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Journal of Clinical Virology 48 (2010) 234–238
Contents lists available at ScienceDirect
Journal of Clinical Virology
journal homepage: www.elsevier.com/locate/jcv
Validation of the Cepheid Xpert Flu A Real Time RT-PCR detection panel for
Emergency Use Authorization
Anthony R. Sambola,∗
, Peter C. Iwena
, Maura Pierettib
, Samik Basuc
, Michael H. Levic
,
Kimberly D. Gilonsked
, Kimberly D. Mosesd
, Jamie L. Marolae
, Preveen Ramamoorthye
a
Department of Pathology & Microbiology, University of Nebraska Medical Center, 42nd and Emile Str., Omaha, NE 68198-5900, United States
b
BayCare Health System, Clearwater, FL 33756, United States
c
Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY 10467, United States
d
Mid America Clinical Laboratories, Indianapolis, IN 46219, United States
e
National Jewish Health, Denver, CO 80216, United States
a r t i c l e i n f o
Article history:
Received 15 March 2010
Received in revised form 26 May 2010
Accepted 2 June 2010
Keywords:
Cepheid Xpert
Emergency Use Authorization
Clinical Laboratory Improvement
Amendment
2009 Influenza A/H1N1
RT-PCR
a b s t r a c t
Background: In April 2009, the United States Secretary of the Department of Health and Human Services
declared a public health emergency concerning the 2009 influenza H1N1 outbreak. This declaration
allowed the FDA to issue Emergency Use Authorization (EUA) of approved in vitro diagnostics to detect
the 2009 influenza H1N1 in clinical specimens.
Objectives: This report outlines the validation testing of the Cepheid Xpert Flu A Panel for the qualitative
detection of 2009 H1N1 viral RNA.
Study design: This study was a multi-site, dual-method clinical evaluation comparing the results of testing
between the Xpert Panel assay to the FDA-cleared Luminex Molecular Diagnostics xTAGTM
Respiratory
Viral Panel (Luminex RVP) assay and the EUA-granted Focus Diagnostics Influenza A/H1N1 (2009) Real
Time RT-PCR (Focus H1N1) assay.
Results: When compared to Luminex RVP (n = 300) for influenza A detection, the Xpert Panel had a sensi-
tivity of 91.2% (95% CI: 85.1–95.4), specificity of 99.4% (95% CI: 96.7–100), positive predictive value (PPV)
of 99.2% (95% CI: 95.6–100), and a negative predictive value (NPV) of 93.1% (95% CI: 88.3–96.4). When
compared to the Focus H1N1 (n = 258) for detection of H1N1, the Xpert Panel had a sensitivity of 92.1%
(95% CI: 82.4–97.4), specificity of 100% (95% CI: 98.5–100), PPV of 100% (95% CI: 95.0–100), and a NPV of
97.5% (95% CI: 94.3–99.2).
Conclusions: The results show the Cepheid Xpert Flu A Panel to be comparable to both the Luminex RVP
and the Focus H1N1 assays. The Cepheid Xpert Panel was granted an EUA on 24 Dec 2009.
Published by Elsevier B.V.
1. Background
Rapid influenza diagnostic tests (RIDTs) are used routinely in
clinical laboratories, physician offices, and emergency rooms to
screen clinical specimens for the influenza A and B viruses. Unfor-
tunately, performances of these immunoassays have been shown
to vary widely in both sensitivity and specificity. These variations
are due, in part, to the prevalence of the disease in the population at
Abbreviations: UNMC, University of Nebraska Medical Center; U.S. FDA, United
States Food and Drug Administration; CDC, Centers for Disease Control and Pre-
vention; CLIA, Clinical Laboratory Improvement Amendment; EUA, Emergency Use
Authorization.
∗ Corresponding author at: Department of Pathology & Microbiology, University
of Nebraska Medical Center, Durham Research Center 2, Room 8030, Omaha,
NE 68198-5900, United States. Tel.: +1 402 559 3032; fax: +1 402 559 7799.
E-mail address: asambol@unmc.edu (A.R. Sambol).
the time, specimen collection type, and adequate training of testing
personnel.1,2
To address these issues, additional testing of patient specimens
is often necessary to confirm screening assay results. However, sen-
sitive and specific confirmatory tests, such as direct fluorescent
antibody and viral culture, are labor intensive and require skilled
and highly trained personnel. Viral cultures can take up to 5 days
to receive final results, although positive culture results are often
available within 2 days.3 By contrast, real time reverse transcrip-
tase polymerase chain reaction assays (RT-PCR) may be used as a
rapid initial or confirmatory test for influenza.
In preparation for an outbreak of a novel strain of influenza virus,
the United States Food and Drug Administration (FDA) published
a draft guidance document in 2008 for the detection and differen-
tiation of influenza viruses.4 Shortly after the appearance of novel
H1N1 virus in North America in the spring of 2009,5 an emergency
was declared by the Secretary of the Department of Health and
1386-6532/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.jcv.2010.06.001

3. Author's personal copy
A.R. Sambol et al. / Journal of Clinical Virology 48 (2010) 234–238 235
Human Services. This allowed the FDA to rapidly grant Emergency
Use Authorization (EUA) to manufacturers of 2009 H1N1 RT-PCR
tests upon submission and review of accelerated clinical valida-
tion studies that were designed in compliance with the 2008 draft
guidance.6
Following the emergency declaration, a number of H1N1 RT-PCR
assays received EUA status.7,8 Cepheid (Sunnyvale, CA) was granted
an EUA from the FDA for its Xpert(R) Flu A Panel test, which was
the only 2009 H1N1 test to have received the Clinical Laboratory
Improvement Amendments (CLIA) classification of “moderate com-
plexity”. This RT-PCR assay is performed using the GeneXpert(R)
System (Cepheid) and allows the identification of the 2009 H1N1
influenza virus in less than 1 h.
2. Objectives
This paper presents the EUA validation process for the perfor-
mance of the Cepheid Xpert Flu A Panel for the qualitative detection
of 2009 H1N1 viral RNA.
3. Study design
The study was designed to evaluate the results of the Xpert Panel
assay compared to the FDA-cleared Luminex Molecular Diagnostics
xTAGTM Respiratory Viral Panel (Luminex RVP; Luminex, Austin,
TX) and the EUA-granted Focus Diagnostics Influenza A/H1N1
(2009) Real Time RT-PCR (Focus H1N1; Focus Diagnostics, Cypress,
CA) for the detection of influenza A and, specifically, H1N1. The
detection of influenza B was not addressed in this study.
3.1. Site selection
The three clinical study sites chosen for this study were Bay-
Care Health System, Clearwater, FL; Albert Einstein College of
Medicine, Montefiore Medical Center, Bronx, NY; and the Uni-
versity of Nebraska Medical Center, Omaha, NE. All study sites
were assessed by Cepheid Clinical Affairs staff prior to the start
of the study to ensure that each site had Institutional Review Board
(IRB) approval or waiver prior to initiating study procedures and
providing specimen samples. The other participating institutions
provided testing service only and were enrolled by the sponsor.
3.2. Method comparison
To determine the clinical performance of the Xpert Panel,
influenza-positive and influenza-negative specimens were tested
using the Xpert Panel and each comparator assay. In the Luminex
RVP evaluation, 300 specimens were tested and included 47
“seasonal” influenza A/H1, 23 influenza A/H3, 65 influenza A “unty-
peable” (not “seasonal” H1 or H3), 164 influenza-negative and 1
specimen that was reported as indeterminate. In the Focus H1N1
comparison, 258 specimens were tested and included 26 “seasonal”
influenza A/H1, 7 were influenza A/H3, 62 were influenza A “unty-
peable”, 162 were negative and 1 specimen that was reported as
indeterminate.
Performance of the Xpert Panel was compared individually to
the results of testing with Luminex RVP or Focus H1N1 assays. In the
event of an indeterminate finding by the Xpert Panel, a single retest
of the sample was performed and the results reported accordingly.
The Luminex RVP detects the presence of multiple influenza strains;
however, for purposes of this part of the study, only influenza A
was considered in the assessment of the Xpert Panel performance
versus the Luminex RVP. The results from the Focus H1N1 were
used to assess the Xpert Panel performance relative to 2009 H1N1
detection.
3.3. Reproducibility and assay complexity
To demonstrate the intra-site reproducibility and the assay com-
plexity, a panel of 4 samples comprised of 3 different influenza A
strains, prepared as a low-positive, high-positive and negative sam-
ples. These samples were tested on 3 different days by 6 different
operators (3 with prior PCR experience and 3 without). One lot of
the Xpert Panel was used and the assays were performed according
to the Xpert Panel procedure.
3.4. Specimen collection, storage, and transport
Specimens were collected as a part of standard care for patients
with influenza-like illness. Specimens were either a nasal aspi-
rate/wash (NA/W) or a nasopharyngeal swab (NP). The samples
were placed into viral culture medium and following routine test-
ing were stored at refrigerator temperatures for no longer than
24 h prior to aliquoting. A minimum of 1 ml for each specimen
was required to provide sufficient sample volume for inclusion in
the study. The samples were subsequently aliquoted into 500 ␮L
samples and stored at −70 ◦C prior to additional testing.
The selection of transport media, specimen volumes, shipping
conditions, and interval from specimen collection time to testing
were followed per each manufacturer’s guidelines. Specimens were
tested by both the Xpert Panel and the comparative assays within
24 h of thawing. Samples were stored at 2–8 ◦C between assays.
3.5. Comparator assay testing and discrepant results
It should be noted that only 258 of the 300 samples had sufficient
volume for all 3 required assays. Duplicate samples from any one
patient were excluded from the analyses.
Comparator assay testing was conducted in accordance with the
corresponding package labeling by all operators. For the purpose
of the EUA submission, the test results of the Xpert Panel were
compared independently to either the Luminex RVP or the Focus
H1N1 assays. Each of these comparator tests was designated as
the gold standard by which to compare the Xpert Panel results.
No discrepant testing was performed when the results obtained
with the Xpert Flu A Panel did not correlate with the results of the
corresponding comparator test.
3.6. Data and statistical analysis
Data was tabulated for the Xpert Panel versus the corresponding
comparator methods for sensitivity (percent positive agreement),
specificity (percent negative agreement), positive predictive value
(PPV), and negative predictive value (NPV). Confidence intervals
were calculated using Minitab v15 (Minitab, Inc., State College, PA).
The Fisher’s exact test for poolability was assessed across sample
types for both the influenza A and 2009 H1N1 viruses.
4. Results
4.1. Specimen accountability
In the first part of the clinical studies, a total of 346 patient spec-
imens were evaluated for inclusion. Of these specimens, 312 were
eligible for inclusion and 34 were excluded (21 were of unspec-
ified specimen type, 11 were an incorrect specimen type, and 2
were “extra” specimens that were not tested by any method). Of
the 312 eligible specimens, 12 (3.8%) were excluded for indetermi-
nate results (“invalid” or “error” based on internal assay controls),
leaving 300 included in the final dataset used for the analysis of
performance compared to the Luminex RVP assay. Forty-four of

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236 A.R. Sambol et al. / Journal of Clinical Virology 48 (2010) 234–238
Fig. 1. Specimen selection and accountability.
the specimens included in the final dataset for the analyses of per-
formance of Xpert Panel versus the Luminex RVP were not sent for
Focus H1N1 testing due to insufficient sample volumes. Therefore,
in the second part of the study, a total of 258 specimens were avail-
able for analysis of performance of Xpert Panel versus Focus H1N1.
Specimen accountability is summarized in Fig. 1.
4.2. Assay reproducibility and laboratory operator experience
An evaluation was conducted to ascertain whether there was
a difference in assay performance between Xpert Panel operators
in this study with and without prior laboratory experience. The
influenza strains and concentrations for the reproducibility and
assay complexity study are shown in Table 1. At each of the 3
participating sites, 1 of the 2 operators had previous laboratory
experience and 1 did not. The results demonstrated that the inexpe-
rienced operator performed as well as the experienced operators in
that 162 of 162 (100%) tests conducted were in complete agreement
(data not shown).
Table 1
Influenza A virus strains used to prepare analytical precision study samples.
Influenza A virus strains High-positivea
Low-positive
Solomon/Islands/2/2006 H1b
5.64 × 102
5.64 × 100
Brisbane/10/07 H3N2b
2.45 × 103
2.45 × 100
W1/629-D00015/2009 H1N1c
1.12 × 103
1.12 × 100
a
Units are in tissue culture infective doses (TCID)50/mL.
b
Influenza strains supplied by Zeptomextrix, Buffalo, NY.
c
Influenza strain supplied by Dr. Nathan Ledeboer, Dynacare Laboratories, Med-
ical College of Wisconsin.
Table 2
Cepheid Xpert Flu A Panel performance compared with the Luminex RVP assay.
Xpert Flu A Panel Luminex RVP
Positive Negative Total
Positive 124 1 125
Negative 12 163 175
Total 136 164 300
% Sensitivity: 91.2% (95% CI: 85.1; 95.4); % specificity: 99.4% (95% CI: 96.7; 100);
positive predictive value: 99.2% (95% CI: 95.6; 100); negative predictive value: 93.1%
(95% CI: 88.3; 96.4).
4.3. Comparative results
The overall performance of the Xpert Panel relative to the
Luminex RVP for detection of influenza A is shown in Table 2. Sen-
sitivity and specificity were 91.2% and 99.4%, respectively, while
the PPV was 99.2% and NPV was 93.1%. The overall performance of
Table 3
Cepheid Xpert Flu A Panel performance compared to Focus 2009 H1N1 assay.
Xpert Flu A Panel Focus 2009 H1N1
Positive Negative Total
Positive 58 0 58
Negative 5a
195 200
Total 63 195 258
% Sensitivity: 92.1% (95% CI: 82.4; 97.4); % specificity: 100% (95% CI: 98.5; 100);
positive predictive value: 100% (95% CI: 95.0; 100); negative predictive value: 97.5%
(95% CI: 94.3; 99.2).
a
None of the 5 discordant results tested positive for the influenza A virus matrix
sequence by the Xpert Flu A Panel.

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A.R. Sambol et al. / Journal of Clinical Virology 48 (2010) 234–238 237
Table 4
Cepheid Xpert Flu A Panel performance compared with comparator assays by specimen type.
Comparator % Diagnostic accuracy (95% confidence interval)
Sensitivity Specificity PPV NPV
Luminex (influenza A) (n = 300)
NA/W (n = 90) 94.6(81.8; 99.3) 98.1(89.9; 100) 97.2(85.5; 99.9) 96.3(87.3; 99.6)
NP (n = 210) 89.9(82.2; 95.1) 100(97.3; 100) 100(96.7; 100) 91.7(85.3; 96.0)
Combineda
91.2(85.1; 95.4) 99.4(96.7; 100) 99.2(95.6; 100) 93.1(88.3; 96.4)
Focus (2009 H1N1) (n = 258)
NA/W (n = 81) 100 (85.4; 100) 100 (95.3; 100) 100(85.4; 100) 100(95.3; 100)
NP (n = 177) 88.6(75.4; 96.2) 100(97.8; 100) 100(92.6; 100) 96.4(91.8; 98.8)
Combinedb
92.1(82.4; 97.4) 100(98.5; 100) 100(95.0; 100) 97.5(94.3; 99.2)
Abbreviations: PPV = positive predictive value; NPV = negative predictive value; Focus = Focus Diagnostics Influenza A/H1N1 (2009) Real Time RT-PCR test; Luminex = Luminex
Molecular Diagnostics xTAGTM
Respiratory Viral Panel; NA/W = nasal aspirate/wash specimens; NP = nasopharyngeal swab specimens.
a
The Luminex poolability results were: sensitivity p = 0.512; specificity p = 0.323; overall agreement p = 0.761.
b
Focus poolability results were: sensitivity p = 0.311; specificity p = 1.000; overall agreement p = 0.329.
the Xpert Panel relative to the Focus H1N1 for detection of 2009
H1N1 is shown in Table 3. Sensitivity and specificity were greater
than 91.2% and 100%, respectively, while the PPV was 100% and
NPV was 97.5%.
Table 4 shows the performance values of the Xpert Panel ver-
sus either the Luminex RVP or the Focus H1N1 when specimen
types were differentiated between NA/W and NP. Poolability was
assessed across sample types for both influenza A and 2009 H1N1.
The results of the analysis show no significant difference in sensi-
tivity or specificity by sample type, thus supporting the combining
of results.
5. Discussion
The capability of the laboratory to provide timely and accurate
results when testing clinical specimens for influenza is paramount
for proper treatment and care of afflicted individuals. In the US,
human testing for influenza virus may only be conducted using
FDA-approved assays and any assay used without this approval
must undergo an extensive in-house validation process. Unfortu-
nately, these criteria do not allow for the rapid development of
assays during a pandemic situation. In 2008, the FDA developed
new guidelines that established the minimum performance char-
acteristics for the emergency approval of in vitro diagnostic devices
for detection and differentiation of influenza viruses.
One criterion for EUA approval is the demonstration of ana-
lytical sensitivity as well as operator reproducibility of the new
assay. In this study, both experienced and inexperienced laboratory
operators of the test were able to reproduce the same test results;
thus, the Xpert Flu A assay was performed successfully by person-
nel who were inexperienced with PCR testing, which supports its
designation as a “moderately complex” test.
EUA approval also requires that specimen collection and accep-
tance criteria, cross-reactivity, sensitivity, specificity, PPV, and NPV
of a new assay be compared to assays already marketed. In this
study, we compared assay results for the Cepheid Xpert Flu A Panel
to the results of the FDA-cleared Luminex Molecular Diagnostics
xTAGTM Respiratory Viral Panel (Luminex RVP) assay for the detec-
tion of influenza A, and to the results of the EUA-granted Focus
Diagnostics Influenza A/H1N1 (2009) Real Time RT-PCR (Focus
H1N1) assay for detection of 2009 H1N1. The overall performance
of the Xpert Panel relative to the approved comparative assays was
≥91.2% for sensitivity and ≥99.4% for specificity.
For these analyses, the number of positive samples selected
for study was enriched to meet the FDA requirements for EUA
approval; thus, the prevalence rate of 45% was greater than that
experienced by testing sites during the pandemic. As a result,
the PPV and NPVs obtained for the Xpert Panel relative to the
Luminex RVP and relative to the Focus H1N1 should not be consid-
ered to be representative of the assay’s performance in an infected
population.
At the beginning of the pandemic influenza outbreak, there were
no FDA-cleared 510(k) assays that would definitely detect and iden-
tify the 2009 H1N1 strain of influenza A. The availability of an EUA
process allowed for new assays to be developed, approved, and
commercialized rapidly. This helped to alleviate the problem of
limited rapid and reliable means for determination of infection in
patients. Cepheid cartridges are designed to be run on-demand.
The encouraging sensitivity and specificity profile for the Xpert Flu
A panel combined with the reproducibility of assay results and ease
of use may allow this assay to fill an important gap in performance
between RIDTs and the new gold standard of PCR testing.
Funding
Cepheid, Inc. provided Xpert Flu A reagents and financial support
for this study.
Ethical approval
All study sites were granted waivers of informed consent by
their IRBs for this study.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
Dana El-Hajjar and David Moran of the University of Nebraska
Medical Center, David Hinkle of Mid America Clinical Laboratories,
Kathleen Schulte of National Jewish Health and Carolyn Dowell and
Jelena Mulin of BayCare Laboratories for their work in the compar-
ative testing of the specimens used in this study.
References
1. Ginocchio CC, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, et al. Evaluation
of multiple test methods for the detection of the novel 2009 influenza A (H1N1)
during the New York City outbreak. J Clin Virol 2009;45:191–5.
2. Sambol AR, Abdalhamid B, Lyden ER, Aden TA, Noel RK, Hinrichs SH. Use of rapid
influenza diagnostic tests under field conditions as a screening tool during an
outbreak of the 2009 novel influenza virus: practical considerations. J Clin Virol
2010;47:229–33.
3. Hurt AC, Alexander R, Hibbert J, Deed N, Barr IG. Performance of six influenza
rapid tests in detection human influenza in clinical specimens. J Clin Virol
2007;39:132–5.
4. United States Food and Drug Administration. Establishing performance charac-
teristics of in vitro diagnostic devices for detection and differentiation of influenza
viruses. Draft guidance; 2008. February 15.

6. Author's personal copy
238 A.R. Sambol et al. / Journal of Clinical Virology 48 (2010) 234–238
5. Centers for Disease Control and Prevention. Outbreak of swine-like origin
influenza A (H1N1) virus infection—Mexico, March–April 2009. Morb Mortal
Wkly Rep 2009;58:467–70.
6. Memorandum. Determination Pursuant to § 564 of the Federal Food, Drug, and
Cosmetic Act; 26 April 2009.
7. Centers for Disease Control and Prevention. CDC protocol of real time
RTPCR for Influenza A (H1N1). April 2009. http://www.who.int/csr/resources/
publications/swineflu/CDCrealtimeRTPCRprotocol 20090428.pdf [accessed 29
September 2009].
8. Food and Drug Administration Website: http://www.cdc.gov/h1n1flu/guidance/
diagnostic tests.htm.