The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Infinity Systems, is an automated multiplexed real-time reverse transcriptase chain reaction (RT-PCR) test intended for use in viro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.

The Xpert Xpress CoV-2/Flu/RSV plus is intended for use in the differential detection of SARS-CoV-2, influenza B and or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Xpert Xpress COV-2/Flu/RSV plus test may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza B, and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Device Description

The Xpert Xpress CoV-2/Flw/RSV plus test is an automated in vitro diagnostic test for the simultaneous qualitative detection and differentiation of SARS-CoV-2, Flu A, Flu B, and RSV viral RNA in nasopharyngeal swab (NPS) and anterior nasal swab (NS) specimens collected from individuals showing signs and symptoms of respiratory viral infection.

The Xpert Xpress CoV-2/Flu/RSV plus test is performed on GeneXpert Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 systems), which consist of an instrument, computer, and preloaded software for running tests and viewing the results. The GeneXpert Instrument Systems automate and integrate sample preparation, nucleic acid extraction and amplification, and detection of the target sequences in simple or complex samples using real-time reverse transcription (RT)-polymerase chain reaction (PCR) and PCR assays. Depending on the instrument, the GeneXpert Instrument Systems can have from 1 and up to 80 randomly accessible modules, each capable of performing separate sample preparation and real-time RT-PCR and 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 real-time RT-PCR and PCR as well as detection. The systems require the use of single-use disposable cartridges that hold the RT-PCR reagents and host sample purification, nucleic acid amplification, and detection of the target sequences. Because the cartridges are selfcontained, cross-contamination between samples is minimized.

The Xpert Xpress CoV-2/Flu/RSV plus test includes reagents for the detection of SARS-CoV-2, Flu A. Flu B and RSV viral RNA from NPS and NS specimens. The primers and probes in the Xpert Xpress CoV-2/Flu/RSV plus test are designed to amplify and detect unique sequences in the genes that encode the following proteins: SARS-CoV-2 nucleocapsid (N), SARS-CoV-2 envelope (E), SARS-CoV-2 RNA-dependent RNA polymerase (RdRP), influenza A matrix (M), influenza A basic polymerase (PB2), influenza A acidic protein (PA), influenza B matrix (M), influenza B non-structural protein (NS), and the RSV A and RSV B nucleocapsid. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge utilized by the GeneXpert instrument. The SPC is present to control for adequate processing of the sample and to monitor for the presence of potential inhibitor(s) in the RT-PCR reaction. The SPC also ensures that the RT-PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the RT-PCR reagents are functional. The PCC verifies reagent rehydration, PCR tube filling, and confirms that all reaction components are present in the cartridge including monitoring for probe integrity and dye stability.

AI/ML Overview

Acceptance Criteria and Study Details for Xpert Xpress CoV-2/Flu/RSV plus

The Xpert Xpress CoV-2/Flu/RSV plus test is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for the qualitative detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and/or Respiratory Syncytial Virus (RSV) viral RNA in nasopharyngeal swab (NPS) and anterior nasal swab (NS) specimens.

The performance of the device was evaluated through analytical and clinical studies.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device are implicitly derived from the observed clinical performance, demonstrating acceptable Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a U.S. FDA-cleared molecular reference method.

Target	Specimen Collection	Performance Metric	Reported Performance (95% CI)
NPS Specimens
SARS-CoV-2	Overall	PPA	97.1 (95.1 - 98.2)
	Overall	NPA	98.2 (97.5 - 98.7)
Flu A	Overall	PPA	99.0 (96.3 - 99.7)
	Overall	NPA	99.1 (98.7 - 99.4)
Flu B	Overall	PPA	96.6 (88.5 - 99.1)
	Overall	NPA	100.0 (99.9 - 100.0)
RSV	Overall	PPA	98.6 (92.5 - 99.8)
	Overall	NPA	100.0 (99.9 - 100.0)
NS Specimens
SARS-CoV-2	Overall	PPA	98.2 (96.6 - 99.1)
	Overall	NPA	98.8 (98.3 - 99.2)
Flu A	Overall	PPA	98.0 (95.0 - 99.2)
	Overall	NPA	99.3 (99.0 - 99.6)
Flu B	Overall	PPA	100.0 (89.8 - 100.0)
	Overall	NPA	99.9 (99.7 - 100.0)
RSV	Overall	PPA	95.8 (88.5 - 98.6)
	Overall	NPA	100.0 (99.9 - 100.0)

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

Clinical Performance Study:
- SARS-CoV-2: A total of 5051 specimens (2536 NPS and 2515 NS) yielded valid results and were included in the performance evaluation.
  - NPS: 462 True Positives, 37 False Positives, 2023 True Negatives, 14 False Negatives.
  - NS: 448 True Positives, 24 False Positives, 2035 True Negatives, 8 False Negatives.
- Flu A/B/RSV: A total of 5954 specimens (3011 NPS and 2943 NS) yielded valid results and were included in the performance evaluation.
  - NPS:
    - Flu A: 191 True Positives, 24 False Positives, 2794 True Negatives, 2 False Negatives.
    - Flu B: 57 True Positives, 0 False Positives, 2952 True Negatives, 2 False Negatives.
    - RSV: 71 True Positives, 0 False Positives, 2939 True Negatives, 1 False Negative.
  - NS:
    - Flu A: 196 True Positives, 18 False Positives, 2725 True Negatives, 4 False Negatives.
    - Flu B: 34 True Positives, 3 False Positives, 2906 True Negatives, 0 False Negatives.
    - RSV: 69 True Positives, 0 False Positives, 2871 True Negatives, 3 False Negatives.
Data Provenance:
- Prospective Data: Fresh (98.9%) and frozen (1.1%) specimens (Category I) were prospectively collected and tested in 2022 from 33 geographically diverse sites in the United States.
- Retrospective Data: Archived prospectively collected frozen clinical specimens (Category II) from the 2016-2017 influenza season were used to supplement the sample size for Flu/RSV. These were primarily collected from the US.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth. Instead, the ground truth was established by comparing the Xpert Xpress CoV-2/Flu/RSV plus test results to results from a U.S. FDA-cleared molecular respiratory panel for SARS-CoV-2 and a U.S. FDA-cleared molecular Flu A/B/RSV assay for their respective targets.

4. Adjudication Method for the Test Set

Discrepant results between the Xpert Xpress CoV-2/Flu/RSV plus test and the comparator method were investigated as follows:

SARS-CoV-2 target: Discrepant results were investigated using a U.S. FDA EUA SARS-CoV-2 molecular test.
Flu A/B/RSV targets: Discrepant results were investigated using a U.S. FDA-cleared molecular respiratory panel.

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 conducted as the device is an in-vitro diagnostic (IVD) test, not an AI-powered diagnostic imaging device involving human readers.

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

Yes, the studies presented are generally standalone performance evaluations of the device (an automated test) without human-in-the-loop adjustment of the result. The device automates sample preparation, nucleic acid extraction, amplification, and detection, and provides results automatically ("algorithm only"). The clinical performance compares the device's output directly to accepted reference methods.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

The ground truth for the clinical performance study was established using U.S. FDA-cleared molecular reference methods for viral detection:

A U.S. FDA-cleared molecular respiratory panel for SARS-CoV-2.
A U.S. FDA-cleared molecular Flu A/B/RSV assay for Flu A, Flu B, and RSV.
For discrepant results, additional U.S. FDA EUA SARS-CoV-2 molecular test or U.S. FDA-cleared molecular respiratory panel was used for adjudication.

8. The Sample Size for the Training Set

The document does not specify a distinct "training set" for the clinical performance study as is typical for AI/ML models. For IVD devices like this, the development process (which would involve internal analytical studies and optimization) constitutes the "training" equivalent. The analytical studies describe the evaluation of analytical sensitivity, inclusivity (wet-testing of multiple strains), and exclusivity, which would directly inform the device's design and parameters. For example:

Analytical sensitivity (LoD) testing involved 2 reagent lots and 20 replicates per virus/lot combination.
Analytical reactivity (inclusivity wet-testing) involved testing 102 respiratory viral strains (18 SARS-CoV-2, 69 influenza, 15 RSV) at ~3x LoD, with 3 replicates per strain.

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

For the analytical "training" phase:

Analytical Sensitivity (LoD): Ground truth was established by using known concentrations of inactivated viruses and international standards (e.g., NATtrol SARS-CoV-2, 1st WHO International Standard, cultured Flu A, Flu B, and RSV strains) spiked into negative clinical matrices. The LoD was determined by Probit regression analysis and verified as the lowest concentration yielding 95% positive results.
Analytical Reactivity (Inclusivity): Ground truth was established by testing known viral strains at specified concentrations (~3x LoD) and expecting a positive detection for the corresponding target.
Analytical Specificity (Exclusivity): Ground truth was established by testing a panel of known microorganisms and expecting negative results for all targets, thus confirming no cross-reactivity. These were either cultured microorganisms or synthetic RNA/genomic DNA at known concentrations.

Summary

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Date: August 17, 2023

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

Cepheid % Yen Nguyen Senior Manager, Regulatory Affairs Cepheid 904 Caribbean Drive Sunnyvale, California 94089

Re: K231481

Trade/Device Name: Xpert Xpress CoV-2/Flu/RSV plus Regulation Number: 21 CFR 866.3981 Regulation Name: Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The SARS-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test Regulatory Class: Class II Product Code: QOF, OOI Dated: May 22, 2023 Received: May 23, 2023

Dear Yen Nguyen:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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.

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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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Joseph Briggs -S

Joseph Briggs, Ph.D. Deputy Branch Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K231481

Device Name Xpert Xpress CoV-2/Flu/RSV plus

Indications for Use (Describe)

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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Image /page/3/Picture/0 description: The image shows the Cepheid company logo, which includes a stylized blue wing-like design above the company name. Below the logo, the text "Xpert® Xpress CoV-2/Flu/RSV plus" is displayed. This text likely refers to a diagnostic test developed by Cepheid, indicating it is a rapid test for COVID-19, influenza, and respiratory syncytial virus.

510(k) Summary for Xpert® Xpress CoV-2/Flu/RSV plus

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Image /page/4/Picture/0 description: The image shows the Cepheid logo and the text "Xpert® Xpress CoV-2/Flu/RSV plus". The Cepheid logo is a blue graphic with the word "Cepheid." next to it. The text "Xpert® Xpress CoV-2/Flu/RSV plus" is written in a smaller font below the logo.

5.	510(K) SUMMARY	3
5.1.	DEVICE DESCRIPTION	4
5.2.	DEVICE INTENDED USE	5
5.3.	SUBSTANTIAL EQUIVALENCE	6
5.4.	PERFORMANCE STUDIES	9
5.4.1.	ANALYTICAL PERFORMANCE	9
5.4.2.	CLINICAL PERFORMANCE	28
5.5.	CONCLUSIONS	34

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Image /page/5/Picture/0 description: The image shows the Cepheid logo and the text "Xpert® Xpress CoV-2/Flu/RSV plus". The Cepheid logo is a blue graphic with the word "Cepheid" next to it. The text "Xpert® Xpress CoV-2/Flu/RSV plus" is below the logo. The image is likely an advertisement for a medical test.

5. 510(k) Summary

As required by 21 CFR Section 807.92(c).

Submitted by:	Cepheid904 Caribbean DriveSunnyvale, CA 94089Phone number: (669) 246-2271
Contact:	Yen H. Nguyen, Ph.D.
Date of Preparation:	May 22, 2023
Device:
Trade name:	Xpert® Xpress CoV-2/Flu/RSV plus
Common name:	Xpert Xpress CoV-2/Flu/RSV plus
Type of Test:	Qualitative real-time reverse transcription polymerase chainreaction (RT-PCR) and detection test
Regulation number:	21 CFR 866.3981
Classification name:	Multi-Target Respiratory Specimen Nucleic Acid TestIncluding SARS-CoV-2 and Other Microbial Agents
Primary Product code:	QOF
Secondary Product code:	OOI
ClassificationAdvisory Panel:	Microbiology (83)
Prescription Use:	Yes
Predicate DeviceAssay:	BioFire Respiratory Panel 2.1 (DEN200031)

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Device Description 5.1.

The Xpert Xpress CoV-2/Flu/RSV plus test is designed for use with NPS or NS specimens collected with nylon flocked swabs and placed into viral transport medium (VTM), Universal Transport Medium (UTM), or eNAT®. The ancillary specimen collection kits, swabs and transport media validated for use with the Xpert Xpress CoV-2/Flu/RSV plus test included:

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. Nasopharyngeal Sample Collection Kit for Viruses

Copan UTM® 3C057N (Flexible Minitip Flocked Swab with UTM® Medium о without Beads)
Copan eNAT® Molecular Collection and Preservation Medium P/N O 6U074S01 (Flexible Minitip Flocked Swab with eNAT® Medium)
Becton Dickinson Universal Viral Transport Kit P/N 220531 (Flexible Minitip O Flocked Swab with UVT Medium)
Nasal Sample Collection Kit for Viruses .
- Copan UTM® 3C064N (Regular Flocked Swab with UTM® Medium without o Beads)
- Copan eNAT® Molecular Collection and Preservation Medium P/N O 6U073S01 (Regular Flocked Swab with eNAT® Medium)
Alternatively, swabs and transport media can be obtained separately: ●
- Nylon flocked swab (Copan P/N 502CS01, 503CS01) O
- Viral transport medium, 3 mL (Copan P/N 330C, 3C047N, BD Universal o Transport Medium, Remel M4RT or Remel M5)

These ancillary reagents allow NPS and NS specimens from patients to be collected, preserved and transported to laboratory prior to analysis with the Xpert Xpress CoV-2/Flu/RSV plus test.

5.2. Device Intended Use

The Xpert® Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert® Dx and GeneXpert® Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.

The Xpert Xpress CoV-2/Flu/RSV plus is intended for use in the differential detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the presence of the identified virus, but do not rule out

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bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza A, influenza B, and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

5.3. Substantial Equivalence

Table 5-1 shows the similarities and differences between Xpert Xpress CoV-2/Flu/RSV plus and the predicate device, BioFire Respiratory Panel 2.1 (DEN20031).

Attribute	Investigational Device	Predicate Device - DEN200031
	Cepheid	BioFire Diagnostics, LLC
	Xpert® Xpress CoV-2/Flu/RSV plus	BioFire Respiratory Panel 2.1
		21 CFR 866.3981
Regulation	Same	Devices to detect and identify nucleic acid targets in respiratory samples from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-analyte test
Product Code/ClassificationName	Same	QOFMulti-target respiratory specimen nucleic acid test including SARS-CoV-2 and other microbial agents
Device Class	Same	II (Special Controls)
Technology/Detection	Real-time reverse transcription polymerase chain reaction (RT-qPCR)	Nested multiplex RT-PCR followed by high resolution melting analysis to confirm identity of amplified product
Intended Use	The Xpert® Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert® Dx and GeneXpert® Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract	The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch Systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19.Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral
Attribute	Investigational Device	Predicate Device - DEN200031
	Cepheid	BioFire Diagnostics, LLC
	Xpert® Xpress CoV-2/Flu/RSV plus	BioFire Respiratory Panel 2.1
	infection due to SARS-CoV-2, influenzaA, influenza B, and RSV can be similar.The Xpert Xpress CoV-2/Flu/RSV plus isintended for use in the differentialdetection of SARS-CoV-2, influenza A,influenza B and/or RSV RNA and aids inthe diagnosis of COVID-19, influenzaand/or RSV infections if used inconjunction with other clinical andepidemiological information, andlaboratory findings. SARS-CoV-2,influenza A, influenza B, and RSV viralRNA are generally detectable innasopharyngeal swab and anterior nasalswab specimens during the acute phase ofinfection.Positive results are indicative of thepresence of the identified virus, but do notrule out bacterial infection or co-infectionwith other pathogens not detected by thetest. The agent(s) detected by the XpertXpress CoV-2/Flu/RSV plus test may notbe the definite cause of disease.Negative results do not preclude SARS-CoV-2, influenza A, influenza B and/orRSV infection. The results of this testshould not be used as the sole basis fordiagnosis, treatment, or other patientmanagement decisions.	and bacterial nucleic acids from individualsexhibiting signs and/or symptoms ofrespiratory infection is indicative of thepresence of the identified microorganismand aids in the diagnosis of respiratoryinfection if used in conjunction with otherclinical and epidemiological information.The results of this test should not be used asthe sole basis for diagnosis, treatment, orother patient management decisions.The following organism types and subtypesare identified using the BioFire RP2.1:• Adenovirus• Coronavirus 229E• Coronavirus HKU1• Coronavirus NL63• Coronavirus OC43• Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2)• Human Metapneumovirus• Human Rhinovirus/Enterovirus• Influenza A, including subtypes• H1, H3 and H1-2009• Influenza B• Parainfluenza Virus 1• Parainfluenza Virus 2• Parainfluenza Virus 3• Parainfluenza Virus 4• Respiratory Syncytial Virus• Bordetella parapertussis• Bordetella pertussis• Chlamydia pneumoniae• Mycoplasma pneumoniaeNegative results in the setting of arespiratory illness may be due to infectionwith pathogens that are not detected by thistest, or lower respiratory tract infection thatmay not be detected by an NPS specimen.Positive results do not rule out co-infectionwith other organisms. The agent(s) detectedby the BioFire RP2.1 may not be thedefinite cause of disease. Additionallaboratory testing (e.g., bacterial and viralculture, immunofluorescence, andradiography) may be necessary whenevaluating a patient with possiblerespiratory tract infection.
Attribute	Investigational DeviceCepheidXpert® Xpress CoV-2/Flu/RSV plus	Predicate Device – DEN200031BioFire Diagnostics, LLCBioFire Respiratory Panel 2.1
Assay Targets	SARS-CoV-2, Influenza A, Influenza B, RSV viral RNA	• Adenovirus• Coronavirus 229E• Coronavirus HKU1• Coronavirus NL63• Coronavirus OC43• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)• Human Metapneumovirus• Human Rhinovirus/Enterovirus• Influenza A, including subtypes• H1, H3 and H1-2009• Influenza B• Parainfluenza Virus 1• Parainfluenza Virus 2• Parainfluenza Virus 3• Parainfluenza Virus 4• Respiratory Syncytial Virus• Bordetella parapertussis• Bordetella pertussis• Chlamydia pneumoniae• Mycoplasma pneumoniae
Specimen Type	• Nasopharyngeal swab (NPS)• Anterior nasal swab (NS)	• Nasopharyngeal swab (NPS)
TransportMedia	• Universal Transport Medium (UTM) / Viral Transport Medium (VTM)• eNAT	• Universal Transport Medium (UTM)/ Viral Transport Medium (VTM)• Saline
Test Format	Same	Single Use
Automation	Same	Automated Nucleic Acid Extraction, Detection and Results Interpretation
Assay Results	Same	Qualitative
InternalControl	Sample Processing Control (SPC)Probe Check Control (PCC)	Sample Processing ControlPCR and Melt Analysis Control
InstrumentSystems	Cepheid GeneXpert® Instrument Systems	BioFire® FilmArray® 2.0 orBioFire® FilmArray® Torch Systems
Time to Result	~ 36 min for sample preparation and RT-PCR	~ 45 minutes

Table 5-1: Comparison of Similarities and Differences Between Xpert Xpress CoV-2/Flu/RSV plus and the Predicate Device

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Image /page/9/Picture/0 description: The image shows the Cepheid logo with the text "Cepheid." next to it. Below the logo is the text "Xpert® Xpress CoV-2/Flu/RSV plus". The text appears to be advertising a product or service related to CoV-2, Flu, and RSV.

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Image /page/10/Picture/0 description: The image shows the Cepheid logo and the text "Xpert® Xpress CoV-2/Flu/RSV plus". The Cepheid logo is a blue, stylized wing-like shape. The text is in a sans-serif font and is black.

The following performance data (analytical and clinical) were provided in support of the substantial equivalence determination.

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Image /page/11/Picture/0 description: The image displays the Cepheid logo, which includes a stylized blue graphic above the company name. Below the logo, the text "Xpert® Xpress CoV-2/Flu/RSV plus" is printed in a clear, legible font. The text indicates a product or test related to CoV-2, Flu, and RSV, suggesting it is a diagnostic tool.

5.4. Performance Studies

5.4.1. Analytical Performance

Analytical Sensitivity - Clinical Nasopharyngeal Swab (NPS) Matrix

The analytical sensitivity of the Xpert Xpress CoV-2/Flu/RSV plus test was first estimated by using 2 reagent lots and testing limiting dilutions of viruses (NATtrol SARS-CoV-2, 1st World Health Organization (WHO) International Standard for SARS-CoV-2. Flu A H1. Flu A H3 , Flu B Victoria linage, Flu B Yamagata lineage, RSV A and RSV B) in pooled negative clinical NPS-UTM/VTM matrix, following the guidance in Clinical and Laboratory Standards Institute (CLSI) document EP17-A2. The LoD is defined as the lowest concentration for each strain at which 95% (19/20) of replicates yield a positive result. The estimated LoD values as determined by Probit regression analysis were verified using 2 lots of Xpert Xpress CoV-2/Flu/RSV plus reagents, by testing 20 replicates per virus/lot combination. The highest (least sensitive) LoD value for the two lots was reported as the final, verified LoD. The verified LoD values for the viruses tested are summarized in Table 5-2.

Virus/Strain	LoD Concentration
USA-WA1/2020 (NATtrol)	138 copies/mL
1st WHO International Standard	94 IU/mL
Flu A/Idaho/07/2018	0.007 TCID50/mL
Flu A/California/07/2009	0.0022 TCID50/mL
Flu A/Hong Kong/45/2019	0.44 FFU/mL
Flu A/Victoria/361/2011	0.05 TCID50/mL
Flu B/Washington/2/2019	12.9 CEID50/mL
Flu B/Wisconsin/10/2016	2.4 TCID50/mL
RSV A/2/Australia/61	0.33 TCID50/mL
RSV A/Long/MD/56	0.17 TCID50/mL
RSV B/9320/MA/77	0.37 TCID50/mL
RSV B/Wash/18537/62	0.2 TCID50/mL

Table 5-2. Xpert Xpress CoV-2/Flu/RSV plus Limit of Detection in Clinical NPS-UTM/VTM Matrix

Analytical Sensitivity - Clinical Anterior Nasal Swab (NS) Matrix

The analytical sensitivity of the Xpert Xpress CoV-2/Flu/RSV plus test in clinical anterior nasal swab (NS) matrix was first estimated by using 2 lots and testing limiting dilutions of viruses (NATtrol SARS-CoV-2, 1st World Health Organization (WHO) International Standard for SARS-CoV-2, Flu A H1, Flu A H3, Flu B Victoria linage, Flu B Yamagata lineage, RSV A and RSV B) in pooled negative clinical NS UTM/VTM matrix, following the guidance in Clinical and Laboratory Standards Institute (CLSI) document EP17-A2. The estimated LoD values as determined by Probit regression analysis were verified using 2 lots of Xpert Xpress CoV-2/Flu/RSV plus reagents, by testing 20 replicates per virus/lot

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combination. The highest (least sensitive) LoD value for the two lots was reported as the final, verified LoD. The verified LoD values for the viruses tested are summarized in Table 5-3.

Virus/Strain	LoD Concentration
USA-WA1/2020 (NATtrol)	64 copies/mL
1st WHO International Standard	143 IU/mL
Flu A/Idaho/07/2018	0.012 TCID50/mL
Flu A/California/07/2009	0.0028 TCID50/mL
Flu A/Hong Kong/45/2019	0.49 FFU/mL
Flu A/Victoria/361/2011	0.065 TCID50/mL
Flu B/Washington/2/2019	26.3 CEID50/mL
Flu B/Wisconsin/10/2016	2.41 TCID50/mL
RSV A/2/Australia/61	0.28 TCID50/mL
RSV A/Long/MD/56	0.22 TCID50/mL
RSV B/9320/MA/77	0.27 TCID50/mL
RSV B/Wash/18537/62	0.4 TCID50/mL

Table 5-3. Xpert Xpress CoV-2/Flu/RSV plus Limit of Detection in Clinical NS-UTM/VTM Matrix

Analytical Reactivity (Inclusivity)

SARS-CoV-2 in silico Analyses

The inclusivity of Xpert Xpress CoV-2/Flu/RSV plus was evaluated using in silico analysis of the assay amplicons in relation to SARS-CoV-2 sequences available in the GISAID gene database as of June 15, 2022. The sequences were separated into the lineages of interest based on the Pango Lineage assigned to each genome by GISAID, and those with ambiguous nucleotides were removed. Thus, the following inclusivity analyses focus on the combined, non-ambiguous sequences from the variants of interest and variants of concern as of June 15, 2022. These constituted 10.310.839 sequences for the E target. 10.428.014 sequences for the N2 target, and 10,178,602 sequences for the RdRP target. Table 5-4 summarizes the effective predicted inclusivity for E, N2 and RdRP amplicons for the variants of interests and concern.

Table 5-4. Predicted Inclusivity for E, N2 and RdRP Amplicons for
SARS-CoV-2 Variants of Interests and Concern

Amplicon	Exact Match	1 Mismatch	2 or More Mismatches	% Total <2 Mismatches
CEP-COV-E-PLUS	10,262,080 of 10,310,839 (99.5%)	47,959 (0.5%)	800 (0.01%)	100%
CEP-COV-N2	10,228,739 of 10,428,014 (98.1%)	194,319 (1.9%)	4,956 (0.05%)	99.9%
CEP-COV-RDRP	10,092,873 of 10,178,602 (99.2%)	84,595 (0.8%)	1,134 (0.01%)	100%

Single-nucleotide mismatches are predicted to not impact the performance of the test. a.

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Based on the built-in redundancy of the Xpert Xpress CoV-2/Flu/RSV plus test's SARS-CoV-2 amplification system (i.e., 3 independent targets, only 1 of 3 must be detected to assign a positive result), it is not anticipated that any of the evaluated SARS-CoV-2 sequences would be missed by the Xpert Xpress CoV-2/Flu/RSV plus test.

SARS-CoV-2, Flu A, Flu B, and RSV Inclusivity Wet-Testing

In addition to the in silico analysis of the SARS-CoV-2 primers and probes for inclusivity. the inclusivity of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated by bench testing against multiple strains of SARS-CoV-2, 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 Yamagata lineages), and respiratory syncytial virus subgroups A and B (RSV A and RSV B) at concentrations of ~3x LoD in simulated matrix. A total of 102 respiratory viral strains comprised of 18 SARS-CoV-2 strains, 69 influenza viruses (48 influenza A and 21 influenza B) and 15 RSV strains were evaluated for analytical reactivity (inclusivity) with the Xpert Xpress CoV-2/Flu/RSV plus test. Three replicates were tested for each strain. All SARS-CoV-2, Flu and RSV strains tested positive in all 3 replicates. Results are shown in Table 5-5.

			Result
VirusStrain		Target Conc.	SARS-CoV-2	Flu A	Flu B	RSV
	NATtrol SARS-CoV-2 USA-WA1/2020	412 copies/mL	POS	NEG	NEG	NEG
	SARS-CoV-2/HongKong/VM20001061/2020	0.03 TCID50/mL	POSa	NEG	NEG	NEG
	SARS-CoV-2/Italy-INM11	1 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2/Africa/KRISPK005325/2020 (Beta)	0.025 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2/England/204820464/2020	0.05 TCID50/mL	POS	NEG	NEG	NEG
SARS-CoV-2	SARS-CoV-2 (NY-Wadsworth-21033899-01/2021) P1_2021 (Gamma)	0.01 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (NY-Wadsworth-21006055-01/2021) P2_2021(Zeta)	0.03 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (NYWadsworth-21025952-01/2021) B.1.526_2021 (Iota)	0.1 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (NY-Wadsworth-103677-01/2020) B.1_2020	0.003 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (NY-Wadsworth-33126-01/2020) B.1.595_2020	0.0015 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (USA/CA-Stanford-15_S02/2021) B.1.617.1 (Kappa)	1.7 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (USA/PHC658/2021)B.1.617.2 (Delta)	0.01 TCID50/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 (USA/MDHP01542/2021) B.1.351 (Beta)	100 (genomeequivalents/mL)	POS	NEG	NEG	NEG
	SARS-CoV-2 (USA/GA-EНС-2811C/20221) B.1.1.529 (Omicron)	100 (genomeequivalents/mL)	POS	NEG	NEG	NEG

					Table 5-5. Analytical Reactivity (Inclusivity) of the Xpert Xpress CoV-2/Flu/RSV plus Test
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Xpert® Xpress CoV-2/Flu/RSV plus

Virus	Strain	Target Conc.	ResultSARS-CoV-2	Flu A	Flu B	RSV
	SARS-CoV-2 RNA, USA/WA2/2020 (C09)b	100 copies/mL	POS	NEG	NEG	NEG
	SARS-CoV-2RNA,England/205041766/2020 (C14) (alpha)b	100 copies/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 RNA, England/MILK-9E05B3/2020 (C15) (alpha)b	200 copies/mL	POS	NEG	NEG	NEG
	SARS-CoV-2 RNA /Japan (Brazil)/IC-0564/2021 (C17) (gamma)b	100 copies/mL	POS	NEG	NEG	NEG
	A/swine/Iowa/15/30	10 TCID50/mL	NEG	POS	NEG	NEG
	A/WS/33	0.6 CEID50/mL	NEG	POS	NEG	NEG
	A/PR/8/34	1.25 CEID50/mL	NEG	POS	NEG	NEG
	A/Mal/302/54	0.156 CEID50/mL	NEG	POS	NEG	NEG
	A/Denver/1/57	1.5 CEID50/mL	NEG	POS	NEG	NEG
Flu A H1N1(pre-2009)	A/New Jersey/8/76	5 CEID50/mL	NEG	POS	NEG	NEG
	A/New Caledonia/20/1999	0.10 TCID50/mL	NEG	POS	NEG	NEG
	A/New York/55/2004	9 TCID50/mL	NEG	POS	NEG	NEG
	A/Solomon Island/3/2006	0.0159TCID50/mL	NEG	POS	NEG	NEG
	A/Taiwan/42/06	0.002 TCID50/mL	NEG	POS	NEG	NEG
	A/Brisbane/59/2007	0.008 TCID50/mL	NEG	POS	NEG	NEG
	A/Swine/NY/02/2009	3.2 TCID50/mL	NEG	POS	NEG	NEG
	A/Colorado/14/2012	0.04 TCID50/mL	NEG	POS	NEG	NEG
	A/Michigan/45/2015	15 CEID50/mL	NEG	POS	NEG	NEG
	A/Iowa/53/2015	6 CEID50/mL	NEG	POS	NEG	NEG
Flu A H1N1(pdm 2009)	A/Michigan/272/2017	0.07 TCID50/mL	NEG	POS	NEG	NEG
	A/Idaho/07/2018	0.0159TCID50/mL	NEG	POS	NEG	NEG
	A/Wisconsin/505/2018	0.08 TCID50/mL	NEG	POS	NEG	NEG
	A/Hawaii/66/2019	100 CEID50/mL	NEG	POS	NEG	NEG
	A/Indiana/02/2020	NAc	NEG	POS	NEG	NEG
	A/Aichi/2/68	2 CEID50/mL	NEG	POS	NEG	NEG
	A/Hong Kong/8/68	0.25 CEID50/mL	NEG	POS	NEG	NEG
	A/Port Chalmers/1/73	8 CEID50/mL	NEG	POS	NEG	NEG
	A/Hawaii/15/2001	33 CEID50/mL	NEG	POS	NEG	NEG
	A/Wisconsin/67/05c	0.22 TCID50/mL	NEG	POS	NEG	NEG
	A/Brisbane/10/2007	0.003 TCID50/mL	NEG	POS	NEG	NEG
	A/Minnesota/11/2010	2.4 CEID50/mL	NEG	POS	NEG	NEG
	A/Indiana/08/2011	0.02 TCID50/mL	NEG	POS	NEG	NEG
	A/Texas/50/2012	0.008 TCID50/mL	NEG	POS	NEG	NEG
	A/Alaska/232/2015	2 CEID50/mL	NEG	POS	NEG	NEG
	A/Singapore/INFIMH-16-0019/2016	2.5 CEID50/mL	NEG	POS	NEG	NEG
Flu AH3N2	A/Texas/71/2017	1 FFU/mL	NEG	POS	NEG	NEG
	A/Kansas/14/2017	0.15 FFU/mL	NEG	POS	NEG	NEG
	A/Wisconsin/04/2018d	0.15 FFU/mL	NEG	POS	NEG	NEG
	A/Arizona/45/2018	2 FFU/mL	NEG	POS	NEG	NEG
	A/Hong Kong/45/2019	0.8 FFU/mL	NEG	POS	NEG	NEG
	A/Mallard/NY/6750/78 (H2N2)	< 1 pg/uL	NEG	POS	NEG	NEG
AvianFlu Ae	A/duck/Hunan/795/2002 (H5N1)	< 1 pg/uL	NEG	POS	NEG	NEG
	A/Vietnam/1194/2004 (H5N1)	< 1 pg/uL	NEG	POS	NEG	NEG
	A/Anhui/01/2005 (H5N1)	< 1 pg/uL	NEG	POS	NEG	NEG
	Strain		Result
Virus		Target Conc.	SARS-CoV-2	Flu A	Flu B	RSV
	A/Japanese white eye/HongKong/1038/2006 (H5N1)	< 1 pg/uL	NEG	POS	NEG	NEG
	A/mallard/WI/34/75 (H5N2)	< 1 pg/uL	NEG	POS	NEG	NEG
	A/turkey/Massachusetts/3740/1965 (H6N2)	0.1 fg/uL	NEG	POS	NEG	NEG
	A/duck/LTC-10-82743 (H7N2)	5 fg/uL	NEG	POS	NEG	NEG
	A/chicken/New Jersey/15086/3(H7N3)	4 fg/uL	NEG	POS	NEG	NEG
	A/Anhui/1/2013 (H7N9)	0.612 ng/uL	NEG	POS	NEG	NEG
	A/Shanghai/1/2013 (H7N9)	NA	NEG	POS	NEG	NEG
	A/chicken/New Jersey/12220/1997(H9N2)	0.05 pg/uL	NEG	POS	NEG	NEG

	B/Lee/40	0.08 PFU/mL	NEG	NEG	POS	NEG
	B/Allen/45	0.25 CEID50/mL	NEG	NEG	POS	NEG
Flu B	B/GL/1739/54	0.50 CEID50/mL	NEG	NEG	POS	NEG
	B/Maryland/1/59	0.2 CEID50/mL	NEG	NEG	POS	NEG
	B/Taiwan/2/62	0.7 CEID50/mL	NEG	NEG	POS	NEG
	B/Hong Kong/5/72	1 CEID50/mL	NEG	NEG	POS	NEG
	B/Panama/45/90	0.125 TCID50/mL	NEG	NEG	POS	NEG
	B/Malaysia/2506/04	0.001 TCID50/mL	NEG	NEG	POS	NEG
	B/Florida/02/06	0.004 TCID50/mL	NEG	NEG	POS	NEG
	B/Brisbane/60/2008	0.005 TCID50/mL	NEG	NEG	POS	NEG
Flu B (VictoriaLineage)	B/Maryland/15/2016	0.06 TCID50/mL	NEG	NEG	POS	NEG
	B/Colorado/6/2017	0.01 TCID50/mL	NEG	NEG	POS	NEG
	B/Hawaii/01/2018	1 TCID50/mL	NEG	NEG	POS	NEG
	B/Missouri/12/2018 (NA D197E)	1.2 TCID50/mL	NEG	NEG	POS	NEG
	B/Washington/02/2019	60 TCID50/mL	NEG	NEG	POS	NEG

	B/Florida/07/2004	0.03 TCID50/mL	NEG	NEG	POS	NEG
Flu B(YamagataLineage)	B/Florida/04/06	0.03 TCID50/mL	NEG	NEG	POS	NEG
	B/Wisconsin/01/2010	0.025 CEID50/mL	NEG	NEG	POS	NEG
	B/Wisconsin/10/2016	2 TCID50/mL	NEG	NEG	POS	NEG
	B/Indiana/17/2017	0.5 TCID50/mL	NEG	NEG	POS	NEG
	B/Oklahoma/10/2018	1 TCID50/mL	NEG	NEG	POS	NEG

	RSV-A/NY	0.386 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A/WI-629.8.2/2007	0.50 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A/WI/629-11-1 2008	0.50 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A, Strain: 4/2015 Isolate #1	0.03 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A (2014, Isolate 342)	0.38 IU/mL	NEG	NEG	NEG	POS
	RSV-A (A2 cpts-248 mutant)	1600 copies/mL	NEG	NEG	NEG	POS
RSV A	RSV-A (2000/3-4)	0.0015TCID50/mL	NEG	NEG	NEG	POS
	RSV-A (2001/3-12)	0.28 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A (1997/12-35)	0.5 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A (Homosapiens/ARG/177/2006)	0.089 TCID50/mL	NEG	NEG	NEG	POS
	RSV-A (1998/3-2)	0.0089TCID50/mL	NEG	NEG	NEG	POS

	RSV-B/WV14617/85	0.04 TCID50/mL	NEG	NEG	NEG	POS
RSV B	RSV-B-CH93(18)-18-01	0.004 TCID50/mL	NEG	NEG	NEG	POS
	RSV-B (12/2014, Isolate #1)	0.008 TCID50/mL	NEG	NEG	NEG	POS
Virus	Strain	Target Conc.	Result
	RSV-B (cp23 Clone 1A2)	4200 copies/mL	SARS-CoV-2NEG	Flu ANEG	Flu BNEG	RSVPOS

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Xpert® Xpress CoV-2/Flu/RSV plus

One of three replicates was Invalid. The run was successfully repeated to obtain three valid replicates. a.

b. In vitro transcripts from Twist Biosciences.

Influenza A/Indiana/02/2020 virus was without titer and the stock was diluted 48,000-fold in simulated matrix for C. testing.

d. One of three replicates yielded an ERROR result. The run was successfully repeated to obtain three valid replicates.

Purified viral RNA in TE and diluted in simulated matrix was tested due to biosafety regulations. e.

Inactivated avian influenza A (H7N9) viral RNA without viral titer was diluted 100,000-fold in simulated matrix for f. testing due to biosafety regulations.

Analytical Specificity (Exclusivity)

In silico Analyses

An in silico analysis for possible cross-reactions with all the organisms listed in Table 5-6 was conducted by mapping the SARS-CoV-2 oligonucleotides and amplicons in the Xpert Xpress CoV-2/Flu/RSV plus test individually to the sequences downloaded from the GISAID database. E gene primers and probes are not specific for SARS-CoV-2 and will detect Human and Bat SARS-coronavirus.

In silico exclusivity analysis using Flu A, Flu B and RSV B primer and probe oligonucleotides against the GenBank database (which encompasses essentially all species) did not identify matches, with at least 80% similarity to each oligonucleotide, for any nontarget organism expected to be found in a human respiratory tract sample. RSV A primer and probe oligonucleotides exhibited >80% homology with two Pangolin RSV A isolates. Therefore, the RSV A primer and probe may cross-react with Pangolin RSV A if the strain is circulating in a human population and present in a sample tested with the Xpert Xpress CoV-2/Flu/RSV plus test. While there was some homology ≥80% to human genomic DNA, the matches were to different chromosomal regions, and there were no cases where a forward and reverse primer for a specific target matched to the same human genomic DNA fragment.

In silico exclusivity analysis using the five Flu amplicons (Flu A MP, Flu A PB2, Flu A PA, Flu B MP and Flu B NS) against the GenBank database produced no significant matches to non-influenza-related sequences. Similarly, no matches to RSV isolates from other species or to genomic sequences from non-RSV species were observed with the RSV B amplicon. While no matches of the RSV A amplicon to genomic sequences from non-RSV species of >80% homology were observed, the RSV A amplicon shared a 95% identity with two Pangolin RSV A isolates.

No cross reactivity with non-SARS-CoV-2, non-influenza and non-RSV viruses listed in Table 5-6 is expected based on the in silico analysis.

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Table 5-6. Microorganisms Analyzed in the in silico Analysis for the SARS-CoV-2 Target

Microorganisms from the Same Genetic Family	High Priority Organisms
Human coronavirus 229E	Viruses
Human coronavirus OC43	Adenovirus (e.g., C1 Ad. 71)
Human coronavirus HKU1	Cytomegalovirus
Human coronavirus NL63	Enterovirus (e.g., EV68)
SARS-coronavirus	Epstein-Barr virus
MERS-coronavirus	Human Metapneumovirus (hMPV)
Bat coronavirus	Influenza A & B
	Measles
	Mumps
	Parainfluenza virus 1-4
	Parechovirus
	Respiratory syncytial virus
	Rhinovirus
	Bacteria
	Bacillus anthracis
	Bordetella pertussis
	Bordetella parapertussis
	Candida albicans
	Chlamydia pneumoniae
	Chlamydia psittaci
	Corynebacterium diphtheriae
	Coxiella burnetii (Q-Fever)
	Escherichia coli
	Fusobacterium necrophorum
	Haemophilus influenzae
	Lactobacillus sp.
	Legionella non-pneumophila
	Legionella pneumophila
	Leptospira
	Moraxella catarrhalis
	Mycobacterium tuberculosis
	Mycoplasma genitalium
	Mycoplasma pneumoniae
	Neisseria elongata
	Neisseria meningitidis
	Pneumocystis jirovecii (PJP)
	Pseudomonas aeruginosa
	Staphylococcus aureus
	Staphylococcus epidermidis
	Staphylococcus salivarius
	Streptococcus pneumoniae
	Streptococcus pyogenes

Microorganisms from the Same Genetic Family	High Priority Organisms
	Fungi
	Aspergillus sp

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Wet Testing

In addition to the in silico analysis of the SARS-CoV-2, influenza A, influenza B, and RSV oligonucleotides and amplicons for cross-reactivity, the analytical specificity of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated by bench testing a panel of 48 microorganisms, comprising 4 human coronaviruses, 1 MERS coronavirus and 43 common respiratory pathogens or those potentially encountered in the nasopharynx. The panel was tested in different pools of microorganisms in simulated matrix; if a pool produced a positive result, then each member of the pool would have been tested individually. Three replicates of each pool were tested. A sample was considered negative if all three replicates were negative. The bacterial and yeast strains were tested at concentrations of ≥ 1 x 10° CFU/mL except for Chlamydia pneumoniae which was tested at 1.2 x 106 IFU/mL and Lactobacillus reuteri which was tested at 5 x 107 copies/mL of genomic DNA. Viruses were tested at concentrations of ≥ 1 x 105 TCID50/mL. The analytical specificity was 100%. Results are shown in Table 5-7.

Count	Strain	Tested Concentration	SARS-CoV-2	Flu A	Flu B	RSV
0	Negative Control	NA	NEG	NEG	NEG	NEG
00	Positive Control (NATFRC-6C)	NA	POS	POS	POS	POS
1	Human coronavirus NL63	1.17e5 TCID50/mL	NEG	NEG	NEG	NEG
2	MERS-coronavirus	1.17e5 TCID50/mL	NEG	NEG	NEG	NEG
3	Human coronavirus 229E	1.21e5 TCID50/mL	NEG	NEG	NEG	NEG
4	Human coronavirus OC43	1.02e5 TCID50/mL	NEG	NEG	NEG	NEG
5	Human coronavirus HKU1a	1.23e6 copies/mL	NEG	NEG	NEG	NEG
6	Adenovirus Type 1	4.07e5 TCID50/mL	NEG	NEG	NEG	NEG
7	Adenovirus Type 7	1.15e5 TCID50/mL	NEG	NEG	NEG	NEG
8	Cytomegalovirus	1.0e5 TCID50/mL	NEG	NEG	NEG	NEG
9	Echovirus	1.14e5 TCID50/mL	NEG	NEG	NEG	NEG
10	Enterovirus	2.80e5 TCID50/mL	NEG	NEG	NEG	NEG
11	Epstein Barr Virus	5.60e6 TCID50/mL	NEG	NEG	NEG	NEG
12	HSV	1.97e5 TCID50/mL	NEG	NEG	NEG	NEG
13	Human metapneumovirus	4.07e5 TCID50/mL	NEG	NEG	NEG	NEG
14	Human parainfluenza Type 1	1.0e5 TCID50/mL	NEG	NEG	NEG	NEG
15	Human parainfluenza Type 2	1.2e5 TCID50/mL	NEG	NEG	NEG	NEG
16	Human parainfluenza Type 3	1.2e5 TCID50/mL	NEG	NEG	NEG	NEG
17	Human parainfluenza Type 4	1.19e6 TCID50/mL	NEG	NEG	NEG	NEG
18	Measles	1.2e5 TCID50/mL	NEG	NEG	NEG	NEG
19	Mumps virus	1.2e5 TCID50/mL	NEG	NEG	NEG	NEG
20	Rhinovirus Type 1A	1.0e5 TCID50/mL	NEG	NEG	NEG	NEG
21	Acinetobacter baumannii	1.30e7 CFU/mL	NEG	NEG	NEG	NEG
22	Bordetella pertussis	6.40e7 CFU/mL	NEG	NEG	NEG	NEG
23	Burkholderia cepacia	1.90e8 CFU/mL	NEG	NEG	NEG	NEG
24	Candida albicans	6.30e6 CFU/mL	NEG	NEG	NEG	NEG

Table 5-7. Respiratory Microorganisms and Human Coronavirus Tested, Concentrations and Xpert Xpress CoV-2/Flu/RSV plus Test Results

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Image /page/19/Picture/0 description: The image shows the Cepheid logo, which includes a blue graphic and the word "Cepheid." The text below the logo reads "Xpert® Xpress CoV-2/Flu/RSV plus." This text likely refers to a diagnostic test or product offered by Cepheid, possibly related to COVID-19, influenza, and respiratory syncytial virus.

Count	Strain	Tested Concentration	SARS-CoV-2	Flu A	Flu B	RSV
25	Candida parapsilosis	1.45e6 CFU/mL	NEG	NEG	NEG	NEG
26	Citrobacter freundii	1.73e8 CFU/mL	NEG	NEG	NEG	NEG
27	Corynebacterium sp.	1.27e7 CFU/mL	NEG	NEG	NEG	NEG
28	Enterococcus faecalis	5.87e7 CFU/mL	NEG	NEG	NEG	NEG
29	Escherichia coli	1.55e8 CFU/mL	NEG	NEG	NEG	NEG
30	Hemophilus influenzae	6.62e6 CFU/mL	NEG	NEG	NEG	NEG
31	Lactobacillus reuteri b	5.0e7 copies/mL	NEG	NEG	NEG	NEG
32	Legionella pneumophila	1.42e8 CFU/mL	NEG	NEG	NEG	NEG
33	Moraxella catarrhalis	2.46e6 CFU/mL	NEG	NEG	NEG	NEG
34	Mycoplasma pneumoniae	2.7e6 CFU/mL	NEG	NEG	NEG	NEG
35	Neisseria meningitides	4.2e6 CFU/mL	NEG	NEG	NEG	NEG
36	Neisseria mucosa	1.0e8 CFU/mL	NEG	NEG	NEG	NEG
37	Propionibacterium acnes	8.25e7 CFU/mL	NEG	NEG	NEG	NEG
38	Pseudomonas aeruginosa	1.05e7 CFU/mL	NEG	NEG	NEG	NEG
39	Staphylococcus haemolyticus	2.66e6 CFU/mL	NEG	NEG	NEG	NEG
40	Staphylococcus aureus	5.87e7 CFU/mL	NEG	NEG	NEG	NEG
41	Staphylococcus epidermidis	2.47e7 CFU/mL	NEG	NEG	NEG	NEG
42	Streptococcus agalactiae	1.75e7 CFU/mL	NEG	NEG	NEG	NEG
43	Streptococcus pneumoniae	2.26e7 CFU/mL	NEG	NEG	NEG	NEG
44	Streptococcus pyogenes	9.0e6 CFU/mL	NEG	NEG	NEG	NEG
45	Streptococcus salivarius	4.19e6 CFU/mL	NEG	NEG	NEG	NEG
46	Streptococcus sanguinis	8.67e6 CFU/mL	NEG	NEG	NEG	NEG
47	Chlamydia pneumoniae	1.20e6 CFU/mL	NEG	NEG	NEG	NEG
48	Mycobacterium tuberculosis(avirulent)	1.20e6 CFU/mL	NEG	NEG	NEG	NEG

NA - Not Applicable

ªLive virus was not available. Synthetic RNA was used.

bLive organism was not available. Genomic DNA was used.

Microbial Interference

Microbial interference of the Xpert Xpress CoV-2/Flu/RSV plus test caused by the presence of bacterial or viral strains that might be encountered in human upper respiratory tract specimens, was evaluated by testing a panel of 10 potentially interfering microorganisms, consisting of 7 viral strains and 3 bacterial strains. Contrived samples consisted of SARS-CoV-2, Flu A, Flu B, RSV A, or RSV B viruses seeded at 3x the Limit of Detection (LoD) into simulated nasopharyngeal swab (NPS)/nasal swab (NS) matrix in the presence of Adenovirus Type 1C, Human Coronavirus OC43, Rhinovirus Type 1A, Human metapneumovirus, Human parainfluenza Types 1, 2, and 3 (each seeded at 1x109 TCID50mL). Hemophilus influenzae (seeded at 1x100 CFU/mL), Staphylococcus aureus or Staphylococcus epidermidis (each seeded at 1x10' CFU/mL).

Eight (8) replicates of positive samples were tested for each target virus (SARS-CoV-2, Flu A, Flu B, RSV A, or RSV B) and each potential microbial interference strain combination. For each target, all 8 of 8 replicate samples were correctly identified using the Xpert Xpress CoV-2/Flu/RSV plus test. No microbial interference by the viral or bacterial strains was reported.

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Image /page/20/Picture/0 description: The image shows the Cepheid logo with the text "Xpert® Xpress CoV-2/Flu/RSV plus" below it. The Cepheid logo is a stylized blue graphic element. The text is in a sans-serif font, with "Xpert" having a registered trademark symbol.

Competitive Interference

Competitive interference of the Xpert Xpress CoV-2/Flu/RSV plus caused by co-infections were evaluated by testing contrived samples of individual SARS-CoV-2, Flu A. Flu B or RSV strains at 3x LoD in the presence of different target strains at a higher concentration in a simulated background matrix. The concentration at 3x LoD was 414 copies/mL for SARS-CoV-2 (inactivated USA-WA1/2020); 0.021 TCIDso/mL for Flu A/Idaho/072018, 38.7 CEIDso/mL for Flu B/Washington/2/2019; 0.99 TCIDso/mL for RSV A/2/Australia/61), and 1.11 TCID50/mL for RSV B/9320/MA/77. The competitive strains were evaluated at >108 RNA copies/mL, as determined by droplet digital PCR (ddPCR).

Replicates of 3 were tested for each target strain and each competitive strain combination. The virus at high concentration shows no competitive inhibitory effects if 3 of 3 replicates for the target strain report positive results. If the results reported less than 3 of 3 positive replicates, the concentration of the competing virus was reduced by 10-fold increments until no interference was observed. The results for competitive interference study are presented in Tables 5-8 through 5-12 for high concentration of Flu A, Flu B, RSV A, RSV B and SARS-CoV-2, respectively.

Table 5-8. Summary of Competitive Interference Study with Flu A at High Concentration

Test Viruses at3X LoD	InterferentVirus	Correct Calls (n/3)
		at 1.7e8RNAcopies/mL	at 1.7e7RNAcopies/mL	at 1.7e6RNAcopies/mL	at 1.7e5RNAcopies/mL
Flu B	Flu A	0/3	0/3	2/3	3/3
RSV A	Flu A	0/3	0/3	3/3	Not tested
RSV B	Flu A	3/3	Not tested	Not tested	Not tested
SARS-CoV-2	Flu A	3/3	Not tested	Not tested	Not tested

				Table 5-9. Summary of Competitive Interference Study with Flu B at High Concentration

Test Virusesat 3X LoD	InterferentVirus	Correct Calls(n/3)at 1.4e5RNA copies/mL
Flu A		3/3
RSV A	Flu B	3/3
RSV B		3/3
SARS-CoV-2		3/3

Table 5-10. Summary of Competitive Interference Study with RSV A at High Concentration
Interferent	Concentration	% Inhibition
RSV B	100 ng/mL	0%
Influenza A	100 ng/mL	0%
hMPV	100 ng/mL	0%

Test Virusesat 3X LoD	InterferentVirus	Correct Calls(n/3)at 4.6e6RNA copies/mL
Flu A		3/3
Flu B	RSV A	3/3
SARS-CoV-2		3/3

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Test Viruses at3X LoD	InterferentVirus	Correct Calls (n/3)at 1.9e5RNA copies/mL
Flu A		3/3
Flu B	RSV B	3/3
SARS-CoV-2		3/3

			Table 5-11. Summary of Competitive Interference Study with RSV B at High Concentration

Table 5-12. Summary of Competitive Interference Study with SARS-CoV-2 at High Concentration

Test Virusesat 3X LoD	InterferentVirus	Correct Calls (n/3)
		at 1e6RNA copies/mL	at 1e5RNA copies/mL
Flu A	SARS-CoV-2	3/3	Not tested
Flu B	SARS-CoV-2	1/3	3/3
RSV A	SARS-CoV-2	3/3	Not tested
RSV B	SARS-CoV-2	3/3	Not tested

The study showed that Flu A/Idaho/07/2018 at concentrations above 1.7e5 RNA copies/mL inhibited detection of Flu B at 3x LoD, and at concentrations above 1.7e6 RNA copies/mL inhibited detection of RSV A at 3x LoD (Table 5-8). In addition, SARS-CoV-2 at concentrations above 1e5 RNA copies/mL inhibited detection of Flu B at 3x LoD (Table 5-12). No other competitive interference was observed for the potential co-infections evaluated in the study at the concentrations tested.

Potentially Interfering Substances

Substances that are normally found in or may be introduced into clinical NPS or NS matrix that could potentially interfere with accurate detection of SARS-CoV-2, Flu A. Flu B and RSV were evaluated with direct testing on the Xpert Xpress CoV-2/Flu/RSV plus.

Potentially interfering substances in the nasal passage and 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. Positive and negative samples were prepared in simulated nasopharyngeal swab (NPS)/ nasal swab (NS) matrix. Negative samples (N = 8) were tested in the presence of each substance to determine the effect on the performance of the sample processing control (SPC). Positive samples (N = 8) were tested per substance with viruses spiked at 3x the LoD determined for each strain. Positive samples tested with the Xpert Xpress CoV-2/Flu/RSV plus included one SARS-CoV-2, two influenza A HIN1, two influenza A H3N2, two influenza B and two RSV (RSV A and RSV B) strains. The substances, with active ingredients and test concentrations, that were evaluated are listed in Table 5-13.

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Substance ID	Substance/Class	Substance/Active Ingredient	Concentrations Tested
No substance	Control	Simulated NPS/NS Matrix	100% (v/v)
Albuterol Sulfate	Beta-adrenergicbronchodilator	Albuterol Sulfate (5mg/mL)	0.83 mg/mL (equivalentto 1 dose per day)
Afrin	Nasal Spray	Oxymetazoline, 0.05%	15% (v/v)
BD UniversalTransport Medium	Transport Media	N/A	100% (v/v)
Blood	Blood	Blood (Human)	2% (v/v)
Copan Swab M	Transport Media	N/A	100% (v/v)
FluMist Quadrivalent	Vaccine	Live attenuated influenzaviruses	6.7e-4% (v/v)6.7e-6% (v/v)6.7e-7% (v/v)
FluticasonePropionate NasalSpray	Nasal corticosteroid	Fluticasone Propionate	5 µg/mL
Human peripheralblood mononuclearcells	Human cells	PBMC	1 x 106 cells/mL0.5 x 106 cells/mL0.25 x 106 cells/mL
Ibuprofen	Nonsteroidal anti-inflammatory drug	Ibuprofen 200 mg/tablet	5% w/v
Menthol	Throat lozenges, oralanesthetic and analgesic	Benzocaine, Menthol	1.7 mg/mL
Mucin	Mucin	Purified Mucin protein (Bovineor porcine submaxillary gland)	0.1 (w/v)
Mupirocin	Antibiotic, nasalointment	Mupirocin (20 mg/g = 2%)	10 mg/mL
PHNY	Nasal Drops	Phenylephrine, 1%	15% (v/v)
Remel M4RT	Transport Media	N/A	100% (v/v)
Remel M5	Transport Media	N/A	100% (v/v)
Saline	Saline Nasal Spray	Sodium Chloride (0.65%)	15% (v/v)
Snuff	Tobacco product	Nicotine	1% (w/v)0.5% (w/v)0.25% (w/v)0.1% (w/v)
Tamiflu	Anti-viral drugs	Zanamivir	7.5 mg/mL
Tobramycin	Antibacterial, systemic	Tobramycin	4 µg/mL
Zicam	Nasal Gel	Luffa opperculata, Galphimiaglauca, Histaminumhydrochloricum Sulfur (0.05%)	15% (w/v)7.5% (w/v)
Zinc	Zinc supplement	Zinc Gluconate	0.1 µg/mL

	Table 5-13. Potentially Interfering Substances Tested
--	-------------------------------------------------------	--	--	--

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Image /page/23/Picture/0 description: The image shows the Cepheid logo, which includes a blue graphic and the word "Cepheid." in black text. Below the logo is the text "Xpert® Xpress CoV-2/Flu/RSV plus" in black text. The text describes a test for CoV-2, Flu, and RSV.

The results from the study (Table 5-14) show that for most cases, 8 out of 8 replicates reported positive results for each combination of virus and substance tested and no interference was observed. In the presence of FluMist at 6.7e-4% (v/v), interfering effects were observed when testing SARS-CoV-2, RSV A and RSV B strains. Inhibitory effects were not observed when testing these viruses in the presence of FluMist at 6.7e-6% (v/v) except for RSV A/Long/MD/56. For RSV A/Long/MD/56, the inhibitory effect was not observed when FluMist concentration was further reduced to 6.7e-7% (v/v). In the presence of human PBMC at 1 x 10° cells/mL, interfering effects were observed when testing Flu B/Washington /2/2019. Inhibitory effects were not observed when the PBMC concentration was reduced to 2.5 x 10° cells/mL. In the presence of snuff at 1% (w/v), interfering effects were observed when testing Flu A /California/07/2009 and Flu B/Washington/2/2019. Inhibitory effects were not observed when testing the viruses at a snuff concentration of 0.1% (w/v). In the presence of Zicam at 15% (w/v), interfering effects were observed when testing Flu A, Flu B and RSV A strains. Inhibitory effects were not observed when testing the viruses in the presence of Zicam at 7.5% (w/v).

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Cepheid.

Substance	ConcentrationTested	No Virus Control	SARS-CoV-2USA-WA-1	Flu ACalifornia/7/2009	Flu A Idaho/07/2018	Flu AHong Kong/ 45/2019	Flu A/Victoria/361/2011	Flu BWisconsin/10/2016	Flu BWashington/02/2019	RSV A2/Australia/61	RSV ALong/MD/56	RSV B9320/MA/77	RSV BWA/18537/62
ControlSimulatedNPS/NS Matrix(No substance)	100% (v/v)	32/32a	24/24	24/24	16/16	16/16	24/24b	24/24	32/32	32/32b	32/32	24/24	24/24
AlbuterolSulfate	0.83 mg/mL	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Afrin	15% (v/v)	16/16	8/8	8/8b	8/8	8/8	8/8	8/8	8/9c	8/8	8/8	8/8	8/8
BD UniversalTransportMedium	100% v/v	16/16	8/8	8/8	8/8	8/8	8/8b	8/8	8/8	8/8	8/8	8/8	8/8
Blood	2% (v/v)	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8a	8/8b	8/8b	8/8	8/8
Copan Swab M	100% (v/v)	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
FluMist	6.7% (v/v)	8/8	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
	6.7e-4% (v/v)	N/A	7/8	N/A	N/A	N/A	N/A	N/A	N/A	0/8	0/8	2/8	0/8
	6.7e-6% (v/v)	N/A	8/8	N/A	N/A	N/A	N/A	N/A	N/A	8/8	7/8	8/8b	8/8
	6.7e-7% (v/v)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	8/8a	N/A	N/A
FluticasonePropionateNasal Spray	5 µg/mL	16/16	8/8	8/8	8/8	8/8	8/8	8/8a, b	8/8	8/8d	8/8	8/8a	8/8
Humanperipheral bloodmononuclearcells	1e6 cells/mL	8/8	8/8	8/8b	8/8b	8/8	8/8	8/8	6/8	8/8b	8/8	8/8b	8/8b
	0.5e6 cells/mL	N/A	N/A	N/A	N/A	N/A	N/A	N/A	7/8	N/A	N/A	N/A	N/A
	0.25e6cells/mL	N/A	N/A	N/A	N/A	N/A	N/A	N/A	8/8	N/A	N/A	N/A	N/A
Ibuprofen	5% (w/v)	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Substance	ConcentrationTested	No Virus Control	SARS-CoV-2USA-WA-1	Flu ACalifornia/7/2009	Flu A Idaho/07/2018	Flu AHong Kong/45/2019	Flu A/Victoria/361/2011	Flu BWisconsin/10/2016	Flu BWashington/02/2019	RSV A2/Australia/61	RSV ALong/MD/56	RSV B9320/MA/77	RSV BWA/18537/62
Menthol	1.7 mg/mL	16/16a	8/8	8/8	8/8	8/8a	8/8	8/8b	8/8	8/8	8/8b	8/8	8/8
Mucin	0.1% (w/v)	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8a, b	8/8	8/8
Mupirocin	10 mg/mL	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
PHNY	15% (v/v)	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Remel M4RT	100% (v/v)	16/16a	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Remel M5	100% (v/v)	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Saline	15% (v/v)	16/16	8/8	8/8a	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8a
Snuff	1% (w/v)	8/8	8/8	6/8	8/8	8/8b	8/8	8/8	4/8b	8/8	8/8	8/8	8/8e
	0.5% (w/v)	N/A	N/A	7/8	N/A	N/A	N/A	N/A	3/8	N/A	N/A	N/A	N/A
	0.25 % (w/v)	N/A	N/A	8/8	N/A	N/A	N/A	N/A	7/8	N/A	N/A	N/A	N/A
	0.1 % (w/v)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	8/8	N/A	N/A	N/A	N/A
Tamiflu	7.5 mg/mL	16/16a	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Tobramycin	4 $ \mu $ g/mL	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Zicam	15% (w/v)	16/16	8/8	7/8	8/8	8/8	8/8	8/8a	5/8	7/8	8/8	8/8	8/8
Zicam	7.5% (w/v)	N/A	N/A	8/8	N/A	N/A	N/A	N/A	8/8	8/8	N/A	N/A	N/A
Zinc	0.1 $ \mu $ g/mL	16/16	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8

Table 5-14. Number of Correct Results for Xpert Xpress CoV-2/Flu/RSV plus Targets Tested in the Presence of Potentially Interfering Substances

510(k) Summary

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Image /page/25/Picture/0 description: The image shows the Cepheid logo. The logo consists of a blue graphic element resembling three curved lines or feathers, positioned above the word "Cepheid" in a blue, sans-serif font. A small circle with a dot in the center is placed to the right of the word "Cepheid".

Xpert® Xpress CoV-2/Flu/RSV plus

BOLD: False negative or INVALID results indicating interference from the substance

a. One replicate reported NO RESULT. The run was successfully repeated to obtain the required number of valid replicates.

b. One replicate reported ERROR. The run was successfully repeated to obtain the required number of valid replicates

c. One of 8 replicates reported a Flu B NEGATIVE result. The Flu B Probe check signals were reduced in this sample suggesting an issue with the EZR beat. The test was repeated and gave a Flu B positive result.

d. One of 8 replicates reported INVALID. The run was successfully repeated to obtain 8 valid replicates

e. Two of 8 replicates reported ERROR. The 2 runs were successfully repeated to obtain 8 valid replicates.

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Image /page/26/Picture/0 description: The image shows the Cepheid logo and the text "Xpert® Xpress CoV-2/Flu/RSV plus". The Cepheid logo is a stylized blue image. The text is in a sans-serif font.

Carryover Contamination

A study was conducted to assess whether the single-use, self-contained Xpert Xpress CoV-2/Flu/RSV plus cartridge prevents specimen and amplicon carryover by testing a negative sample immediately after testing of a very high positive sample in the same GeneXpert module. The negative sample used in this study consisted of simulated NPS/NS matrix and the positive sample consisted of high Flu B and high SARS-CoV-2 virus concentrations (Flu B/Wisconsin/10/2016 at 1.0e6 TCID50/mL and inactivated SARS-CoV-2 USA-WA1/2020 at 1e4 copies/mL) seeded into simulated NPS/NS matrix. The negative sample was tested in a GeneXpert module at the start of the study. Following the initial testing of the negative sample, the high positive sample was processed in the same GeneXpert module immediately followed by another negative sample. This was repeated 20 times in the same module, resulting in 20 positives and 21 negatives for the module. The study was repeated using a second GeneXpert module for a total of 40 positive and 42 negative samples. All 40 positive samples were correctly reported as SARS-CoV-2 POSITIVE; Flu A NEGATIVE; Flu B POSITIVE; RSV NEGATIVE. All 42 negative samples were correctly reported as SARS-CoV-2 NEGATIVE; Flu A NEGATIVE; Flu B NEGATIVE; RSV NEGATIVE with the Xpert Xpress CoV-2/Flu/RSV plus test. No specimen or amplicon carry-over contamination was observed in this study.

Single-Site Precision

The precision of the Xpert Xpress CoV-2/Flu/RSV plus test was established at a single site using a 9-member panel including one negative sample, 4 low positive (~1.5x LoD) samples and 4 moderate positive (~3x LoD) samples. The negative sample consisted of simulated matrix without target microorganism or target RNA. The positive samples were contrived using inactivated NATtrol SARS-CoV-2 (ZeptoMetrix, Buffalo, NY, catalog number NATSARS(COV2)-ST), and cultured viruses Influenza A/Idaho/07/2018, Influenza B/Wisconsin/10/2016, and RSV B/Wash/18537/62 in a simulated NPS/NS matrix.

Testing was conducted over 20 days, using 1 lot of Xpert Xpress CoV-2/Flu/RSV plus cartridges at a single site and with 1 operator to yield a total of 80 observations per panel member (1 Site x 1 Operator x 1 Lot x 20 Days x 2 Runs x 2 Replicates = 80 observations/panel member). The results from the study are summarized in Table 5-15.

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Panel Member	Agreement	% Agreement (95% CI)
Negative	80/80	100%(95.4%-100%)
SARS-CoV-2Low Positive (~1.5x LoD)	79/80	98.8%(93.3%-99.8%)
SARS-CoV-2Moderate Positive (~3x LoD)	80/80	100%(95.4%-100%)
Flu ALow Positive (~1.5x LoD)	78/80	97.5%(91.3%-99.3%)
Flu AModerate Positive (~3x LoD)	80/80	100%(95.4%-100%)
Flu BLow Positive (~1.5x LoD)	77/80	96.3%(89.5%-98.7%)
Flu BModerate Positive (~3x LoD)	80/80	100%(95.4%-100%)
RSVLow Positive (~1.5x LoD)	78/80	97.5%(91.3%-99.3%)
RSVModerate Positive (~3x LoD)	80/80	100%(95.4%-100%)

Table 5-15. Summary of the Precision Results by Panel Member - % Agreement

Reproducibility

The reproducibility of the Xpert Xpress CoV-2/Flu/RSV plus test was established at 3 sites (2 external and 1 internal) using a 9-member panel including 1 negative sample, 4 low positive (~1.5x LoD) and 4 moderate positive (~3x LoD) samples. The negative sample consisted of simulated matrix without target microorganism or target RNA. The positive samples were contrived using inactivated NATtrol SARS-CoV-2 (ZeptoMetrix), cultured viruses Influenza A/ Idaho/07/2018, Influenza B/Wisconsin/ 10/2016, and RSV B/Wash/18537/62 in a simulated NPS/NS matrix. Testing was conducted over 6 days, using 3 lots of Xpert Xpress CoV-2/Flu/RSV plus cartridges at 3 participating sites each with 2 operators to yield a total of 144 observations per panel member (3 Sites x 2 Operators x 3 Lots x 2 Days/Lot x 2 Runs x 2 Replicates = 144 observations/panel member). The results from the study are summarized in Tables 5-16 and 5-17.

The percent agreement of the correct results compared to the expected results analyzed by each of the 6 operators and each site is shown in Table 5-17. In addition, the overall percent agreement for each sample (% total agreement) and the two-sided Wilson Score confidence intervals (CI) are presented in the last column.

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Image /page/28/Picture/0 description: The image shows the Cepheid logo. The logo consists of a stylized blue wing-like design on the left, followed by the word "Cepheid" in a lowercase, sans-serif font. A small circle is present to the right of the word, likely a registration mark.

Xpert® Xpress CoV-2/Flu/RSV plus

		Site 1				Site 2				Site 3
Sample	Op 1	Op 2	Site	Op 1	Op 2	Site	Op 1	Op 2	Site
Negative	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]
SARS-CoV-2Low Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]
SARS-CoV-2Mod Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]
Flu ALow Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]
Flu AMod Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]
Flu BLow Pos	100%24/24	100%24/24	100%48/48	95.8%23/24	95.8%23/24	95.8%46/48	100%24/24	100%24/24	100%48/48	98.6% (142/144)[95.1-99.6]
Flu BMod Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%23/23	95.8%23/24	97.9%46/47	99.3% (142/143a)[96.1-99.9]
RSVLow Pos	100%24/24	100%24/24	100%48/48	95.8%23/24	100%24/24	97.9%47/48	100%24/24	100%24/24	100%48/48	99.3% (143/144)[96.2-99.9]
RSVMod Pos	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100%24/24	100%24/24	100%48/48	100% (144/144)[97.4-100.0]

Table 5-16. Summary of the Reproducibility Results - % Agreement

a. One replicate was excluded because it was run on external positive control produced an incorrect result but was inadvertently not reested.

The evaluation of reproducibility and within-laboratory precision of the underlying analyte Ct values for the Xpert Xpress COV-2/Flu/RSV plus test was analyzed using nested Analysis of Variance (ANOVA). The mean Ct, standard deviation (SD), and coefficient of variation (%CV) between-sites, between-lots, between-days, between-runs and within-run for each panel member are presented in Table 5-17.

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Image /page/29/Picture/9 description: The image shows the Cepheid logo. The logo consists of a blue graphic element resembling stylized wings or feathers, positioned to the left of the word "Cepheid." The word "Cepheid" is written in a simple, sans-serif font, and there is a small circle or dot to the right of the word.

Xpert® Xpress CoV-2/Flu/RSV plus

				Variance Source
				Site		OP		Lot		Day		Run		Within-Run		Total
Sample	Analyte	N	Mean Ct	SD	CV(%)	SD	CV(%)	SD	CV(%)	SD	CV(%)	SD	CV(%)	SD	CV(%)	SD	CV(%)
Negative	SPC	144	30.8	0.1	0.4	0.0	0.0	0.9	2.9	0.5	1.5	0.0	0.0	1.3	4.2	1.6	5.3
SARS-CoV-2Low Pos	SARS-CoV-2	144	37.4	0.0	0.0	0.2	0.5	0.1	0.2	0.0	0.0	0.3	0.7	0.4	1.1	0.5	1.4
SARS-CoV-2Mod Pos	SARS-CoV-2	144	36.2	0.0	0.1	0.1	0.3	0.0	0.0	0.1	0.3	0.2	0.4	0.4	1.0	0.4	1.2
Flu ALow Pos	Flu A1	144	35.7	0.2	0.6	0.0	0.0	0.1	0.2	0.0	0.0	0.0	0.0	0.6	1.6	0.6	1.7
Low Pos	Flu A2	135b	37.9	0.3	0.8	0.0	0.0	0.2	0.5	0.0	0.0	0.4	1.1	0.9	2.5	1.1	2.9
Flu AMod Posa	Flu A1	144	34.7	0.0	0.0	0.1	0.2	0.0	0.0	0.1	0.3	0.0	0.0	0.4	1.2	0.4	1.3
	Flu A2	144	36.6	0.0	0.1	0.0	0.0	0.2	0.4	0.0	0.0	0.0	0.0	0.5	1.5	0.6	1.5
Flu BLow Pos	Flu B	144	36.3	0.3	0.8	0.0	0.1	0.0	0.0	0.1	0.2	0.3	0.7	0.7	2.1	0.8	2.3
Flu BMod pos	Flu B	142c	35.1	0.0	0.0	0.1	0.4	0.1	0.3	0.3	0.8	0.0	0.0	0.7	2.0	0.8	2.2
RSVLow Pos	RSV	144	35.8	0.1	0.2	0.0	0.0	0.2	0.4	0.0	0.0	0.0	0.0	0.6	1.7	0.6	1.8
RSVMod Pos	RSV	144	34.8	0.1	0.2	0.0	0.0	0.1	0.4	0.0	0.0	0.2	0.5	0.5	1.4	0.5	1.5

Table 5-17. Summary of Nested ANOVA by Coefficient of Variation

One replicate from Site 2 used the reagent lot 102 instead of the intended reagent lot 401. In the nested ANOVA analysis, this replicated under the a. intended lot 401 following the principle of "analyze as intended".

Nine replicates were excluded due to zero Flu A2 Ct values. b.

One replicate was excluded due to the B C. value; one replicate was excluded due to the incorrect external positive control result preceding the retest. C.

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5.4.2. Clinical Performance

The clinical performance of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated in a multi-site, observational and method comparison study that included 33 geographically diverse sites in the United States (US) using specimens collected from individuals showing signs and symptoms of respiratory infection. Of the 33 sites participated in specimen collection only, 27 performed Xpert testing and specimen collection, and 1 site performed Xpert testing as well as comparator and discrepant testing.

Specimens tested included prospective clinical NPS and NS specimens collected in UTM/VTM. Prospectively collected fresh clinical specimens (Category I) tested in the study were from a larger US specimen collection protocol. Fresh (98.9%) and frozen (1.1%) specimens meeting the eligibility criteria were prospectively collected and tested in 2022. Due to low prevalence of Flu/RSV in 2022, archived prospectively collected frozen clinical specimens (Category II) collected during the 2016-2017 influenza season were used to supplement the sample size. These specimens represent contemporary Flu/RSV strains. Since these specimens were collected prior to the COVID-19 pandemic, they were expected to be negative for SARS-CoV-2 and therefore tested only for the Flu A, Flu B, and RSV targets. Available demographic data from the individuals from whom Category I and Category II specimens were collected are presented in Table 5-18.

Prospectively CollectedFresh and Frozen Specimensfrom 2022(Category I and Category II)	NP Swab(N=2672)	NS(N=2659)	Overall(N=5331)
Gender
Female	1568 (58.7%)	1634 (61.5%)	3202 (60.1%)
Male	1104 (41.3%)	1025 (38.5%)	2129 (39.9%)
Age Group (Years)
≤5	9 (0.3%)	183 (6.9%)	192 (3.6%)
6-21	623 (23.3%)	562 (21.1%)	1185 (22.2%)
22-59	1676 (62.7%)	1553 (58.4%)	3229 (60.6%)
≥60	364 (13.6%)	361 (13.6%)	725 (13.6%)
Race
American Indian or AlaskaNative	5 (0.2%)	6 (0.2%)	11 (0.2%)
Asian	73 (2.7%)	80 (3.0%)	153 (2.9%)
Asian, White	8 (0.3%)	1 (0.0%)	9 (0.2%)
Black or African American	734 (27.5%)	730 (27.5%)	1464 (27.5%)
Black or African American,White	10 (0.4%)	12 (0.5%)	22 (0.4%)
Native Hawaiian or Other PacificIslander	6 (0.2%)	2 (0.1%)	8 (0.2%)
Other Mixed (N≤3)	4 (0.1%)	5 (0.2%)	9 (0.2%)

				Table 5-18. Demographic Summary for Category I and II Specimens
--	--	--	--	-----------------------------------------------------------------

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Image /page/31/Picture/0 description: The image contains the logo for Cepheid. The logo consists of a stylized blue wing-like graphic on the left, followed by the word "Cepheid." in a sans-serif font. A small circle is present to the right of the word.

Xpert® Xpress CoV-2/Flu/RSV plus

Prospectively CollectedFresh and Frozen Specimensfrom 2022(Category I and Category II)	NP Swab(N=2672)	NS(N=2659)	Overall(N=5331)
White	1685 (63.1%)	1641 (61.7%)	3326 (62.4%)
Participant Declined to Answer,or Unknown	147 (5.5%)	182 (6.8%)	329 (6.2%)
Ethnicity
Hispanic	228 (8.5%)	213 (8.0%)	441 (8.3%)
Non-Hispanic	2333 (87.3%)	2323 (87.4%)	4656 (87.3%)
Participant Declined to Answer,or Unknown	111 (4.2%)	123 (4.6%)	234 (4.4%)
Specimen Testing
Fresh	2641 (98.8%)	2631 (98.9%)	5272 (98.9%)
Frozen	31 (1.2%)	28 (1.1%)	59 (1.1%)
COVID-19 Vaccination Status
Vaccinated	1969 (73.7%)	1865 (70.1%)	3834 (71.9%)
Not Vaccinated	665 (24.9%)	764 (28.7%)	1429 (26.8%)
Unknown	38 (1.4%)	30 (1.1%)	68 (1.3%)
Testing Environment
CLIA Waiver	1603 (60.0%)	1619 (60.9%)	3222 (60.4%)
Laboratory/NPT	1069 (40.0%)	1040 (39.1%)	2109 (39.6%)
Prospectively Collected FrozenSpecimens from 2016-2017Influenza Season(Category II)	NPS(N=422)	NS(N=368)	Overall(N=790)
Gender
Female	211 (50.0%)	223 (60.6%)	434 (54.9%)
Male	211 (50.0%)	145 (39.4%)	356 (45.1%)
Age Group (Years)
<5	164 (38.9%)	144 (39.1%)	308 (39.0%)
6-21	85 (20.1%)	72 (19.6%)	157 (19.9%)
22-59	134 (31.8%)	111 (30.2%)	245 (31.0%)
>60	39 (9.2%)	41 (11.1%)	80 (10.1%)

Specimens were tested using Xpert Xpress CoV-2/Flu/RSV plus side-by-side with a U.S.FDA-cleared molecular respiratory panel that includes SARS-CoV-2 and a U.S. FDAcleared molecular Flu A/B/RSV assay, in a randomized and blinded fashion.

Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA), and nondeterminate rate were determined by comparing the results of the Xpert Xpress CoV-2/Flu/RSV plus test relative to the results of a U.S. FDA-cleared molecular respiratory panel for the SARS-CoV-2 target, and a U.S.FDA-cleared molecular Flu A/B/RSV assay for the Flu A, Flu B, and RSV targets, respectively.

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Discrepant results between Xpert Xpress CoV-2/Flu/RSV plus and the comparator for the SARS-CoV-2 target were investigated using a U.S. FDA EUA SARS-CoV-2 molecular test. Discrepant results between the Xpert Xpress CoV-2/Flu/RSV plus and the comparator for the Flu A/B/RSV targets were investigated using a U.S. FDA-cleared molecular respiratory panel.

A total of 5051 specimens, including 2536 NPS and 2515 NS specimens that vielded valid results by both the Xpert Xpress CoV-2/Flu/RSV plus and the U.S. FDA-cleared molecular respiratory panel were included in the performance evaluation for SARS-CoV-2. A total of 5954 specimens, including 3011 NPS and 2943 NS (specimens that yielded valid results by both the Xpert Xpress CoV-2/Flu/RSV plus and the U.S. FDA-cleared molecular Flu A/B/RSV assay were included in the performance evaluation for Flu A, Flu B, and RSV targets.

For the NPS specimens (both fresh and frozen specimens, combined), Xpert Xpress CoV-2/Flu/RSV plus demonstrated a PPA and NPA of 97.1% and 98.2% for SARS-CoV-2, respectively: 99.0% and 99.1% for Flu A. respectively: 96.6% and 100.0% for Flu B, respectively; 98.6% and 100.0% for RSV, respectively (Table 5-19). The initial nondeterminate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 2.4% (74/3094). On repeat testing, 66 specimens yielded valid results. The final non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 0.3% (8/3094).

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Target	SpecimenCollection	Numbers ofSpecimens	TP	FP	TN	FN	PPA(%)	95%CI	NPA(%)	95%CI
SARS-CoV-2	Fresh	2505	454	37a	2000	14b	97	95.0 - 98.2	98.2	97.5 - 98.7
	Frozen	31	8	0	23	0	100	67.6 - 100.0	100	85.7- 100.0
	Overall	2536	462	37	2023	14	97.1	95.1 - 98.2	98.2	97.5-98.7
Flu A	Fresh	2562	98	11c	2451	2d	98	93.0 - 99.5	99.6	99.2 - 99.8
	Frozen	449	93	13e	343	0	100	96.0 - 100.0	96.3	93.9 - 97.9
	Overall	3011	191	24	2794	2	99.0	96.3 - 99.7	99.1	98.7 - 99.4
Flu B	Fresh	2562	0	0	2562	0	NA	NA	100	99.9 - 100.0
	Frozen	449	57	0	390	2f	96.6	88.5 - 99.1	100	99.0 - 100.0
	Overall	3011	57	0	2952	2	96.6	88.5 - 99.1	100	99.9 - 100.0
RSV	Fresh	2562	12	0	2550	0	100	75.8 - 100.0	100	99.8 - 100.0
	Frozen	449	59	0	389	1g	98.3	91.1 - 99.7	100	99.0 - 100.0
	Overall	3011	71	0	2939	1	98.6	92.5 - 99.8	100	99.9 - 100.0

Table 5-19. Xpert Xpress CoV-2/Flu/RSV plus Performance Results for NPS Specimens

TP: True Positive; FP: False Positive; TN: True Negative; C1: 95% two-sided Confidence Interval

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 15/37 SARS-CoV-2 positive; a. 22/37 SARS-CoV-2 negative

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 3/14 SARS-CoV-2 positive; b. 10/14 SARS-CoV-2 negative; 1/14 invalid result

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 8/11 Flu A positive; 3/11 Flu A C. negative

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 1/2 Flu A d. negative

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 13/13 tests not performed due e. to specimens being stored for a longer duration than recommended per the package insert

f. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/2 test not performed due to specimens being stored for a longer duration than recommended per the package insert

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 1/1 test not performed due to g. specimens being stored for a longer duration than recommended per the package insert

For the NS specimens (both fresh and frozen specimens, combined), Xpert Xpress CoV-2/Flu/RSV plus demonstrated a PPA and NPA of 98.2% and 98.8% for SARS CoV-2, respectively; 98.0% and 99.3% for Flu A, respectively; 100.0% and 99.9% for Flu B, respectively; 95.8% and 100.0% for RSV, respectively (Table 5-20). The initial nondeterminate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 2.4% (74/3027). On repeat testing, 57 specimens gave valid results upon retest. The final non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 0.6% (17/3027).

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Target	SpecimenCollection	Numbers ofSpecimens	TP	FP	TN	FN	PPA(%)	95%CI	NPA(%)	95%CI
	Fresh	2489	442	23a	2017	7b	98.4	96.8 - 99.2	98.9	98.3 - 99.2
SARS-CoV-2	Frozen	26	6	1c	18	1d	85.7	48.7 - 97.4	94.7	75.4 - 99.1
	Overall	2515	448	24	2035	8	98.2	96.6 - 99.1	98.8	98.3 - 99.2
	Fresh	2553	130	6e	2413	4f	97.0	92.6 - 98.8	99.8	99.5 - 99.9
Flu A	Frozen	390	66	12g	312	0	100	94.5 - 100.0	96.3	93.6 - 97.9
	Overall	2943	196	18	2725	4	98.0	95.0 - 99.2	99.3	99.0 - 99.6
	Fresh	2553	0	0	2553	0	NA	NA	100	99.8 - 100.0
Flu B	Frozen	390	34	3h	353	0	100	89.8 - 100.0	99.2	97.6 - 99.7
	Overall	2943	34	3	2906	0	100	89.8 - 100.0	99.9	99.7 - 100.0
	Fresh	2553	14	0	2538	1i	93.3	70.2 - 98.8	100	99.8 - 100.0
RSV	Frozen	390	55	0	333	2j	96.5	88.1 - 99.0	100	98.9 - 100.0
	Overall	2943	69	0	2871	3	95.8	88.5 - 98.6	100	99.9 - 100.0

Table 5-20. Xpert Xpress CoV-2/Flu/RSV plus Performance Results for NS Specimens

TP: True Positive; FP: False Positive; TN: True Negative; C1: 95% two-sided Confidence Interval

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 6/23 SARS-CoV-2 positive; a. 14/23 SARS-CoV-2 negative; 2/23 invalid result; 1/23 discrepant testing was inadvertently not performed

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 2/7 SARS-CoV-2 positive; 5/7 b. SARS-CoV-2 negative

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 1/1 SARS-CoV-2 positive C.

d. Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 1/1 SARS-CoV-2 negative
e. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 5/6 Flu A negative
f. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/4 Flu A negative
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 12/12 tests not performed due g. to specimens being stored for a longer duration than recommended per the package insert

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 3/3 tests not performed due to h. specimens being stored for a longer duration than recommended per the package insert

i. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 1/1 RSV positive

j. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/2 test not performed due to specimens being stored for a longer duration than recommended per the package insert

The number of specimens with positive results for more than one target as detected by Xpert Xpress CoV-2/Flu/RSV plus is presented in Table 5-21 and Table 5-22, where bolded values indicate concordant results.

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Infection		Comparator Results					Total	Co-InfectionRate (%)
		SARS-CoV-2only	SARS-CoV-2 andFlu A	Flu A only	RSV only	Negative
Xpert Xpress CoV-2/Flu/RSV plus	SARS-CoV-2 only	876	1	0	0	57	934
	SARS-CoV-2 and Flu A	0	2	1	0	0	3
	Flu A only	0	0	220	0	17	237	0.3
	RSV only	0	0	0	26	0	26
	Negative	22	0	5	1	3693	3721
	Total	898	3	226	27	3767	4921
	Co-Infection Rate (%)	0.3

Table 5-21. Multi-Target Detection by Xpert Xpress CoV-2/Flu/RSV plus for Specimens Collected in 2022

As presented in Table 5-21, a total of 4921 Category I specimens collected in 2022 yielded valid results for SARS-CoV-2, Flu A, and RSV targets for both the Xpert Xpress CoV-2/Flu/RSV plus test and the comparator test. The co-infection rate for Xpert Xpress CoV-2/Flu/RSV plus was 0.3% (3/1200) and the rate of co-infection by the comparator was 0.3% (3/1154).

Table 5-22. Flu A, Flu B, and RSV Multi-Target Detection by Xpert Xpress CoV-2/Flu/RSV plus for Specimens Collected in 2016-2017 and 2022

Infection		Comparator						Total	Co-InfectionRate (%)
		Flu A only	Flu B Only	RSV Only	Flu A and Flu B	Flu A and RSV	Flu B and RSV			Negative
Xpert Xpress CoV-2/Flu/RSV plus	Flu A only	381	0	0	1	1	0	36	419
	Flu B Only	0	85	0	0	0	0	2	87
	RSV Only	0	0	135	0	0	0	0	135
	Flu A and Flu B	0	4	0	1	0	0	1	6
	Flu A and RSV	0	0	1	0	3	0	0	4	1.7
	Flu B and RSV	0	0	0	0	0	1	0	1
	Negative	6	1	3	0	0	0	5292	5302
Total		387	90	139	2	4	1	5331	5954
Co-InfectionRate (%)		1.1

As presented in Table 5-22, of the 5954 Category I and II specimens evaluated for Flu A, Flu B and RSV targets, the co-infection rate for Xpert Xpress CoV-2/Flu/RSV plus was 1.7% (11/652) and the rate of co-infection by the comparator was 1.1% (7/623).

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Image /page/36/Picture/0 description: The image shows the Cepheid logo and the text "Xpert® Xpress CoV-2/Flu/RSV plus". The Cepheid logo is a blue stylized image of a bird in flight. The text is in black and is located below the logo.

5.5. Conclusions

The results of the analytical and clinical performance studies summarized above demonstrated that the Xpert Xpress CoV-2/Flu/RSV plus test is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.3981 Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test.

(a)
Identification. A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
e.g., BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
e.g., when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
e.g., recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
e.g., regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
e.g., saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads 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.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
i.e., H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.

Acceptance Criteria and Study Details for Xpert Xpress CoV-2/Flu/RSV plus

1. Table of Acceptance Criteria and Reported Device Performance

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

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

4. Adjudication Method for the Test Set

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

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

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

8. The Sample Size for the Training Set

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

Re: K231481

Joseph Briggs -S

Indications for Use

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510(k) Summary for Xpert® Xpress CoV-2/Flu/RSV plus

TABLE OF CONTENTS

5. 510(k) Summary

Device Description 5.1.

. Nasopharyngeal Sample Collection Kit for Viruses

5.2. Device Intended Use

5.3. Substantial Equivalence

Table 5-1: Comparison of Similarities and Differences Between Xpert Xpress CoV-2/Flu/RSV plus and the Predicate Device

5.4. Performance Studies

5.4.1. Analytical Performance

Analytical Sensitivity - Clinical Nasopharyngeal Swab (NPS) Matrix

Analytical Sensitivity - Clinical Anterior Nasal Swab (NS) Matrix

Table 5-3. Xpert Xpress CoV-2/Flu/RSV plus Limit of Detection in Clinical NS-UTM/VTM Matrix

Analytical Reactivity (Inclusivity)

SARS-CoV-2 in silico Analyses

SARS-CoV-2, Flu A, Flu B, and RSV Inclusivity Wet-Testing

Xpert® Xpress CoV-2/Flu/RSV plus

Xpert® Xpress CoV-2/Flu/RSV plus

Analytical Specificity (Exclusivity)

In silico Analyses

Table 5-6. Microorganisms Analyzed in the in silico Analysis for the SARS-CoV-2 Target

Wet Testing

Microbial Interference

Competitive Interference

Potentially Interfering Substances

Table 5-14. Number of Correct Results for Xpert Xpress CoV-2/Flu/RSV plus Targets Tested in the Presence of Potentially Interfering Substances

Xpert® Xpress CoV-2/Flu/RSV plus

Carryover Contamination

Single-Site Precision

Reproducibility

Xpert® Xpress CoV-2/Flu/RSV plus

Table 5-16. Summary of the Reproducibility Results - % Agreement

Xpert® Xpress CoV-2/Flu/RSV plus

Table 5-17. Summary of Nested ANOVA by Coefficient of Variation

5.4.2. Clinical Performance

Xpert® Xpress CoV-2/Flu/RSV plus

Table 5-19. Xpert Xpress CoV-2/Flu/RSV plus Performance Results for NPS Specimens

Table 5-20. Xpert Xpress CoV-2/Flu/RSV plus Performance Results for NS Specimens

Table 5-21. Multi-Target Detection by Xpert Xpress CoV-2/Flu/RSV plus for Specimens Collected in 2022

5.5. Conclusions

§ 866.3981 Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test.