XPERT FLU
Device Facts
| Record ID | K123191 |
|---|---|
| Device Name | XPERT FLU |
| Applicant | Cepheid |
| Product Code | OCC · Microbiology |
| Decision Date | Dec 21, 2012 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Indications for Use
The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
Device Story
The Xpert Flu Assay is an automated, in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B, and 2009 H1N1 viral RNA. It utilizes single-use, multi-chambered fluidic cartridges containing PCR reagents. The user loads the specimen (nasal aspirate/wash or nasopharyngeal swab) into the cartridge and inserts it into the GeneXpert Instrument System. The system automates sample preparation, nucleic acid extraction, and real-time RT-PCR amplification and detection. Each module features a syringe drive for fluid movement, an ultrasonic horn for cell lysis, and an I-CORE thermocycler. The process is completed in 75 minutes. Results are provided via the system user interface to healthcare providers to aid in influenza diagnosis. The device is intended for use in clinical settings by laboratory personnel (CLIA moderate or high complexity).
Clinical Evidence
Retrospective clinical study of 166 archived clinical samples (NP swabs and NA/W) compared to the predicate device. Results demonstrated high positive and negative percent agreement for Influenza A, 2009 H1N1, and Influenza B. Contrived specimens (n=10) spiked at LoD were also tested and correctly identified. Analytical performance included LoD studies for 12 strains and analytical specificity testing against 40 common respiratory pathogens/flora, showing 100% specificity.
Technological Characteristics
Multiplex real-time RT-PCR assay; integrated sample preparation and detection in single-use fluidic cartridges; utilizes GeneXpert® Instrument Systems (1-80 modules); ICORE® thermocycler; ultrasonic horn for cell lysis; automated fluidic movement via syringe drive/rotary valve; software-controlled; qualitative output.
Indications for Use
Indicated for patients with signs and symptoms of respiratory infection; used as an aid in the diagnosis of influenza A, influenza B, and 2009 H1N1 influenza viral RNA; requires clinical and epidemiological risk factor assessment.
Regulatory Classification
Identification
A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.
Special Controls
*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.
Predicate Devices
- Cepheid Xpert® Flu Assay (K120911)
Related Devices
- K103766 — XPERT FLU, GENEXPERT DX SYSTEMS, GENEXPERT INFINITY SYSTEM, GENEXPERT SYSTEM SOFTWARE VERISIONS 2.1 AND 4.0 · Cepheid · Apr 21, 2011
- K120911 — XPERT FLU GENEXPERT DX SYSTEMS (GX-I, GX-IV) · Cepheid · May 18, 2012
- K162456 — Xpert Xpress Flu, Xpert Nasopharyngeal Sample Collection Kit, GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI), GeneXpert Infinity-48 System and GeneXpert Infinity-48s System, GeneXpert Infinity-80 System · Cepheid · Feb 13, 2017
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number: K123191
B. Purpose for Submission: The submission is for a modification to the previously cleared Xpert® Flu Assay (K103766, K120911). The modification is the addition of a reverse primer that targets the matrix gene segment of the influenza A virus strain A/Victoria/361/2011. The primer was added to improve the Xpert® Flu Assay detection of this virus strain which is predicted to be the dominant circulating H3N2 virus type during the 2012-2013 influenza season. The A/Victoria/361/2011 virus is also included in the trivalent influenza vaccine for the 2012-2013.
C. Measurand: Influenza A and/or influenza B nucleic acid isolated from nasal aspirates/washes or nasopharyngeal swabs and amplified by real-time RT-PCR.
D. Type of Test: The Xpert® Flu Assay is a real-time RT-PCR assay that measures the presence of influenza A and/or influenza B nucleic acid.
E. Applicant: Cepheid
F. Proprietary and Established Names:
Xpert® Flu, Xpert® Flu Assay
GeneXpert® Instrument Systems
GeneXpert® Dx System
GeneXpert® Infinity-48 System
GeneXpert® Infinity-80 System
G. Regulatory Information:
1. Regulation section:
866.3980, Respiratory viral panel multiplex nucleic acid assay
2. Classification: Class II
3. Product code: OCC, OQW, OOI
4. Panel: Microbiology (83)
H. Intended Use:
1. Intended use(s):
The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative
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detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.
Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary.
If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
2. Indication(s) for use:
Same as intended use.
3. Special conditions for use statement(s):
Prescription use only.
4. Special instrument requirements: The Xpert Flu Assay requires the use of either the GeneXpert® Dx System (Software version 4.3 or higher), or the GeneXpert® Infinity-48 System (Software version 4.3 or higher), or the GeneXpert® Infinity-80 System (Software version 6.0 or higher) from Cepheid.
I. Device Description:
The Xpert Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1. The assay is performed on the Cepheid GeneXpert® Instrument Systems. The GeneXpert® Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR assays. The systems require the use of
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single-use disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.
The Xpert Flu Assay includes reagents for the detection and differentiation of influenza A, influenza B, and influenza A, subtype 2009 H1N1 directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients suspected of having influenza. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.
The liquid specimen (NA/W) or swab specimen (NP) is collected according to the institution's standard procedures and placed into Universal Transport Medium (3mL UTM tubes). Following a brief mixing by inverting the UTM tube five times, the eluted material and one single-use reagent (Reagent 1), that is provided with the assay, are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert® Flu cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert® instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fully automated and completely integrated.
The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time PCR for detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 in 75 minutes. The GeneXpert® Instrument Systems, which consist of the GeneXpert® Dx System, the GeneXpert® Infinity-48 System, and the GeneXpert® Infinity-80 System, have 1 to 80 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary ICORE® thermocycler for performing real-time PCR and detection.
J. Substantial Equivalence Information:
1. Predicate device name(s): Cepheid Xpert® Flu Assay
2. Predicate 510(k) number(s): K120911
3. Comparison with predicate:
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| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| 510(K) | K123191 | K120911 |
| Regulation | Same | 866.3332 and 866.3980 |
| Product Code | Same | OQW, OCC, OOI |
| Device Class | Same | II |
| Technology/Detection | Same | Multiplex real time RT/PCR |
| Intended Use | The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert® Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert® Flu Assay is intended as an aid in the diagnosis of influenza.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.
Performance characteristics for influenza A were established during the | The Cepheid Xpert® Flu Assay is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert® Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert® Flu Assay is intended as an aid in the diagnosis of influenza.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.
Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 |
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| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary.
If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. | influenza was the predominant influenza A virus in circulation. When other influenza A viruses are emerging, performance characteristics may vary.
If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. |
| Indication for Use | Same | Patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. |
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| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Assay Targets | Same | Influenza A, influenza B, and influenza A, subtype 2009 H1N1 |
| Specimen Types | Same | Nasal aspirates/washes (NA/W) and Nasopharyngeal (NP) swabs |
| Technological Principles | Same | RT-PCR |
| Nucleic Acid Extraction | Same | Yes |
| Extraction Methods | Same | Sample preparation integrated in GeneXpert® Cartridge and Gene Xpert® Instrumentation System |
| Assay Results | Same | Qualitative |
| Instrument System | Same | Cepheid® GeneXpert® Instrument Systems |
| Assay Controls | Same | Encapsulated (armored) RNA pseudovirus as a sample processing control. Available but not provided are inactivated virus controls for Flu A/B and Flu A 2009 H1N1 as external positive controls and Coxsackie virus as an external negative control. |
| Rapid Test Results | Same | Total 75 minutes for sample preparation and real-time RT-PCR |
| Primers and Probes for influenza A, influenza B and influenza A subtype H1N1 | Same plus an additional primer for Influenza A. | Original primers and probes to detect the presence of nucleic acid sequences of influenza A, influenza B, and influenza A, subtype 2009 H1N1 |
| Laboratory Users | Same | CLIA Moderate or High Complexity |
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K. Standard/Guidance Document Referenced (if applicable):
N/A
L. Test Principle:
The modified Xpert® Flu Assay detects the presence of influenza A and/or influenza B through detection and amplification of nucleic acid. Nucleic acid is isolated from nasal aspirates/washes or nasopharyngeal swabs and amplified by real-time RT-PCR.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
The addition of one primer (oligonucleotide) would not impact the Precision/Reproducibility of this assay. See K120911 for additional information.
b. Linearity/assay reportable range:
The addition of one primer (oligonucleotide) would not impact the Linearity/assay reportable range of this assay. See K120911 for additional information.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
The addition of one primer (oligonucleotide) would not impact the Traceability, Stability, Expected values (of the controls, calibrators, or methods) of this assay. See K120911 for additional information.
d. Detection limit:
A Limit of Detection study was performed for the following twelve influenza virus strains:
Seasonal influenza A H1N1: A/Brisbane/59/07, A/New Caledonia/20/1999
Seasonal influenza A H3N2: A/Brisbane/10/07, A/Wisconsin/67/05, A/Victoria/361/2011
Influenza A 2009 H1N1: A/SwineNY/01/2009, A/SwineNY/02/2009, A/SwineNY/03/2009, A/SwineCanada/6294, A/WI/629-S1/2009(D00015)
Influenza B: B/Florida/07/04, B/Florida/02/06
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Each virus was tested with the modified Xpert® Flu Assay in 20 replicates at four concentrations to obtain the LoD. The LoD was determined to be the lowest concentration at which there were 19/20 or 20/20 positive test results. The LoDs reported in this study were equivalent to or better than those reported for the predicate device.
# e. Analytical specificity:
Influenza viruses were diluted in a simulated background matrix of $2.5\%$ porcine mucin and $1\%$ human whole blood in $0.85\%$ sodium chloride in 1X PBS solution with $15\%$ glycerol diluted with UTM at a 1:6 ratio. A representative virus was chosen for each test analyte, influenza A, influenza A 2009 H1N,1 and influenza B. A total of 40 strains of viruses (18), bacteria (21), and yeast (1) were then spiked into the test sample to determine the specificity of the Xpert Flu Assay in the presence of contaminants that are commonly found in the nasopharyngeal flora.
Table 1: Analytical Specificity Determination for Xpert® Flu Assay
| Strain | Concentration (per Cartridge) | Influenza A | Influenza A 2009 H1N1 | Influenza B |
| --- | --- | --- | --- | --- |
| Positive Control 1 – Influenza A/Influenza B | N/A* | + | - | + |
| Positive Control 2 – Influenza A2009 H1N1 | N/A* | + | + | - |
| Negative Control Coxsackie virus | N/A* | - | - | - |
| Adenovirus Type 7A | 1.1 x 105TCID50/mL | - | - | - |
| Adenovirus Type 1 | 1.0 x 107TCID50/mL | - | - | - |
| Human Coronavirus 229E | 2.5 x 104TCID50/mL | - | - | - |
| Human Coronavirus OC43 | 5.6 x 104TCID50/mL | - | - | - |
| Cytomegalovirus* | 4.7 x 107Copies/mL | - | - | - |
| Enterovirus Type 71 | 3.5 x 105TCID50/mL | - | - | - |
| Epstein-Barr Virus | 7.1 x 108TCID50/mL | - | - | - |
| Parainfluenzavirus Type 1 | 1.1 x 105TCID50/mL | - | - | - |
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| Parainfluenzavirus Type 2 | 3.1 x 10^{7} TCID_{50}/mL | - | - | - |
| --- | --- | --- | --- | --- |
| Parainfluenzavirus Type 3 | 1.9 x 10^{6} TCID_{50}/mL | - | - | - |
| Measles Virus | 6.3 x 10^{4} TCID_{50}/mL | - | - | - |
| Human Metapneumovirus | 3.8 x 10^{5} TCID_{50}/mL | - | - | - |
| Mumps | 6.3 x 10^{6} TCID_{50}/mL | - | - | - |
| Respiratory Syncytial Virus A | 5.3 x 10^{7} TCID_{50}/mL | - | - | - |
| Respiratory Syncytial Virus B | 1.2 x 10^{7} TCID_{50}/mL | - | - | - |
| HSV | 3.1 x 10^{6} TCID_{50}/mL | - | - | - |
| Human Rhinovirus | 1.2 x 10^{5} TCID_{50}/mL | - | - | - |
| Echovirus | 3.3 x 10^{8} TCID_{50}/mL | - | - | - |
| Bordetella pertussis | 500 ng/mL | - | - | - |
| Chlamydia pneumonia | 5 x 10^{6} CFU/mL | - | - | - |
| Corynebacterium xerosis | 1 x 10^{6} CFU/mL | - | - | - |
| Esherichia coli | 1 x 10^{6} CFU/mL | - | - | - |
| Proteus vulgaris | 1 x 10^{6} CFU/mL | - | - | - |
| Proteus mirabilis | 1 x 10^{6} CFU/mL | - | - | - |
| Klebsiella pneumonia | 1 x 10^{6} CFU/mL | - | - | - |
| Haemophilus influenza | 1 x 10^{6} CFU/mL | - | - | - |
| Lactobacillus crispatus | 1 x 10^{6} CFU/mL | - | - | - |
| Legionella pneumophila | 1 x 10^{6} CFU/mL | - | - | - |
| Moraxella catarrhalis | 1 x 10^{6} CFU/mL | - | - | - |
| Mycobacterium tuberculosis (BCG strain) | 1 x 10^{6} CFU/mL | - | - | - |
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| Mycoplasm pneumonia | 1 x 10^{6} CFU/mL | - | - | - |
| --- | --- | --- | --- | --- |
| Nisseria meningitides | 1 x 10^{6} CFU/mL | - | - | - |
| Nisseria cinerea | 1 x 10^{6} CFU/mL | - | - | - |
| Pseudomonas aeruginosa | 1 x 10^{6} CFU/mL | - | - | - |
| Staphylococcus aureus | 1 x 10^{6} CFU/mL | - | - | - |
| Staphylococcus epidermidis | 1 x 10^{6} CFU/mL | - | - | - |
| Streptococcus pneumoniae | 1 x 10^{6} CFU/mL | - | - | - |
| Streptococcus pyogenes | 1 x 10^{6} CFU/mL | - | - | - |
| Streptococcus salivarius | 1 x 10^{6} CFU/mL | - | - | - |
| Candida albicans | 1 x 10^{6} CFU/mL | - | - | - |
*Concentration not available because these are inactivated viruses
^Nucleic acid was tested for Cytomegalovirus and Bordetella pertussis
The results of this study show that the modified Xpert® Flu Assay has 100% analytical specificity under the test conditions used. This is the same analytical specificity that was reported for the predicate.
f. Analytical reactivity:
All virus strains were tested at either the LoD or the lowest concentration where all three replicates generated appropriate positive results for Flu A, 2009 H1N1, or Flu B. All influenza viruses were diluted in a simulated background matrix of 2.5% porcine mucin and 1% human whole blood in 0.85% sodium chloride in 1X PBS solution with 15% glycerol diluted with UTM at a 1:6 ratio. All dilutions made for testing were prepared on the day of the test and kept on ice prior to testing. The analytical reactivity data for the modified Xpert® Flu Assay is acceptable. All virus strains were detected and the titers tested are appropriate for evaluating reactivity.
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Table 2: Summary Table of Analytical Inclusivity Results of the Xpert® Flu (Flu A Add) Assay
| Viral Strain (n=3) | Target | Concentration (TCID50/mL) | Flu A | 2009 H1N1 | Flu B |
| --- | --- | --- | --- | --- | --- |
| A/Denver/1/57 | Seasonal H1N1 | 250 | POS | NEG | NEG |
| A/Hawaii/15/2001 | Seasonal H1N1 | 50 | POS | NEG | NEG |
| A/Mal/302/54 | Seasonal H1N1 | 50 | POS | NEG | NEG |
| A/New Jersey/8/76 | Seasonal H1N1 | 250 | POS | NEG | NEG |
| A/NWS/33 | Seasonal H1N1 | 5 | POS | NEG | NEG |
| A/PR/8/34 | Seasonal H1N1 | 50 | POS | NEG | NEG |
| A/Taiwan/42/06 | Seasonal H1N1 | 50 | POS | NEG | NEG |
| A/Swine/1976/31 (Swine H1N1) | Seasonal H1N1 | 250 | POS | NEG | NEG |
| A/Swine/Iowa/15/30 (Swine H1N1) | Seasonal H1N1 | 50 | POS | NEG | NEG |
| A/Brisbane/59/07* | Seasonal H1N1 | 1 | POS | NEG | NEG |
| A/NewCaledonia/20/1999* | Seasonal H1N1 | 5 | POS | NEG | NEG |
| A/Brisbane/10/07* | Seasonal H3N2 | 1.25 | POS | NEG | NEG |
| A/Victoria/361/2011* | Seasonal H3N2 | 1.25 | POS | NEG | NEG |
| A/Victoria/3/75 | Seasonal H3N2 | 250 | POS | NEG | NEG |
| A/Aichi2/68 | Seasonal H3N2 | 50 | POS | NEG | NEG |
| A/Hong Kong/8/68 | Seasonal H3N2 | 50 | POS | NEG | NEG |
| A/NewYork/55/2004 | Seasonal H3N2 | 50 | POS | NEG | NEG |
| A/Port Chalmers/1/73 | Seasonal H3N2 | 50 | POS | NEG | NEG |
| A/Wisconsin/67/05* | Seasonal H3N2 | 10 | POS | NEG | NEG |
| A/Perth/16/2009 | Seasonal H3N2 | 50 | POS | NEG | NEG |
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| Viral Strain (n=3) | Target | Concentration (TCID50/mL) | Flu A | 2009 H1N1 | Flu B |
| --- | --- | --- | --- | --- | --- |
| A/SwineNY/01/2009* | 2009 H1N1 | 25 | POS | POS | NEG |
| A/SwineNY/02/2009* | 2009 H1N1 | 1.25 | POS | POS | NEG |
| A/SwineNY/03/2009* | 2009 H1N1 | 1.2505 | POS | POS | NEG |
| A/California/7/2009 | 2009 H1N1 | 5 | POS | POS | NEG |
| A/Canada/6294* | 2009 H1N1 | 50 | POS | POS | NEG |
| A/Wisconsin/629-S1 | 2009 H1N1 | 1 | POS | POS | NEG |
| A/Mallard/WI/34/75 | H5N2 | 3 pg/μL^^ | POS | NEG | NEG |
| A/Anhui/02/2005/PR8-IBCDC-RG5 | H5N1 | 0.122 pg/μL^^ | POS | NEG | NEG |
| A/chicken/NJ/15086-3/94 | H7N3 | 50 pg/μL^^ | POS | NEG | NEG |
| B/Allen/45 | Flu B | 50 | NEG | NEG | POS |
| B/Florida/02/06* | Flu B | 5 | NEG | NEG | POS |
| B/Florida/04/06 | Flu B | 50 | NEG | NEG | POS |
| B/Florida/07/04* | Flu B | 5 | NEG | NEG | POS |
| B/GL/1739/54 | Flu B | 50 | NEG | NEG | POS |
| B/Hong Kong/5/72 | Flu B | 50 | NEG | NEG | POS |
| B/Lee/40 | Flu B | 50 | NEG | NEG | POS |
| B/Malaysia/2506/04 | Flu B | 50 | NEG | NEG | POS |
| B/Maryland/1/59 | Flu B | 5 | NEG | NEG | POS |
| B/Panama/45/90 | Flu B | 250 | NEG | NEG | POS |
| B/Taiwan/2/62 | Flu B | 50 | NEG | NEG | POS |
*Strains (12) used in analytical LOD study (D15589) and tested at limit of detection
Concentration expressed in picograms/μL
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# g. Interfering Substances:
Ten potentially interfering substances were tested on the Xpert® Flu Assay platform to look for any significant changes in the reported Ct value. Each potentially interfering substance was added to either a negative control reaction, where change in sample processing control Ct was evaluated, or a simulated clinical sample with A/Victoria/361/2011 at a titer of 3.2x LoD, where a change in Flu A Ct was evaluated.
Samples containing blood or mucin and A/Victoria/361/2011 had significantly increased Ct value indicating an inhibitory effect of these two substances. This inhibition is addressed in the Limitations Section of the Package Insert.
Table 3: Effect of Substance on Mean SPC Cts Relative to UTM Control in Flu A Negative Samples
| Substance | Concentration Tested | n | Flu A A/Victoria/361/2011 Sample Processing Control Ct (mean) | §P-values |
| --- | --- | --- | --- | --- |
| UTM (Control) | 100% (w/v) | 8 | 28.0 | |
| Blood | 2% (v/v) | 8 | 27.9 | 1.000 |
| Mucin* | 2.5% (w/v) | 8 | 27.4 | 0.010 |
| Neo-Synephrine® Nasal Drops | 15% (v/v) | 8 | 27.7 | 0.466 |
| Anefrin Nasal Spray | 15% (v/v) | 8 | 28.0 | 1.000 |
| Zicam Nasal gel | 5% (v/v) | 8 | 28.0 | 1.000 |
| Saline Nasal Spray | 15% (v/v) of dose | 8 | 27.9 | 1.000 |
| Lozenges | 1.7 mg/mL menthol | 8 | 27.8 | 0.848 |
| Oseltamivir (TamiFlu) | 7.5 mg/mL | 8 | 27.8 | 0.691 |
| Mupirocin | 10 mg/mL | 8 | 28.2 | 0.520 |
| Tobramycin | 4.0μg/mL | 8 | 27.8 | 0.691 |
$^{*}\mathrm{p} < 0.05$ indicates a statistically significant difference; Ct value is 0.6 cycles earlier relative to UTM control
$\S$ Dunnett Simultaneous Test from one-way ANOVA, comparison with UTM control
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| Substance | Concentration Tested | n | Flu A A/Victoria/361/2011 Sample Processing Control Ct (mean) | §P-values |
| --- | --- | --- | --- | --- |
| UTM (Control) | 100% (w/v) | 8 | 30.6 | |
| Blood* | 2% (v/v) | 8 | 31.6 | 0.001 |
| Mucin* | 2.5% (w/v) | 8 | 31.6 | 0.002 |
| Neo-Synephrine® Nasal Drops | 15% (v/v) | 8 | 31.1 | 0.295 |
| Anefrin Nasal Spray | 15% (v/v) | 8 | 30.7 | 0.997 |
| Zicam Nasal gel | 5% (v/v) | 8 | 31.4 | 0.012 |
| Saline Nasal Spray | 15% (v/v) of dose | 8 | 31.0 | .0440 |
| Lozenges | 1.7 mg/mL menthol | 8 | 31.2 | 0.100 |
| Oseltamivir (TamiFlu) | 7.5 mg/mL | 8 | 31.1 | 0.349 |
| Mupirocin | 10 mg/mL | 8 | 31.1 | 0.248 |
| Tobramycin | 4.0μg/mL | 8 | 30.8 | 0.939 |
* p<0.05 indicates a statistically significant difference
§ Dunnett Simultaneous Test from one-way ANOVA, comparison with UTM control
h. Assay cut-off:
The addition of one primer (oligonucleotide) did not impact the Assay cut-off of this assay. This was confirmed in the clinical study for the modified Xpert® Flu Assay.
i. Validation of Internal Controls
Three internal controls were evaluated in testing with the clinical sample results from the Study Protocol 166. The Sample Processing Control (SPC) was reported as “INVALID” in 0.1% of the 689 total runs. A two-sample t-test indicates that false negative and true negatives occur at the same rate, suggesting that the SPC is functioning properly as an internal control.
The Probe Check Control (PCC) is designed to verify, reagent rehydration, PCR tube filling, probe integrity and dye stability. Eleven test results reported and “ERROR” due to PCC failure (1.6%). This is an adequate control and an acceptable level of error.
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The Maximum Pressure (Max PSI) detects fluidic movement within the cartridge and aborts any run that generates a pressure of over 120PSI which may result in failure of the cartridge integrity. There were no Max PSI errors reported during any runs.
The internal controls for the modified Xpert ® Flu Assay are appropriate and adequate to control for the various steps of sample processing, PCR reaction and detection.
## 2. Comparison studies:
a. Method comparison with predicate device:
The modified Xpert ® Flu Assay was compared to the currently cleared Xpert ® Flu Assay.
b. Matrix comparison:
The matrix comparison study was not needed since device modification did not affect the matrices.
## 3. Clinical studies:
a. Retrospective clinical study:
The modified Xpert ® Flu Assay was compared to the current Xpert ® Flu Assay. The modified assay detected more positive samples than the current assay, detecting not only additional A/Victoria/361-like viruses, but also A/Perth/16/2009-like viruses. The positive and negative percent agreement of based on testing 166 samples are listed in the table below. The modified Xpert ® Flu Assay with the additional A/Victoria/361 primer demonstrated an improved ability of the test to detect this virus. In addition to testing archived clinical samples, ten contrived specimens (5 NP swab and 5 NA/W background matrices) spiked at LoD with A/Victoria/361 were tested. These specimens were tested blinded as part of the clinical specimens and all were reported as Flu A Positive; 2009 H1N1 NOT Detected; Flu B negative.
Table 4: Positive and Negative Agreement of the Modified Xpert ® Flu Assay.
| Analyte | Sample Type | Positive Agreement | Negative Agreement |
| --- | --- | --- | --- |
| Influenza A | Archived NP Swab | 94.8% (95% CI: 90.4-97.6) | 100% (95% CI: 97.9-100) |
| Influenza A, 2009 H1N1 | Archived NP Swab | 98.5% (95% CI: 92.1-100) | 99.6% (95% CI: 98.1-100) |
| Influenza B | Archived NP Swab | 90.0% (95% CI: 85.0-92.0) | 100% (95% CI: 97.9-100) |
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| | | CI: 73.5-97.9) | 98.9-100) |
| --- | --- | --- | --- |
| Influenza A | Archived NA/W | 98.0% (95% CI: 94.2-99.6) | 100% (95% CI: 97.6-100) |
| Influenza A, 2009 H1N1 | Archived NA/W | 98.5% (95% CI: 92.0-100) | 99.1% (95% CI: 97.0-99.9) |
| Influenza B | Archived NA/W | 95.7% (95% CI: 78.1-99.9) | 100% (95% CI: 98.7-100) |
# 4. Clinical cut-off:
The clinical cut-off is reported as Ct value of 37 for the sample processing control and a Ct of 40 for Influenza A, Influenza A, 2009 H1N1, and Influenza B analytes.
# 5. Expected values/Reference range:
In the prospective Cepheid Xpert Flu clinical study there was a total of 639 samples collected. Of the 639 samples, 348 were collected from male subjects and 291 from female subjects. The age distribution among study subjects was: $59.3\%$ (379/639) $< 5$ years; $16.1\%$ (103/639) 6-21 years; $16.9\%$ (108/639) 22-59 years; and $7.7\%$ (49/639) $\geq 60$ years. A total of 342 prospective NA/W specimens were tested for influenza A, influenza A subtype 2009 H1N1 and influenza B by the Xpert Flu Assay and viral culture plus DFA. A total of 297 prospectively collected NP swab specimens were tested for influenza A, influenza A subtype 2009 H1N1 and influenza B by the Xpert Flu Assay and viral culture plus DFA. These samples were collected during the 2010 influenza season at three clinical laboratories in Australia from August through October of 2010 and from a U.S. clinical site from May to mid-August. The number and percentage of influenza A, influenza A subtype 2009 H1N1, and influenza B prospectively collected positive cases as determined positive by the Xpert Flu Assay are: For NA/W prospective samples influenza A is $2.6\%$ (9/342), influenza A subtype 2009 H1N1 is $2.3\%$ (8/342), and influenza B is $2.6\%$ (9/342). For NP swab prospective samples influenza A is $4.0\%$ (12/297), influenza A subtype 2009 H1N1 is $2.7\%$ (8/297), and influenza B is $2.7\%$ (8/297).
# N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
# O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
