Clinical Medicine & Research
Volume 10, Number 4: 215-218
©2012 Marshfield Clinic
Keywords: Detection; Influenza virus; Specimen type
Objective: Examine differences in the detection of influenza by specimen and test type using paired
nasal and nasopharyngeal swabs.
Design: Prospective study
Setting: Enrollment took place between January and March 2007 in a central Wisconsin population.
Participants: Adult patients were screened and enrolled by trained research coordinators
following medical encounters for acute respiratory illnesses of <10 days duration.
Methods: Paired nasal and nasopharyngeal swabs were collected from consenting patients and
tested by both real-time reverse transcriptase polymerase chain reaction (rRT-PCR) and viral
culture. A composite measure of positivity was used as the gold standard; cases included any
positive result by rRT-PCR or viral culture from either specimen type.
Results: Paired samples were collected from 240 adults; 33 (14%) individuals tested positive for
influenza by rRT-PCR. Using rRT-PCR, the sensitivity of the nasal swab was 89% (95% CI, 78%-99%)
and the sensitivity of the nasopharyngeal swab was 94% (95% CI, 87%-100%), compared to a
composite gold standard.
Conclusion: Test sensitivity did not vary significantly by swab type when using a highly sensitive
molecular diagnostic test, but power was limited to detect modest differences.
Received: February 23, 2012
Revised: May 1, 2012
Accepted: May 9, 2012
Financial Disclosure: Funding for this research was provided by a cooperative agreement with the Centers for Disease Control and Prevention, Atlanta, GA
(1 U01 CI000192-01).
Corresponding Author: Stephanie Irving, MHS; Abt Associates; 2200 Century
Parkway, Suite 950; Atlanta, GA 30345; Tel: 404.946.6309; Fax: 404.965.3065;
Stephanie A. Irving, MHS; Mary F. Vandermause, BSMT; David K. Shay, MD, MPH;
and Edward A. Belongia, MD
Comparison of Nasal and Nasopharyngeal Swabs
for Influenza Detection in Adults
Influenza is a major cause of acute respiratory illness
worldwide and accounts for thousands of deaths in the United
States in a typical season.1,2 The 2009 pandemic and increasing
type-specific antiviral resistance have heightened the need for
influenza testing that is accurate, timely, and well-tolerated
A variety of specimens have been used for influenza testing
including the nasopharyngeal (NP) swab, oropharyngeal
swab, nasal wash, and nasal aspirate. The NP wash or aspirate
is generally considered the ‘gold standard’ for virus isolation,
but it is cumbersome to perform and unpleasant for patients.5-7
Swabs are easier and faster to collect and may be preferred by
providers and patients. Comparative data on the sensitivity of
influenza sampling procedures are limited; many studies
focus on pediatric populations or use older diagnostic
methods.5,8-12 Recent literature has focused on NP sampling
compared to oropharyngeal or combined nose-throat
swabs.6,7,10,12 To date, no studies have compared paired nasal
and NP swabs collected from adults.
Several methods for laboratory diagnosis of influenza are
available. Viral culture has historically been considered the
‘gold standard’ diagnostic test, but traditional culture can
CM&R 2012 : 4 (November)
require up to 7 days to obtain a positive result.5 Recent
findings from studies utilizing real-time reverse transcriptase
polymerase chain reaction (rRT-PCR) for the detection of
respiratory pathogens suggest that the use of this current
molecular technology may outweigh potential differences in
sensitivity due to specimen type.7,10
We conducted a prospective study to examine differences in
the detection of influenza by specimen type. Paired nasal
and NP swabs were tested by both viral culture and
rRT-PCR. Study procedures were reviewed and approved
by the Marshfield Clinic Institutional Review Board, and
all participants gave written informed consent for
Enrollment in the study took place between January and
March 2007. Adult patients were enrolled by study staff
following a medical encounter for acute respiratory illness.
Eligible illnesses were <10 days duration and included fever,
chills, or cough.13
Both shallow nasal and NP swabs were collected from
consenting patients. The NP swab was collected using an
aluminum/plastic unishaft swab, inserted half the distance
from the nares to the base of the ear, or to a depth of
approximately 2 inches (Remel, Thermo Fisher Scientific,
As it was less invasive, the nasal swab was collected first,
using a large tipped, plastic shafted Dacron swab. The swab
was inserted approximately 1 centimeter, rubbed along the
septum of the nostril for 3 to 5 seconds, and withdrawn. The
swab was then placed in M4-RT viral transport media for
testing. The more invasive NP swab was then collected using
a smaller Dacron swab on a wire shaft. As per manufacturer’s
protocol, the swab was inserted to the point where the wire
shaft meets the plastic sleeve, rotated, and withdrawn by
scraping the septum.14 The wire-shafted swab was then placed
in a separate M4-RT viral transport tube for testing. All
samples were refrigerated for <24 hours until aliquots could
be taken, which were then frozen until testing.
Real-time reverse transcriptase polymerase chain reaction
(rRT-PCR) and viral cultures were performed at the Marshfield
Clinic Research Foundation. rRT-PCR was performed on
nucleic acid extracts from 200 µl of clinical sample using the
LightCycler Real-Time PCR System (Roche Diagnostics,
Indianapolis, IN), and Invitrogen SuperScript III Platinum
One-Step Quant RT-PCR chemistry (Life Technologies,
Grand Island, NY). All rRT-PCR protocols, primers, and
probes are property of and were provided by the Influenza
Division of the Centers for Disease Control and Prevention
(CDC) (protocols available from the CDC on request).15 The
assay was a TaqMan based, real-time detection of the matrix
1 protein (M1) of influenza A and the non-structural protein 1
(NS1) of influenza B; the sequences of both proteins are
highly conserved. The human RNase P gene primer and probe
set served as an internal positive control for human
Viral culture was performed using Madin-Darby canine
kidney (MDCK) shell vial (Diagnostic Hybrid, Athens, OH).
Cells were inoculated with 200 µl of specimen and 1 ml of
culture media. Inoculated shell vials were centrifuged for one
hour at 2000 rpm to enhance viral contact and more rapid
infection of the MDCK cells. The vials were incubated at 35°
to 37°C. Cultures were microscopically examined daily for
cytopathic effect (CPE). When CPE was observed or after 5
days of incubation with no CPE, the shell vial monolayer was
scraped, and a slide was prepared and stained for Influenza A
and B immunofluorescent identification (D3 Influenza A/
Influenza B DFA Kit, Diagnostic Hybrid, Athens, OH).
Sensitivities and 95% confidence intervals (CI) were
calculated from two-by-two tables. Sensitivity was calculated
as compared to a composite gold standard, which included
any positive result by rRT-PCR or viral culture from either
specimen type. Sensitivities were compared using chi-square;
P values <0.05 were considered statistically significant. Data
analysis was performed using SAS 9.2.
Paired nasal and NP swabs were collected from 240 patients.
The median age of the patients was 60 years (range 47 to 91
Nasal and NP swab comparison
Table 1. Comparison of the sensitivity of nasal and nasopharyngeal swab specimens for the detection of influenza by viral culture
Nasal swabs Nasopharyngeal swabs
95% CI P value†
Viral culture 14 40.0 23.8-56.2 18 51.4 34.9-68.2 0.34
rRT-PCR 31 88.6 78.0-99.1 33 94.3 86.6-100 0.40
*Sensitivity calculated as compared to a composite gold standard. Gold standard cases included any positive result by rRT-PCR or viral culture from either
specimen type (n=35).
†Sensitivity of nasal swab as compared to NP swab, using same diagnostic testing method.
CI, confidence interval; NP, nasopharyngeal; rRT-PCR, real time reverse transcriptase polymerase chain reaction
CM&R 2012 : 4 (November)
Irving et al
years), 151 (63%) were female, 147 (61%) were vaccinated
with the 2006–2007 influenza vaccine, and 109 (45%) had a
chronic medical condition. The mean interval from symptom
onset to specimen collection was 4.8 (range 0–10) days.
A total of 35 swabs tested positive for influenza by either rRT-
PCR or viral culture by either swab type (CGS). Thirty-one
samples were positive for influenza A; four samples were
positive for influenza B. There were 14 (6%) nasal swabs and
18 (8%) NP swabs that tested positive for influenza by viral
culture. There were 31 (13%) nasal swabs and 33 (14%) NP
swabs that tested positive for influenza by rRT-PCR. The
nasal swab had 40.0% sensitivity by viral culture and 88.6%
sensitivity by rRT-PCR, when calculated compared to the
CGS (P<0.0001). The NP swab had 51.4% sensitivity by viral
culture and 94.3% sensitivity by rRT-PCR, compared to the
CGS (P<0.0001). The sensitivity differences by swab type
when using the same diagnostic test were not significant
Of the 18 NP swabs positive for influenza by viral culture, 14
paired nasal swabs tested positive, for a sensitivity of 77.8%
(95 % CI, 58.6%–97.0%) for the nasal swab, as compared to
the NP swab. Of the 33 NP swabs positive by rRT-PCR, 29
paired nasal swabs tested positive, for a sensitivity of 87.9%
(95% CI, 76.7%–99.0%) for the nasal swab, as compared to
the NP swab. The difference in sensitivity of the nasal swab
as compared to the paired NP swab by diagnostic test was not
This study compared the sensitivities of two specimen
collection methods using two diagnostic methods for the
diagnosis of influenza. The nasal swab was less sensitive than
the NP swab, irrespective of diagnostic test, but the difference
in sensitivities between sampling methods was not significant.
Real-time RT-PCR was significantly more sensitive than viral
culture, irrespective of specimen collection method.
Our results, together with findings from the literature, suggest
that less invasive methods of specimen collection may be
acceptable in the era of molecular testing. A larger study of
influenza detection using combined nose and throat swabs
versus NP aspirates found that the combined swabs had a
higher diagnostic yield, but the performance of nasal swabs
alone was not evaluated.7 A recent large pediatric study
reported 88% sensitivity for the detection of influenza A
using NP aspirates, and 84% sensitivity with the nasal swab
when tested by PCR; the difference in sensitivities was not
statistically significant (P=0.72).12 Lambert and colleagues10
compared combined nose-throat swabs with NP aspirates in a
large pediatric population. Reported sensitivities of the nose-
throat swab were 92% for the detection of influenza A and
100% for influenza B.10
We are not aware of any other published studies evaluating
paired nasal and NP swabs from adults using rRT-PCR as the
diagnostic method for detection of influenza. A strength of
our study is the use of consistent recruitment procedures and
standardized sample collection methods. The most important
limitation of the study is the limited power to detect modest
differences in sensitivity; only 35 participants tested positive
for influenza. Sensitivity of sample collection methods may
vary by influenza type/subtype, and our case numbers did not
allow for stratified analyses. The paired specimens in this
analysis were collected from older adults only; thus, we are
unable to generalize our results to younger populations.
Finally, specimens underwent an additional freeze-thaw cycle
between rRT-PCR and culture testing. While this has the
potential to affect the sensitivity of the viral culture, any
impact would have been minor. Additionally, the focus of our
investigation was the agreement between collection methods,
not diagnostic test, as literature has previously demonstrated
increased sensitivity of PCR compared to viral culture.
Traditional specimen collection methods for the detection of
influenza are based on the use of viral culture as the diagnostic
test.5 A ‘gold standard’ sampling method has not been
identified and validated for influenza detection using rRT-
PCR, but emerging evidence suggests that less invasive
samples may have comparable sensitivity to nasopharyngeal
swabs or aspirates when using molecular diagnostic tests.
The authors would like to thank LaShondra Berman, Steve
Lindstrom, Jennifer Meece, Marshfield Clinic Research
Foundation Core Lab staff, Epidemiology Research Center
research coordinators, and 2006-2007 Influenza Vaccine
Effectiveness study participants for their contributions to this
The findings and conclusions in this report are those of the
authors and do not necessarily represent the official position
of the Centers for Disease Control and Prevention.
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L, Meltzer MI, Bresee JS, Shay DK. Estimates of US
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Stephanie A. Irving, MHS,*,†; Mary F. Vandermause, BSMT‡;
David K. Shay, MD, MPH§; Edward A. Belongia, MD*
*Epidemiology Research Center, Marshfield Clinic Research
Foundation, Marshfield, Wisconsin, USA
†Current Affiliation: Senior Analyst, Abt Associates,
Atlanta, Georgia, USA
‡Core Laboratory, Marshfield Clinic Research Foundation,
Marshfield, Wisconsin, USA
§Influenza Division, Centers for Disease Control and
Prevention, Atlanta, Georgia, USA