess the performance of Binax NOW for influenza B.
Performance of DFA in our study is not generalizable due
to the fact that it is not a standardized test and solely
depends on individual expertise.
In conclusion, during the 2006 to 2007 influenza
season, DFA and Binax NOW demonstrated high specifi-
city but failed to identify a substantial proportion of
Detection of influenza virus by Binax NOW and DFA in comparison with
viral culture and RT-PCR
Viral culture Se
Specificity was always 100%.
165M. Rahman et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 162–166
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