The Cepheid Xpert Flu-RSV Xpress Assay, performed on the GeneXpert Xpress System, is an automated, multiplex realtime, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu+RSV Xpress Assay uses nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu+RSV Xpress Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors.

Negative results do not preclude influenza virus or respiratory syncytial 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 2014-2015 influenza season. When other novel 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.

The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens and to preserve and transport nasal aspirate/wash specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu Assay or the Xpert Flu/RSV XC Assay. The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu+RSV Xpress Assay.

Device Description

The Xpert Flu+RSV Xpress Assay is an automated in vitro diagnostic test for qualitative detection and differentiation of influenza A. influenza B, and respiratory syncytial virus (RSV). The assay is performed on the Cepheid GeneXpert Xpress System (GeneXpert Dx System, GX-I). The GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection of the target sequence in simple or complex samples using real-time PCR and reverse transcriptase PCR (RT-PCR) assays. The systems require the use of single-use disposable cartridges (the Xpert Flu+RSV Xpress 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+RSV Xpress Assay includes reagents for the detection and differentiation of influenza A, influenza B, and RSV viral RNA directly from nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge. 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 single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza B and RSV viral RNA in approximately 60 minutes. The GeneXpert Xpress System, comprised of the GeneXpert Dx System GX-I, has one module that is capable of performing separate sample preparation and real-time PCR and RT-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 I-CORE® thermocycler for performing realtime PCR and RT-PCR and detection.

Specimens are collected following the instructions for collecting NP swab specimens provided in Xpert Flu+RSV Xpress Assay package insert for influenza and RSV testing. The Cepheid Xpert Nasopharyngeal Sample Collection Kit (Cepheid catalog #SWAB/B-100) is required but not provided for use with the assay. The NP swab specimen is placed in the Xpert viral transport medium and sent to the GeneXpert® Xpress testing area for processing. When stored in the transport medium, the NP swab specimen is stable for up to 24 hours at 2-30 °C or up to seven days at 2-8 °C. When ready to test the specimen, the user briefly mixes the specimen by inverting the tube five times, transfers the eluted material to the sample chamber in the top of the disposable fluidic cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert Xpress instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of RNA. The results are automatically generated at the end of the process in a report that can be viewed and printed.

AI/ML Overview

1. Table of Acceptance Criteria and Reported Device Performance

Device Performance Metric	Acceptance Criteria (Implicit)	Reported Device Performance
Analytical Sensitivity (LoD)	Reproducibly distinguish negative samples with 95% confidence or at least 19/20 replicates positive at the lowest concentration.	Influenza A 2009 H1N1: A/California/7/2009: 0.3 TCID50/mL (20/20 positive); A/Florida/27/2011: 16.0 TCID50/mL (20/20 positive). Influenza A H3N2: A/Perth/16/2009: 0.3 TCID50/mL (20/20 positive); A/Victoria/361/2011: 0.8 TCID50/mL (20/20 positive). Influenza B: B/Massachusetts/2/2012: 0.5 TCID50/mL (20/20 positive); B/Wisconsin/01/2011: 0.6 TCID50/mL (20/20 positive). RSV A: RSV A/2/Australia/61: 1.2 TCID50/mL (20/20 positive); RSV A/Long/MD/56: 1.0 TCID50/mL (20/20 positive). RSV B: RSV B/Washington/18537/62: 1.8 TCID50/mL (20/20 positive); RSV B/9320/Massachusetts/77: 2.0 TCID50/mL (20/20 positive). Influenza A H7N9: A/Anhui/1/2013: 0.8 TCID50/mL (19/20 positive).
Analytical Specificity	100% specificity against common respiratory pathogens and potentially encountered organisms in the nasopharynx.	100% analytical specificity against a panel of 44 cultures (16 viral, 26 bacterial, 2 yeast strains). No false positives observed.
Analytical Reactivity	Detection of multiple strains of influenza A (H1N1, H3N2, avian), influenza B (Victoria and Yamagata lineages), and RSV (A and B subgroups) at levels near the LoD.	Successfully detected all 64 tested strains (54 influenza and 10 RSV) at levels near the analytical LoD. Results consistently showed Flu A POSITIVE, Flu B POSITIVE, or RSV POSITIVE as expected, with negative results for non-target analytes.
Potentially Interfering Substances	No assay interference in the presence of various substances (e.g., blood, nasal secretions, medications, vaccines) at tested concentrations.	No assay interference observed in the presence of 11 different potentially interfering substances (e.g., Albuterol Sulfate, Human Blood, Mucin, Mupirocin, Phenylephrine, Zanamivir, Tobramycin, FluMist, Fluticasone Propionate, saline nasal spray, various transport media). All positive and negative replicates were correctly identified. FluMist was correctly reported as Flu A POSITIVE; FLU B POSITIVE; RSV NEGATIVE.
Carry-Over Contamination	Single-use, self-contained cartridges prevent carry-over contamination in negative samples following very high positive samples.	All 40 positive samples correctly reported, and all 42 negative samples directly following very high positive samples were correctly reported as negative.
Fresh vs. Frozen Sample Equivalency	No statistically significant effect in performance between fresh and freeze-thawed samples for positive and negative specimens.	All positive and negative replicates were correctly identified after fresh testing, one freeze-thaw cycle, and two freeze-thaw cycles. No statistically significant effect on performance was observed.
Clinical Comparison (PPA/NPA)	Acceptable PPA and NPA relative to an FDA-cleared molecular comparator assay for influenza A, influenza B, and RSV.	Fresh NP Swab Specimens: - Flu A: PPA 100% (98.5-100), NPA 94.8% (93.7-95.7) - Flu B: PPA 100% (94.3-100), NPA 99.5% (99.1-99.8) - RSV: PPA 96.9% (92.3-99.1), NPA 99.6% (99.2-99.8) Pre-selected Frozen NP Swab Specimens: - Flu A: NPA 98.5% (96.1-99.6) (PPA not applicable as no positive collected) - Flu B: PPA 100% (96.4-100), NPA 98.7% (95.5-99.8) - RSV: PPA 97.6% (87.1-99.9), NPA 99.5% (97.5-100)
Reproducibility (Total Agreement)	High percentage of samples yielding expected results across sites, days, and operators, especially for positive samples.	Negative: 100% (88/88) Flu A-Low Pos: 98.9% (89/90) Flu A-Mod Pos: 100% (90/90) Flu B-Low Pos: 98.9% (89/90) Flu B-Mod Pos: 100% (90/90) RSV-Low Pos: 97.8% (87/89) RSV-Mod Pos: 100% (90/90) (Note: High Neg samples show lower agreement, consistent with near cutoff concentrations).
Reproducibility (Ct Variation)	Low variability (SD, CV) in Ct values across sites, days, and operators for detected targets.	SPC: Total Std Dev 1.5, Total CV 4.8% Flu A1 (Low Pos): Total Std Dev 1.6, Total CV 4.5% Flu A1 (Mod Pos): Total Std Dev 1.4, Total CV 4.2% Flu B (Low Pos): Total Std Dev 1.9, Total CV 5.9% Flu B (Mod Pos): Total Std Dev 1.0, Total CV 3.3% RSV (Low Pos): Total Std Dev 1.7, Total CV 5.0% RSV (Mod Pos): Total Std Dev 1.0, Total CV 3.0% (Similar values for corresponding High Neg samples).

2. Sample Size Used for the Test Set and Data Provenance

Clinical Comparison Study (Test Set):
- Total NP Swab Specimens: 2435
- Fresh, Prospectively Collected: 2176
- Pre-selected Frozen, Archived Specimens: 259
- Data Provenance: United States (12 institutions in the U.S.), retrospective for archived samples and prospective for fresh samples. The study was conducted during the 2014-2015 influenza season.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of those Experts

The document does not explicitly state the number of experts used to establish the ground truth for the test set or their specific qualifications (e.g., radiologist with 10 years of experience).

Instead, the ground truth for the clinical comparison study was established using a FDA-cleared molecular comparator assay. For discrepant results between the Xpert Flu+RSV Xpress Assay and the comparator assay, bidirectional sequencing was performed. While sequencing is a definitive molecular method, it's not described as an "expert" review in the traditional sense of a clinical expert.

4. Adjudication Method for the Test Set

Initial Comparison: Xpert Flu+RSV Xpress Assay results were compared to an FDA-cleared molecular comparator assay.
Discrepancy Resolution/Adjudication: For specimens where the Xpert Flu+RSV Xpress Assay and the comparator assay were discrepant, bidirectional sequencing was performed. The results from sequencing were provided for informational purposes only. This implies that while sequencing helped understand discrepancies, the primary comparison was with the FDA-cleared molecular assay.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly mentioned or described in the provided text.
The study focuses on the diagnostic performance of the device itself (algorithm only) against a comparator assay, and its reproducibility under different operator conditions, not on human reader performance with or without AI assistance.
The statement "The study results showed the Xpert Flu+RSV Xpress Assay is acceptable for its intended use with inexperienced lab users" refers to the device's performance when used by such users, not an improvement in human reader performance aided by AI.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, a standalone study (algorithm-only performance) was effectively done. The clinical comparison study evaluates the performance of the Xpert Flu+RSV Xpress Assay (the device/algorithm) against an FDA-cleared molecular comparator assay.
While operators load samples and initiate the test, the entire process of sample extraction, purification, amplification, and detection, and interpretation of the fluorescent signals to yield a positive/negative result, is automated by the device ("GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection..."). The output is a "report that can be viewed and printed," indicating an algorithm-generated result.

7. The Type of Ground Truth Used

Clinical Comparison Study: The primary ground truth was established by an FDA-cleared molecular comparator assay. For discordant results, bidirectional sequencing was used as an additional, more definitive form of molecular ground truth for informational purposes.
Non-Clinical Studies (Analytical Sensitivity, Specificity, Reactivity): The ground truth was based on known concentrations of purified viral cultures (TCID50/mL or pg/µL) or bacterial/yeast cultures (CFU/mL), a laboratory-defined ground truth.

8. The Sample Size for the Training Set

The document does not explicitly specify a sample size for a "training set." This type of in-vitro diagnostic device (PCR assay) typically relies on extensive analytical validation (LoD, specificity, inclusivity, interference) and clinical validation with prospectively and retrospectively collected samples, rather than a distinct "training set" in the machine learning sense. The device's underlying algorithm is based on established molecular biology principles (RT-PCR) calibrated during development, not specifically "trained" on a dataset in the way an AI model would be.

9. How the Ground Truth for the Training Set Was Established

As noted above, the concept of a clear "training set" with ground truth in the context of machine learning is not directly applicable here. For the development and establishment of the assay's performance characteristics, ground truth would have been established through:

Analytical studies: Using known concentrations and strains of target and non-target pathogens to define sensitivity (LoD), specificity, and inclusivity.
Internal validation/optimization: During the assay development phase, characterized clinical samples or contrived samples with confirmed presence/absence of targets via established reference methods (e.g., PCR, sequencing, culture) would have been used to optimize assay parameters (e.g., primer/probe design, cycle thresholds).

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

Cepheid Scott Campbell, PhD, MBA Executive Director, Clinical Affairs 904 Caribbean Drive Sunnyvale, CA 94089

December 3, 2015

Re: K151226

Trade/Device Name: Xpert® Flu+RSV Xpress, Xpert® Nasopharyngeal Sample Collection Kit, GeneXpert Xpress System (GX-I) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: II Product Code: OCC, OOI, JSM Dated: May 5, 2015 Received: May 8, 2015

Dear Dr. Campbell:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Uwe Scherf -S

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K151226

Device Name Xpert Flu+RSV Xpress

Xpert® Nasopharyngeal Sample Collection Kit

Indications for Use (Describe)

Negative results do not preclude influenza virus or respiratory syncytial 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 2014-2015 influenza season. When other novel influenza A viruses are emerging, performance characteristics may vary.

Type of Use (Select one or both, as applicable)
-------------------------------------------------

Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

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5.0 510(k) Summary

As required by 21 CFR Section 807.92(c).

Submitted by:	Cepheid904 Caribbean DriveSunnyvale, CA 90489Phone number: (847) 228-3299Fax number: (847) 593-0233
Contact:	Scott A. Campbell, PhD, MBA
Date of Preparation:	May 5, 2015
Device:
Trade name:	Xpert® Flu+RSV Xpress
Common name:	Xpert Flu+RSV Xpress Assay
Type of Test:	Automated, multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza A,influenza B, and respiratory syncytial virus.
Regulation number/Classification name/	866.3980/Respiratory viral panel multiplex nucleic acid assay/866.2570/Instrumentation for clinical multiplex test systems866.2390/Transport culture medium
Product code(s):	OCC, OOI, JSM
Classification	Class II
Advisory Panel	Microbiology (83)
Prescription Use	Yes
Predicate DevicesName(s):	1) For the detection and differentiation of influenza A,influenza B, and RSV viral RNA in nasopharyngeal swabspecimens:Cepheid Xpert Flu/RSV XC [510(k) #K142045]; and,2) For the Sample Collection Kits:

Cepheid Xpert Nasopharyngeal Sample Collection Kit [510(k) #K142045]

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Device Description:

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Device Intended Use:

Xpert Flu+RSV Xpress Assay:

The Cepheid Xpert® Flu+RSV Xpress Assay, performed on the GeneXpert® Xpress System, is an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu+RSV Xpress Assay uses nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu+RSV Xpress Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus in conjunction with clinical and epidemiological risk factors.

Negative results do not preclude influenza virus or respiratory syncytial 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 2014-2015 influenza season. When other novel 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Ancillary Collection Kits' Indications for Use (the expanded indication is shown in bold):

Xpert Nasopharyngeal Sample Collection Kit

Substantial Equivalence:

The Xpert Flu+RSV Xpress Assay is substantially equivalent to the Cepheid Xpert Flu/RSV XC Assay [510(k) #K142045]. The Xpert Flu+RSV Xpress Assay and the Xpert Flu/RSV XC Assay detect influenza A. influenza B. and RSV from NP swab specimens. Both assays utilize the same technology by determining the presence of the target organisms through realtime RT-PCR amplification and fluorogenic target-specific hybridization detection. A multi

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center clinical study was conducted to determine the performance characteristics of the device with collection of NP swab specimens from each subject relative to a comparator device, the ProFlu+ Assay which is FDA cleared for NP swab specimens. Discordant results between the Xpert Flu+RSV Xpress Assay and the ProFlu+ Assay were analyzed by sequencing using primers different from those used in the Xpert Flu-RSV Xpress Assay. The study results showed the Xpert Flu+RSV Xpress Assay is acceptable for its intended use with inexperienced lab users.

Table 5-1 shows the similarities and differences between the Xpert Flu+RSV Xpress Assay and the predicate assay, Xpert Flu/RSV XC Assay.

Similarities
Item	Device	Predicate Device
	Cepheid Xpert Flu+RSVXpress Assay	Cepheid Xpert Flu/RSV XCAssay
510(k) Number	K151226	K142045
Regulation	866.3980	866.3980
Product Code	OCC, OOI	OCC, OOI
Device Class	Same	II
Technology Principleof Operation	Same	Multiplex real time RT-PCR
Similarities
Item	Device	Predicate Device
	Cepheid Xpert Flu+RSVXpress Assay	Cepheid Xpert Flu/RSV XCAssay
Intended Use	The Cepheid Xpert Flu+RSVXpress Assay, performed onthe GeneXpert Xpress System,is an automated, multiplex real-time, reverse transcriptasepolymerase chain reaction (RT-PCR) assay intended for the invitro qualitative detection anddifferentiation of influenza A,influenza B, and respiratorysyncytial virus (RSV) viralRNA. The Xpert Flu+RSVXpress Assay usesnasopharyngeal swab specimenscollected from patients withsigns and symptoms ofrespiratory infection. The XpertFlu+RSV Xpress Assay isintended as an aid in thediagnosis of influenza andrespiratory syncytial virus inconjunction with clinical andepidemiological risk factors.	The Cepheid Xpert Flu/RSV XCAssay is an automated,multiplex real-time, reversetranscriptase polymerase chainreaction (RT-PCR assayintended for the in vitroqualitative detection anddifferentiation of influenza A,influenza B, and respiratorysyncytial virus (RSV) viralRNA. The Xpert Flu/RSV XCAssay uses nasopharyngealswab and nasal aspirate/washspecimens collected frompatients with signs andsymptoms of respiratoryinfection in conjunction withclinical and epidemiological riskfactors. The Xpert Flu/RSV XCAssay is intended as an aid inthe diagnosis of influenza andrespiratory syncytial virus.
	Negative results do not precludeinfluenza virus or respiratorysyncytial virus infection andshould not be used as the solebasis for treatment or otherpatient management decisions.Performance characteristics forinfluenza A were establishedduring the 2014-2015 influenzaseason. When other novelinfluenza A viruses areemerging, performancecharacteristics may vary.	Negative results do not precludeinfluenza virus or respiratorysyncytial virus infection andshould not be used as the solebasis for treatment or otherpatient management decisions.Performance characteristics forinfluenza A were establishedduring the 2013-2014 influenzaseason. When other novelinfluenza A viruses areemerging, performancecharacteristics may vary.
Similarities
Item	Device	Predicate Device
	Cepheid Xpert Flu+RSVXpress Assay	Cepheid Xpert Flu/RSV XCAssay
Intended Use	If infection with a novelinfluenza A virus is suspectedbased on current clinical andepidemiological screeningcriteria recommended by publichealth authorities, specimensshould be collected withappropriate infection controlprecautions for novel virulentinfluenza viruses and sent tostate or local health departmentfor testing. Viral culture shouldnot be attempted in these casesunless a BSL 3+ facility isavailable to receive and culturespecimens.	If infection with a novelinfluenza A virus is suspectedbased on current clinical andepidemiological screeningcriteria recommended by publichealth authorities, specimensshould be collected withappropriate infection controlprecautions for novel virulentinfluenza viruses and sent tostate or local health departmentfor testing. Viral culture shouldnot be attempted in these casesunless a BSL 3+ facility isavailable to receive and culturespecimens.
Indication for Use	Same	Patients with signs andsymptoms of respiratoryinfection in conjunction withclinical and epidemiological riskfactors.
Assay Targets	Same	Influenza A Virus, Influenza BVirus, and RSV viral RNA
Specimen Types	Nasopharyngeal (NP) swabspecimens	Nasopharyngeal (NP) swabspecimens andNasal aspirate/wash (NA/W)specimens
Nucleic AcidExtraction	Yes	Yes
Extraction Methods	Sample preparation integrated inGeneXpert Cartridge andGeneXpert Xpress System	Sample preparation integrated inGeneXpert Cartridge andGeneXpert Instrument System
Assay Results	Same	Qualitative
Instrument System	Cepheid GeneXpert XpressSystem (instrument modelGX-I); same Cepheid I-coretechnology	Cepheid GeneXpert InstrumentSystems (various instrumentmodels including instrumentmodel GX-I); Cepheid I-coretechnology
Similarities
Item	Device	Predicate Device
	Cepheid Xpert Flu+RSVXpress Assay	Cepheid Xpert Flu/RSV XCAssay
Assay Controls	Same	Encapsulated (armored) RNApseudovirus as a sampleprocessing control.Available but not provided areinactivated virus controls forinfluenza A/B and RSV asexternal positive controls, andCoxsackie virus as an externalnegative control.
Time to obtain testresults	Approximately 60 minutes forsample preparation and real-time RT-PCR.	Approximately 60 minutes orless for sample preparation andreal-time RT-PCR.
Primers and probes	Same	Primers and probes to detect thepresence of nucleic acidsequences of influenza A,influenza B, and RSV.

Table 5-1: Comparison of Similarities and Differences of the Xpert Flu+RSV Xpress Assay with the Predicate Device

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Primary Differences(Differences are also Captured in the Similarities Table above)
	New Device	Predicate Device
Item	Cepheid Xpert Flu+RSVXpress Assay	Cepheid Xpert Flu/RSV XCAssay
Instrument System	Cepheid GeneXpert XpressSystem	Cepheid GeneXpert DxSystems and GeneXpertInfinity Systems
Laboratory Users	Untrained operators with noclinical lab experience in aCLIA-waiver environment.	Operators with no clinical labexperience to experiencedclinical laboratorytechnologists.
Combinatorial AssaySelections	No combinatorial assayselections are available.	Yes, user may select combinedassay with all targets or a Fluonly assay or a RSV onlyassay.
Early assaytermination function	No early assay terminationfunction is available.	Yes, on Flu only or RSV onlyassay selections.

The Xpert Flu+RSV Xpress Assay has the same general intended use as the predicate device and has the same technological characteristics as the predicate device. The differences between the Xpert Flu+RSV Xpress Assay and the predicate device do not raise different questions of safety and effectiveness. The clinical study demonstrates that the Xpert Flu+RSV Xpress Assay is acceptable for its intended use with inexperienced laboratory users and is substantially equivalent to the predicate device described above.

Xpert Nasopharyngeal Sample Collection Kit

The predicate device for the ancillary specimen collection kit, the Xpert® Nasopharyngeal Sample Collection Kit, is the Cepheid Nasopharyngeal Sample Collection Kit, [510(k) # K042970]. The similarities and differences are shown in Table 5-2.

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Sample Collection Kit with the Predicate DeviceSimilarities
	Device	Predicate
Item	Xpert Nasopharyngeal SampleCollection Kit	Xpert Nasopharyngeal SampleCollection Kit
Intended Use(Similarities)	For collection, preservationand transport ofnasopharyngeal swabspecimens and to preserve andtransport nasal aspirate/washspecimens containing virusesfrom patients with signs andsymptoms of respiratoryinfection prior to analysis withthe Xpert Flu Assay and theXpert Flu/RSV XC Assay.For collection, preservation andtransport of nasopharyngealswab specimens containingviruses from patients with signsand symptoms of respiratoryinfection prior to analysis withthe Xpert Flu+RSV XpressAssay.	For collection, preservationand transport ofnasopharyngeal swabspecimens and to preserve andtransport nasal aspirate/washspecimens containing virusesfrom patients with signs andsymptoms of respiratoryinfection prior to analysis withthe Xpert Flu Assay and theXpert Flu/RSV XC Assay.
Single-use Device	Yes	Yes
Medium Formulation	Same	Hank's Balanced Salt SolutionBovine Serum AlbuminL-cysteineGelatinSucroseL-glutamic acidHEPES bufferVancomycinAmphotericin BColistinPhenol red
pH	Same	$7.3 \pm 0.2$

Table 5-2: Comparison of Similarities and Differences of the Xpert Nasopharyngeal
Sample Collection Kit with the Predicate Device

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Similarities
	Device	Predicate
Item	Xpert Nasopharyngeal SampleCollection Kit	Xpert Nasopharyngeal SampleCollection Kit
Storage Temperature	Same	2 - 25°C (refrigeratedand room temperature)
Volume	Same	3 ml
Glass Beads	Same	3 x 3 mm
Container	Same	Plastic (medical-gradepolypropylene)
Product Configuration	Same	Medium Tube in Kit withindividually-wrapped sterileswab.
Differences
Item	Device	Predicate
Intended Use(differences)	For collection, preservation andtransport of nasopharyngealswab specimens containingviruses from patients with signsand symptoms of respiratoryinfection prior to analysis withthe Xpert Flu+RSV XpressAssay.	For collection, preservation andtransport of nasopharyngealswab specimens and to preserveand transport nasalaspirate/wash specimenscontaining viruses from patientswith signs and symptoms ofrespiratory infection prior toanalysis with the Xpert FluAssay and the Xpert Flu/RSVXC Assay.

Both devices have the same general intended use and use the same technology to collect, store, and transport clinical specimens, including viruses, to the laboratory for further testing. The differences between the two devices do not raise new or different questions of safety and effectiveness. The multi-center clinical study of the Xpert Flu+RSV Xpress Assay was conducted using Xpert Nasopharyngeal Sample Collection Kit and demonstrated that the assay and its collection kit are acceptable for their intended use with inexperienced lab users and substantially equivalent to the predicate device.

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Non-Clinical Studies:

Analytical Sensitivity (Limit of Detection)

Studies were performed to determine the analytical limit of detection (LoD) of the Xpert Flu+RSV Xpress Assay with two lots of reagents across three testing days. The higher LoD observed per strain and per lot was selected for verification. Verification of the estimated LoD claim was performed on one reagent lot across a minimum of three testing days. LoD was established using two influenza A H3N2 strains, two influenza A 2009 H1N1 strains, two influenza B strains, two respiratory syncytial virus A (RSV A) strains, two respiratory syncytial virus B (RSV B) strains, and one influenza A H7N9 strain diluted into a negative pooled clinical matrix. The LoD is defined as the lowest concentration (tissue culture infective dose, TCID50/mL) per sample that can be reproducibly distinguished from negative samples with 95% confidence or the lowest concentration at which 19 of 20 replicates were positive. Each strain was tested in replicates of 20 per concentration of virus.

The LoD was determined empirically as the first concentration that had 19/20 or 20/20 positive results. The LoD point estimates for each strain tested are summarized in Tables 5-3 to 5-8.

Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
Influenza A/California/7/2009	0.3 (20/20)
Influenza A/Florida/27/2011	16.0 (20/20)

Table 5-3: Confirmed LoD (TCID50/mL): Influenza A 2009 H1N1

Table 5-4: Confirmed LoD (TCID50/mL): Influenza A H3N2

Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
Influenza A/Perth/16/2009	0.3 (20/20)
Influenza A/Victoria/361/2011	0.8 (20/20)

Table 5.5: Confirmed LoD (TCID50/mL): Influenza B

Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
Influenza B/Massachusetts/2/2012	0.5 (20/20)
Influenza B/Wisconsin/01/2011	0.6 (20/20)

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Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
RSV A/2/Australia/61	1.2 (20/20)
RSV A/Long/MD/56	1.0 (20/20)

Table 5-6: Confirmed LoD (TCID50/mL): Respiratory Syncytial Virus A

Table 5-7: Confirmed LoD (TCID50/mL): Respiratory Syncytial Virus B

Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
RSV B/Washington/18537/62	1.8 (20/20)
RSV B/9320/Massachusetts/77	2.0 (20/20)

Table 5-8: Confirmed LoD (TCID50/mL): Influenza A H7N9

Strain ID	Confirmed LoD(TCID50/mL)(at least 19/20 positive)
Influenza A/Anhui/1/2013	0.8 (19/20)

Although this test has been shown to detect the novel avian influenza A (H7N9) cultured material, the performance characteristics of this device with clinical specimens that are positive for the novel avian influenza A (H7N9) virus have not been established. The Xpert Flu+RSV Xpress Assay can distinguish between influenza A and B viruses, but it cannot differentiate influenza A subtypes.

Analytical Specificity (Exclusivity)

The analytical specificity of the Xpert Flu+RSV Xpress Assay was evaluated by testing a panel of 44 cultures consisting of 16 viral, 26 bacterial, and two yeast strains representing common respiratory pathogens or those potentially encountered in the nasopharynx. Three replicates of all bacterial and yeast strains were tested at concentrations of ≥ 100 CFU/mL with the exception of one strain which was tested at 10' CFU/mL (Chlamydia pneumoniae). Three replicates of each virus were tested at concentrations of ≥ 105 TCID50/mL. The analytical specificity was 100%. Results are shown in Table 5-9.

Table 5-9: Analytical Specificity of Xpert Flu+RSV Xpress Assay

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Organism	Concentration	Influenza A	Influenza B	RSV
No Template Control		NEG	NEG	NEG
Adenovirus Type 1	1.12x107 TCID50/mL	NEG	NEG	NEG
Adenovirus Type 7	1.87x105 TCID50/mL	NEG	NEG	NEG
Human coronavirus OC43	2.85x105 TCID50/mL	NEG	NEG	NEG
Human coronavirus 229E	1x105 TCID50/mL	NEG	NEG	NEG
Cytomegalovirus	7.24x105 TCID50/mL	NEG	NEG	NEG
Echovirus	3.31x107 TCID50/mL	NEG	NEG	NEG
Enterovirus	1x105 TCID50/mL	NEG	NEG	NEG
Epstein Barr Virus	7.16x107 TCID50/mL	NEG	NEG	NEG
HSV	8.9x106 TCID50/mL	NEG	NEG	NEG
Measles	6.3x105 TCID50/mL	NEG	NEG	NEG
Human metapneumovirus	3.8x105 TCID50/mL	NEG	NEG	NEG
Mumps virus	6.31x106 TCID50/mL	NEG	NEG	NEG
Human parainfluenza Type 1	1.15x106 TCID50/mL	NEG	NEG	NEG
Human parainfluenza Type 2	1x105 TCID50/mL	NEG	NEG	NEG
Human parainfluenza Type 3	3.55x107 TCID50/mL	NEG	NEG	NEG
Rhinovirus Type 1A	1.26x105 TCID50/mL	NEG	NEG	NEG
Acinetobacter baumannii	>1x106 CFU/mL	NEGa	NEG	NEG
Burkholderia cepacia	>1x106 CFU/mL	NEG	NEG	NEG
Candida albicans	>1x106 CFU/mL	NEG	NEG	NEG
Candida parapsilosis	>1x106 CFU/mL	NEG	NEG	NEG
Bordetella pertussis	1x108 CFU/mL	NEG	NEG	NEG
Chlamydia pneumoniae	3.16x105 CFU/mL	NEG	NEG	NEG
Organism	Concentration	Influenza A	Influenza B	RSV
Citrobacter freundii	>1x106 CFU/mL	NEG	NEG	NEG
Corynebacterium sp.	>1x106 CFU/mL	NEG	NEG	NEG
Escherichia coli	>1x106 CFU/mL	NEG	NEG	NEG
Enterococcus faecalis	>1x106 CFU/mL	NEG	NEG	NEG
Haemophilus influenzae	1x106 CFU/mL	NEG	NEG	NEG
Lactobacillus reuter	1x106 CFU/mL	NEG	NEG	NEG
Legionella spp.	1x108 CFU/mL	NEG	NEG	NEG
Moraxella catarrhalis	>1x106 CFU/mL	NEG	NEG	NEG
Mycobacterium tuberculosis(avirulent)	1.15x106 CFU/mL	NEG	NEG	NEG
Mycoplasma pneumoniae	1x107 CFU/mL	NEG	NEG	NEG
Neisseria meningitidis	>1x106 CFU/mL	NEG	NEG	NEG
Neisseria mucosa	>1x106 CFU/mL	NEG	NEG	NEG
Propionibacterium acnes	>1x106 CFU/mL	NEG	NEG	NEG
Pseudomonas aeruginosa	>1x106 CFU/mL	NEG	NEG	NEG
Staphylococcus aureus	>1x106 CFU/mL	NEG	NEG	NEG
Staphylococcus epidermidis	>1x106 CFU/mL	NEG	NEG	NEG
Staphylococcus haemolyticus	>1x106 CFU/mL	NEG	NEG	NEG
Streptococcus agalactiae	>1x106 CFU/mL	NEG	NEG	NEG
Streptococcus pneumoniae	>1x106 CFU/mL	NEG	NEG	NEG
Streptococcus pyogenes	>1x106 CFU/mL	NEG	NEG	NEG
Streptococcus salivarius	>1x106 CFU/mL	NEG	NEG	NEG
Streptococcus sanguinis	>1x106 CFU/mL	NEG	NEG	NEG

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Analytical Reactivity (Inclusivity)

The analytical reactivity of the Xpert Flu+RSV Xpress Assay was evaluated against multiple strains of influenza A H1N1 (seasonal pre-2009), influenza A H1N1 (pandemic 2009), influenza A H3N2 (seasonal), avian influenza A (H5N1, H5N2, H6N2, H7N2, H7N3, H2N2, H7N9, and H9N2), influenza B (representing strains from both Victoria and

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Yamagata lineages), and respiratory syncytial virus subgroups A and B (RSV A and RSV B) at levels near the analytical LoD. A total of 64 strains including 54 influenza viruses and 10 RSV strains were tested in this study with the Xpert Flu+RSV Xpress Assay.

Three replicates were tested for each strain. Results are shown in Table 5-10.

Virus	Strain	Concentration	Result
			Flu A	Flu B	RSV
No Template Control			NEG	NEG	NEG
Influenza AH1N1 (pre-2009)	A/swine/Iowa/15/30	32.0 TCID50/mL	POS	NEG	NEG
	A/WS/33	32.0 TCID50/mL	POS	NEG	NEG
	A/PR/8/34	32.0 TCID50/mL	POS	NEG	NEG
	A/Mal/302/54	32.0 TCID50/mL	POS	NEG	NEG
	A/Denver/1/57	32.0 TCID50/mL	POS	NEG	NEG
	A/New Jersey/8/76	32.0 TCID50/mL	POS	NEG	NEG
	A/New Caledonia/20/1999	32.0 TCID50/mL	POS	NEG	NEG
	A/New York/55/2004	32.0 TCID50/mL	POS	NEG	NEG
	A/Soloman Islands/3/2006	32.0 TCID50/mL	POS	NEG	NEG
	A/Taiwan/42/06	32.0 TCID50/mL	POS	NEG	NEG
	A/Brisbane/59/2007	32.0 TCID50/mL	POS	NEG	NEG
	A/California/7/2009	32.0 TCID50/mL	POS	NEG	NEG
Influenza AH1N1(pdm2009)	A/swine/NY/02/2009	32.0 TCID50/mL	POS	NEG	NEG
	A/Florida/27/2011	32.0 TCID50/mL	POS	NEG	NEG
	A/Colorado/14/2012	32.0 TCID50/mL	POS	NEG	NEG
	A/Washington/24/2012	80.0ª TCID50/mL	POS	NEG	NEG

	A/Aichi/2/68	1.6 TCID50/mL	POS	NEG	NEG
	A/HongKong/8/68	1.6 TCID50/mL	POS	NEG	NEG
	A/Port Chalmers/1/73	1.6 TCID50/mL	POS	NEG	NEG
	A/Hawaii/15/2001	1.6 TCID50/mL	POS	NEG	NEG
Influenza AH3N2(Seasonal)	A/Wisconsin/67/05	1.6 TCID50/mL	POS	NEG	NEG
	A/Brisbane/10/2007	1.6 TCID50/mL	POS	NEG	NEG
	A/Perth/16/2009	1.6 TCID50/mL	POS	NEG	NEG
	A/Minnesota/11/2010 (H3N2)v	1.6 TCID50/mL	POS	NEG	NEG
	A/Indiana/08/2011 (H3N2)v	1.6 TCID50/mL	POS	NEG	NEG
	A/Victoria/361/2011	1.6 TCID50/mL	POS	NEG	NEG
	A/Texas/50/2012	1.6 TCID50/mL	POS	NEG	NEG
Virus	Strain	Concentration	Result
			Flu A	Flu B	RSV
	A/duck/Hunan/795/2002 (H5N1)	≤ 1pg/μLb	POS	NEG	NEG
	A/chicken/Hubei/327/2004 (H5N1)	≤ 1pg/μLb	POS	NEG	NEG
	A/Anhui/01/2005 (H5N1)	≤ 1pg/μLb	POS	NEG	NEG
	A/Japanese whiteeye/HongKong/1038/2006 (H5N1)	≤ 1pg/μLb	POS	NEG	NEG
	A/mallard/WI/34/75 (H5N2)	≤ 1pg/μLb	POS	NEG	NEG
	A/chicken/CA431/00 (H6N2)	≤ 1pg/μLb	POS	NEG	NEG
Avianinfluenza A	A/duck/LTC-10-82743/1943 (H7N2)	≤ 1pg/μLb	POS	NEG	NEG
	A/chicken/NJ/15086-3/94 (H7N3)	≤ 1pg/μLb	POS	NEG	NEG
	A/Anhui/1/2013 (H7N9)	N/Ac	POS	NEG	NEG
	A/Shanghai/1/2013 (H7N9)	N/Ac	POS	NEG	NEG
	A/chicken/Korea/38349-p96323/ 1996(H9N2)	≤ 1pg/μLb	POS	NEG	NEG
	A/mallard/NY/6750/78 (H2N2)	≤ 1pg/μLb	POS	NEG	NEG
	B/Lee/40	1.2 TCID50/mL	NEG	POS	NEG
	B/Allen/45	1.2 TCID50/mL	NEG	POS	NEG
	B/GL/1739/54	1.2 TCID50/mL	NEG	POS	NEG
	B/Maryland/1/59	1.2 TCID50/mL	NEG	POS	NEG
	B/Panama/45/90d	3.0 TCID50/mLe	NEG	POS	NEG
	B/Florida/07/2004f	1.2 TCID50/mL	NEG	POS	NEG
	B/Florida/02/06d	1.2 TCID50/mL	NEG	POS	NEG
Influenza B	B/Florida/04/06	1.2 TCID50/mL	NEG	POS	NEG
	B/Wisconsin/01/2011d	1.2 TCID50/mL	NEG	POS	NEG
	B/Massachusetts/2/2012f	1.2 TCID50/mL	NEG	POS	NEG
	B/Hong Kong/5/72	1.2 TCID50/mL	NEG	POS	NEG
	B/Wisconsin/01/2010f	1.2 TCID50/mL	NEG	POS	NEG
	B/Malaysia/2506/04d	1.2 TCID50/mL	NEG	POS	NEG
	B/Taiwan/2/62	1.2 TCID50/mL	NEG	POS	NEG
	B/Brisbane/60/2008d	1.2 TCID50/mL	NEG	POS	NEG
RSV A	RSV-A/Long/MD/56	2.4 TCID50/mL	NEG	NEG	POS
Virus	Strain	Concentration	Result
			Flu A	Flu B	RSV
	RSV-A/2/Australia/61	2.4 TCID50/mL	NEG	NEG	POS
	RSV-A/NY (Clinical unknown)	2.4 TCID50/mL	NEG	NEG	POS
	RSV-A/WI/629-8-2/2007	2.4 TCID50/mL	NEG	NEG	POS
	RSV-A/WI/629-11-1/2008	2.4 TCID50/mL	NEG	NEG	POS
RSV B	RSV-B/Wash/18537/62	4.0 TCID50/mL	NEG	NEG	POS
	RSV-B/9320/MA/77	4.0 TCID50/mL	NEG	NEG	POS
	RSV-B/WV14617/85	4.0 TCID50/mL	NEG	NEG	POS
	RSV-B/CH93(18)-18	20.0 TCID50/mLg	NEG	NEG	POS
	RSV-B/WI/629-5B/0607	4.0 TCID50/mL	NEG	NEG	POS

Table 5-10: Analytical Reactivity (Inclusivity) of Xpert Flu+RSV Xpress Assay

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{19}------------------------------------------------

"Influenza A/Washington/24/2012 was tested at 5X LoD (80.0 TCID56/mL) to obtain 3 of 3 Flu A POSITIVE result calls.

"Purified viral RNA in simulated background matrix was used for avian influenza A viruses due to biosafety regulations.

Shactivated avian influenza A (H7N9) viruses without viral titer was diluted 100,000 fold in simulated background matrix and tested due to biosafety regulations.

4Known Victoria lineage.

SInfluenza B/Panama/45/90 was tested at 5X LoD (3.0 TCID30mL) to obtain 3/3 Flu B POSITIVE result calls. 4Known Yamagata lineage.

6RSV-B/CH93(18)-18 was tested at 10X LoD (20.0 TCID50/mL) to obtain 3/3 RSV POSITIVE result calls.

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Potentially Interfering Substances

In a non-clinical study, potentially interfering substances that may be present in the nasopharynx were evaluated directly relative to the performance of the Xpert Flu+RSV Xpress Assay. Potentially interfering substances in the nasopharynx may include, but are not limited to: blood, nasal secretions or mucus, and nasal and throat medications used to relieve congestion, nasal dryness, irritation, or asthma and allergy symptoms, as well as antibiotics and antivirals. Negative samples (n = 8) were tested per each substance to determine the effect on the performance of the sample processing control (SPC). Positive samples (n = 8) were tested per substance with six influenza (four influenza A and two influenza B) and four RSV (two RSV A and two RSV B) strains spiked at 2X the analytical LoD determined for each strain. All results were compared to positive and negative Universal Transport Medium (UTM) controls.

These evaluated substances are listed in Table 5.11 with active ingredients and concentrations tested shown. There was no assay interference in the presence of the substances at the concentrations tested in this study. All positive and negative replicates were correctly identified using the Xpert Flu+RSV Xpress Assay.

FluMist vaccine samples were correctly reported as Flu A POSITIVE; FLU B POSITIVE; RSV NEGATIVE as expected. Samples containing FluMist may cause false positive results. This is addressed in the device labeling Section 17. Limitations.

Substance/Class	Description/Active Ingredient	Concentration Tested
Beta-adrenergicbronchodilator	Albuterol Sulfate	0.83 mg/mL(equivalent to1 dose per day)
Blood	Blood (Human)	2% (v/v)
BDTM UniversalViral TransportSystem	Transport Media	100% (v/v)
Remel M4	Transport Media	100% (v/v)
Remel M4RT	Transport Media	100% (v/v)
Remel M5®	Transport Media	100% (v/v)
Throat lozenges,oral anestheticand analgesic	Benzocaine, Menthol	1.7 mg/mL
Mucin	Purified Mucin protein(Bovine or porcinesubmaxillary gland)	2.5% (w/v)
Antibiotic, nasalointment	Mupirocin	10 mg/mL
Saline NasalSpray	Sodium Chloride (0.65%)	15% (v/v)
Anefrin Nasal	Oxymetazoline, 0.05%	15% (v/v)

Table 5-11. Potentially Interfering Substances in Xpert Flu+RSV Xpress Assay

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Substance/Class	Description/ActiveIngredient	ConcentrationTested
Spray
Nasal Drops	Phenylephrine, 0.5%	15% (v/v)
Tamiflu®/Anti-viral drugs	Zanamivir	7.5 mg/mL
Antibacterial,systemic	Tobramycin	4 µg/mL
Zicam®/NasalGel	Luffa opperculata,Galphimia glauca,Histaminumhydrochloricum Sulfur	15% (w/v)
FluMist®	Live intranasal influenzavirus vaccine	6.7% (v/v)
Nasalcorticosteroid	Fluticasone Propionate	5 µg/mL

Carry-Over Contamination

A study was conducted to demonstrate that single-use, self-contained GeneXpert cartridges prevent carry-over contamination in negative samples run following very high positive samples in the same GeneXpert module. The study consisted of a negative sample processed in the same GeneXpert module immediately following a very high influenza A sample (approximately 106 TCID5(/test) or a very high RSV A sample (approximately 100 TCID50/test). This testing scheme was repeated 20 times on two GeneXpert modules for a total of 82 runs resulting in 40 positive and 42 negative specimens for each virus type. All 40 positive samples were correctly reported as Flu A POSITIVE; Flu B NEGATIVE; RSV NEGATIVE or Flu A NEGATIVE; Flu B NEGATIVE; RSV POSITIVE. All 42 negative samples were correctly reported as Flu A NEGATIVE; Flu B NEGATIVE; RSV NEGATIVE.

Fresh vs. Frozen Sample Equivalency Study

Fresh and frozen specimen equivalency in the Xpert Flu+RSV Xpress Assay was evaluated by testing individual influenza and RSV strains at three different concentrations representing low positives (2X LoD), moderate positives (5X LoD), and high positives (10X LoD) in simulated background matrix. Negative samples consisted of simulated background matrix only. Fresh and frozen specimen equivalency was determined using one seasonal Flu A H3N2 strain (A/Victoria/361/2011), one Flu B strain (B/Wisconsin/01/11), one RSV A strain (RSV A/Long/MD/56), and one RSV B strain (RSV B/9320/MA/77). Replicates of 20 were tested for each specimen type and concentration. All positive and negative specimens were tested fresh, after one freeze-thaw cycle, and after two freeze-thaw cycles.

There was no statistically significant effect in the performance of the Xpert Flu+RSV Xpress Assay between fresh virus dilutions and two sequential freeze thaw cycles for positive and negative samples. All positive and negative replicates were correctly identified using the Xpert Flu+RSV Xpress Assay.

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Linearity

Not applicable, the Xpert Flu+RSV Xpress Assay is a qualitative assay.

Clinical Studies

Clinical Comparison Study

Performance characteristics of the Xpert Flu+RSV Xpress Assay were evaluated at 12 institutions in the U.S. during the 2014-2015 influenza season. Due to the low prevalence of Flu B and RSV during the sampling time frame of this study, supplementation with preselected archived NP swab specimens known to be positive for Flu B or RSV was required to meet the sample size for these targets.

Subjects included individuals with signs or symptoms of respiratory infection and whose routine care called for collection of NP swab specimens for influenza and/or RSV testing. For eligible subjects, NP swab specimens were obtained for testing with the Xpert Flu+RSV Xpress Assay and the comparator assay, and patient management continued at the site per their standard practice.

The Xpert Flu+RSV Xpress Assay performance was compared to a FDA-cleared molecular comparator assay. Xpert Flu+RSV Xpress Assay results from NP swab specimens were compared to the molecular comparator assay result from the same swab specimens. Bidirectional sequencing was performed on specimens where the Xpert Flu-RSV Xpress Assav and the comparator assay were discrepant, and is provided for informational purposes only.

NP Swab Specimens

A total of 2435 NP swab specimens were tested for influenza A, influenza B and RSV by the Xpert Flu+RSV Xpress Assay and the comparator assay. Of the 2435 NP swab specimens 2176 were fresh, prospectively collected and 259 were pre-selected frozen, archived specimens.

On fresh, prospectively collected NP swab specimens, the Xpert Flu+RSV Xpress Assay demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) for detection of influenza A of 100% and 94.8%, respectively, relative to the comparator assay (Table 5-12). The Xpert Flu+RSV Xpress Assay PPA and NPA for influenza B were 100% and 99.5%, respectively (Table 5-12). The Xpert Flu+RSV Xpress Assay PPA and NPA for RSV were 96.9% and 99.6%, respectively (Table 5-12).

On pre-selected frozen, archived NP swab specimens, the Xpert Flu+RSV Xpress Assay demonstrated an NPA for detection of influenza A of 98.5% relative to the comparator assay (Table 5-12). The Xpert Flu+RSV Xpress Assay PPA and NPA for influenza B were 100% and 98.7%, respectively (Table 5-12). The Xpert Flu+RSV Xpress Assay PPA and NPA for RSV were 97.6% and 99.5%, respectively (Table 5-12).

SpecimenType	Target	n	TP	FP	TN	FN	PPA %(95 CI)	NPA %(95 CI)
Fresh	Flu A	2176	250	101a	1825	0	100(98.5-100)	94.8(93.7-95.7)

Table 5-12: Xpert Flu+RSV Xpress Assay Performance on NP Swab Specimens

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	Flu B	2176	63	10b	2103	0	100(94.3-100)	99.5(99.1-99.8)
	RSV	2176	125	8c	2039	4d	96.9(92.3-99.1)	99.6(99.2-99.8)
Pre-selectedFrozen	Flu A	259	0	4e	255	0	NA	98.5(96.1-99.6)
	Flu B	259	100	2f	157	0	100(96.4-100)	98.7(95.5-99.8)
	RSV	259	40	1g	217	1h	97.6(87.1-99.9)	99.5(97.5-100)

a. Testing results by sequencing: 92 of 101 were Flu A positive: 8 of 101 failed to sequence: 1 of 101 insufficient remaining volume for sequencing.

b. Testing results by sequencing: 9 of 10 were Flu B positive; 1 of 10 insufficient remaining volume for sequencing.

c. Testing results by sequencing: 7 of 8 were RSV positive; 1 of 8 was RSV negative.

d. Testing results by sequencing: 3 of 4 were RSV positive; 1 of 4 was RSV negative.

e. Testing results by sequencing: 4 of 4 insufficient remaining volume for sequencing.

f. Testing results by sequencing: 2 of 2 insufficient remaining volume for sequencing.

g. Testing results by sequencing: 1 of 1 insufficient remaining volume for sequencing.

h. Testing results by sequencing: 1 of 1 insufficient remaining volume for sequencing.

Of the Xpert Flu+RSV Xpress Assay runs performed with eligible specimens, 95.0% (2335/2459) of these specimens were successful on the first attempt. The initial invalid rate was 5.0% (95% CI 4.2-6.0%). One-hundred twenty-four gave invalid results on the first attempt (121 NO RESULT-REPEAT TEST and 3 INSTRUMENT ERROR). One-hundred eighteen of the 124 specimens were retested, of which 107 yielded valid results after a single retest. There were 17 NP swab specimens with invalid results upon retest which were excluded in the analyses.

Reproducibility Study

A panel of 10 specimens with varying concentrations of influenza A. influenza B, and RSV was tested on ten different days by three different operators, at each of three sites (10 specimens x 1 time/day x 10 days x 3 operators x 3 sites). One lot of Xpert Flu-RSV Xpress Assay cartridges was used at each of the 3 testing sites. The Xpert Flu+RSV Xpress Assay was performed according to the Xpert Flu+RSV Xpress Assay procedure. Results are summarized in Table 5-13.

SampleID	Site 1			Site 2			Site 3			% TotalAgreementby Samplea
	Op 1	Op 2	Op 3	Op 1	Op 2	Op 3	Op 1	Op 2	Op 3
Negative	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(9/9)b	100%(9/9)b	100%(10/10)	100%(10/10)	100%(88/88)b

Table 5-13: Summary of Reproducibility Results

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Sample	Site 1			Site 2			Site 3			% Total
Flu A-High Neg	0.0%(0/10)	20.0%(2/10)	60.0%(6/10)	20.0%(2/10)	10.0%(1/10)	50.0%(5/10)	75.0%(6/8)b	20.0%(2/10)	20.0%(2/10)	29.5%(26/88)b
Flu A-Low Pos	100%(10/10)	100%(10/10)	90.0%(9/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	98.9%(89/90)
Flu A-Mod Pos	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100% (90/90)
Flu B-High Neg	50.0%(5/10)	0.0%(0/10)	50.0%(5/10)	20.0%(2/10)	10.0%(1/10)	55.6%(5/9)b	80.0%(8/10)	50.0%(5/10)	20.0%(2/10)	37.1%(33/89)b
Flu B-Low Pos	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	90.0%(9/10)	100%(10/10)	100%(10/10)	98.9%(89/90)
Flu B-Mod Pos	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100% (90/90)
RSV-High Neg	60.0%(6/10)	30.0%(3/10)	66.7%(6/9)b	50.0%(5/10)	60.0%(6/10)	50.0%(5/10)	90.0%(9/10)	33.3%(3/9)b	50.0%(5/10)	54.5%(48/88)b
RSV-Low Pos	88.9%(8/9)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	90.0%(9/10)	100%(10/10)	100%(10/10)	97.8%(87/89)c
RSV-Mod Pos	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100%(10/10)	100% (90/90)

a. Percent of samples yielding expected results – negative for Neg and High Neg samples; positive for Low Pos samples.

b. Seven samples (2 Neg, 2 Flu A High Neg, and 2 RSV High Neg) were indeterminate upon initial and retest.

c. One RSV Low Pos sample was inadvertently not tested.

The reproducibility of the Xpert Flu+RSV Xpress Assay was also evaluated in terms of the fluorescence signal expressed in Ct values for each target detected. The mean, standard deviation (SD), and coefficient of variation (CV) between-sites, between-days, and betweenoperators for each panel member are presented in Table 5-14. One replicate was performed per day per operator, therefore, operator and assay (within-run) precision are confounded.

{25}------------------------------------------------

Sample	AssayChannel(Analyte)	Na	MeanCt	Between-Site		Between-Day		Between-Operator +Within Assay		Total
				SD	CV(%)b	SD	CV(%)b	SD	CV(%)b	SD	CV(%)b
Neg	SPC	88	31.4	0.3	0.9	0.0	0.0	1.5	4.7	1.5	4.8
Flu A -High Neg	Flu A1	62	36.9	0.1	0.3	0.6	1.6	1.6	4.4	1.7	4.7
Flu A -Low Pos	Flu A1	89	34.5	0.2	0.7	0.0	0.0	1.6	4.5	1.6	4.5
Flu A -Mod Pos	Flu A1	90	32.3	0.0	0.0	0.7	2.2	1.2	3.7	1.4	4.2
Flu B -High Neg	Flu B	56	34.6	0.0	0.0	1.4	4.0	2.1	6.0	2.5	7.2
Flu B -Low Pos	Flu B	89	32.6	0.6	2.0	0.7	2.1	1.7	5.1	1.9	5.9
Flu B -Mod Pos	Flu B	90	30.4	0.0	0.0	0.0	0.0	1.0	3.3	1.0	3.3
RSV -High Neg	RSV	40	36.9	1.2	3.1	0.0	0.0	1.7	4.6	2.1	5.6
RSV -Low Pos	RSV	87	35.0	0.7	1.9	0.0	0.0	1.6	4.6	1.7	5.0
RSV -Mod Pos	RSV	90	32.6	0.0	0.0	0.1	0.4	1.0	3.0	1.0	3.0

Table 5-14: Summary of Reproducibility Data

a. Results with non-zero Ct values out of 90.

b. (%) is contribution of variance component to overall CV.

Conclusions

The results of the nonclinical analytical and clinical performance studies summarized above demonstrate that the Xpert Flu+RSV Xpress Assay performs comparably to the predicate device for its intended use with inexperienced users in a CLIA waived environment and is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
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.
(b)
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.