The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Xpress System, 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 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, influera 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 A. 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 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 the disease.

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

Device Description

The Xpert Xpress CoV-2/Flu/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 Xpress System, which consist of a GeneXpert IV instrument that executes sample preparation, nucleic acid amplification and real-time fluorescent signal detection for the tests, and a GeneXpert Hub with preloaded GeneXpert Xpress software for running the tests and viewing the test results. The GeneXpert Hub accessory integrates the computer, touchscreen monitor and barcode scanner. Each of the GeneXpert modules in the GeneXpert IV instrument can perform independent sample preparation and testing. The GeneXpert Xpress System requires 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 self-contained, cross-contamination between samples is minimized.

The Xpert Xpress CoV-2/Flu/RSV plus test cartridge 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 Xpress System. 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.

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 include:

Nasopharyngeal Sample Collection Kit for Viruses
- Copan UTM® 3C057N (Flexible Minitip Flocked Swab with UTM® Medium O without Beads)
- Copan eNAT® Molecular Collection and Preservation Medium P/N 6U074S01 о (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 6U073S01 O (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.

AI/ML Overview

The provided document is a 510(k) Summary for the Cepheid Xpert Xpress CoV-2/Flu/RSV plus test. It details the performance studies conducted to demonstrate substantial equivalence to a predicate device. Here's a breakdown of the acceptance criteria and study proving the device meets them, based on the provided text:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly demonstrated through the reported performance in both analytical and clinical studies, aiming for high levels of agreement with established methods. The summarized performance data serves as the proof that the device meets these (implied) acceptance criteria.

Table of Acceptance Criteria and Reported Device Performance:

Since explicit acceptance criteria values (e.g., "PPA must be >= 95%") are not explicitly stated in the provided text as separate criteria, I will infer them from the reported strong performance and the nature of a 510(k) submission, where substantial equivalence to a predicate device is the goal. The reported performance is the validation that it meets the expected performance for such a device.

Performance Metric Category	Specific Metric (Target)	Acceptance Criteria (Implied / Goal)	Reported Device Performance (Xpert Xpress CoV-2/Flu/RSV plus)
Analytical Sensitivity (Limit of Detection - LoD)	LoD for various viral strains (copies/mL, TCID50/mL, FFU/mL, IU/mL, CEID50/mL) in NPS/NS matrix	Lowest concentration for each strain at which 95% of replicates yield a positive result.	NPS Matrix: SARS-CoV-2: 138 copies/mL (NATtrol), 94 IU/mL (WHO). Flu A: 0.007-0.44. Flu B: 2.4-12.9. RSV: 0.17-0.37. NS Matrix: SARS-CoV-2: 64 copies/mL, 143 IU/mL. Flu A: 0.0028-0.49. Flu B: 2.41-26.3. RSV: 0.22-0.4. (All verified with 20 replicates per virus/lot).
Analytical Reactivity (Inclusivity)	Detection of diverse strains within each target (SARS-CoV-2, Flu A, Flu B, RSV A, RSV B)	100% detection of tested strains at ~3x LoD. In silico: high percentage exact matches/1 mismatch.	In silico: SARS-CoV-2 E: 100%, N2: 99.9%, RdRP: 100% (total <2 mismatches for variants of interest/concern as of 2022). Wet-Testing: 102 respiratory viral strains (18 SARS-CoV-2, 69 Flu, 15 RSV) tested positive in all 3 replicates at ~3x LoD.
Analytical Specificity (Exclusivity)	No cross-reactivity with common respiratory pathogens or human coronaviruses.	100% analytical specificity (no false positives).	In silico: No cross-reactivity expected based on analysis of 48 microorganisms in Table 6. Wet-Testing: 100% analytical specificity (all negative controls and interfering microorganisms tested negative).
Microbial Interference	No inhibition of target detection by common co-present microorganisms.	Correct detection of target viruses (8/8 replicates) in presence of interfering microorganisms.	For each target (SARS-CoV-2, Flu A, Flu B, RSV A, RSV B) at 3x LoD, all 8 of 8 replicates were correctly identified.
Competitive Interference	No inhibition of target detection at 3x LoD by other target strains at higher concentrations.	3/3 replicates for target strain report positive results after concentration adjustments.	Interference Observed & Resolved: Flu A inhibited Flu B (>1.7e5 RNA copies/mL) & RSV A (>1.7e6 RNA copies/mL). SARS-CoV-2 inhibited Flu B (>1e5 RNA copies/mL). Issues resolved by reducing interfering virus concentration. No other competitive interference.
Potentially Interfering Substances	No impact on test performance by substances found in respiratory specimens.	8/8 replicates correctly identified for positive samples (viruses at 3x LoD) and negative samples.	Interference Observed & Resolved: FluMist, human PBMC, snuff, Zicam showed interference at high concentrations for some targets. Inhibitory effects not observed at lower or adjusted concentrations. All other substances (Table 13) showed no interference.
Carryover Contamination	No contamination from high positive to negative samples.	All 40 positive samples correctly detected. All 42 negative samples correctly detected.	All 40 positive samples (high Flu B and SARS-CoV-2) were correctly reported as positive for their respective targets. All 42 negative samples were correctly reported as negative. No carry-over contamination observed.
Reproducibility	Consistent results across sites, operators, and days for various sample concentrations.	High percent agreement across operators and sites for negative, low positive, and moderate positive samples. Low coefficient of variation (CV%) for Ct values.	Clinical Agreement: Negatives: 100%. SARS-CoV-2 Low/Mod Pos: 100%. Flu A Low Pos: 97.8% (88/90). Flu A Mod Pos: 100%. Flu B Low Pos: 97.8%. Flu B Mod Pos: 100%. RSV Low/Mod Pos: 100%. Ct Value Variability: Total CV% for analytes ranged from 0.9% to 3.6%.
Clinical Performance (NPS)	High Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to comparator.	PPA and NPA (typically >90-95% for 510(k) submissions) relative to US FDA-cleared molecular panels.	SARS-CoV-2: PPA 98.7% (96.6-99.5 CI), NPA 98.5% (97.7-99.0 CI). Flu A: PPA 99.1% (96.6-99.7 CI), NPA 98.5% (97.9-99.0 CI). Flu B: PPA 96.6% (88.5-99.1 CI), NPA 99.9% (99.7-100.0 CI). RSV: PPA 97.8% (92.4-99.4 CI), NPA 100% (99.7-100.0 CI).
Clinical Performance (NS)	High Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to comparator.	PPA and NPA (typically >90-95% for 510(k) submissions) relative to US FDA-cleared molecular panels.	SARS-CoV-2: PPA 98.4% (96.2-99.3 CI), NPA 99.3% (98.7-99.6 CI). Flu A: PPA 97.6% (94.6-99.0 CI), NPA 98.9% (98.3-99.2 CI). Flu B: PPA 100% (89.9-100.0 CI), NPA 99.9% (99.6-100.0 CI). RSV: PPA 97.0% (91.6-99.0 CI), NPA 99.9% (99.6-100.0 CI).
Non-Determinate Rate (Wastage/Invalid)	Low initial and final non-determinate rates.	Low percentage (e.g., <5%).	NPS: Initial 2.5%, Final 0.3%. NS: Initial 2.9%, Final 0.8%.
Co-Infection Rate	Test capable of detecting multiple targets simultaneously when present.	Comparable co-infection rates to comparator.	SARS-CoV-2/Flu/RSV (2022 data): Xpert Xpress: 0.1% (1/797). Comparator: 0.3% (2/774). Flu A/B/RSV (Mixed 2016-17 & 2022 data): Xpert Xpress: 2.0% (15/737). Comparator: 1.3% (9/698).

Study Details:

Sample sizes used for the test set and the data provenance:
- Analytical Studies (LoD, Inclusivity, Specificity, Interference, Carryover, Reproducibility): These studies used "replicates" (e.g., 20, 3, 8) of contrived/simulated samples rather than patient samples for their test sets.
  - LoD: 20 replicates per virus/lot combination.
  - Inclusivity (wet-testing): 3 replicates for each of 102 respiratory viral strains.
  - Exclusivity (wet-testing): 3 replicates of each microorganism pool.
  - Microbial Interference: 8 replicates of positive samples for each target virus and microbial interference strain combination.
  - Competitive Interference: 3 replicates for each target strain and competitive strain combination (adjusted as needed).
  - Potentially Interfering Substances: 8 negative and 8 positive samples (pooled) for each substance.
  - Carryover Contamination: 40 positive and 42 negative samples (20 pairs per module, 2 modules).
  - Reproducibility: 90 observations per panel member (3 Sites x 3 Operators x 1 Lot x 5 Days x 1 Run x 2 Replicates).
- Clinical Performance Study:
  - Total Specimens: 4561 (NPS=2300, NS=2261)
  - Test Set for SARS-CoV-2 Performance: 3147 valid specimens (1565 NPS, 1582 NS).
  - Test Set for Flu A, Flu B, RSV Performance: 4310 valid specimens (2175 NPS, 2135 NS).
  - Data Provenance:
    - Fresh (Category I): 3333 specimens (1 frozen specimen in this category). Prospectively collected in 2022 from 23 geographically diverse sites in the United States.
    - Archived Frozen (Category II): 1228 specimens. Prospectively collected during the 2016-2017 influenza season (pre-pandemic), used to supplement Flu/RSV sample size. These were collected in the US.
  - Retrospective/Prospective: Primarily prospective (Category I) with a significant portion of archived prospective (Category II) specimens.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The document implies that the ground truth for the clinical test set was established by U.S. FDA-cleared molecular diagnostic tests (a molecular respiratory panel for SARS-CoV-2 and a molecular Flu A/B/RSV test).
- For discrepant results, additional U.S. FDA EUA SARS-CoV-2 molecular tests or U.S. FDA-cleared molecular respiratory panels were used.
- The document does not specify the number or qualifications of human experts directly involved in establishing the ground truth for the clinical test set. The ground truth relies on the results of the comparator molecular assays.
Adjudication method (e.g. 2+1, 3+1, none) for the test set:
- For the clinical performance study, results were compared "side-by-side" with a U.S. FDA-cleared molecular respiratory panel.
- Discrepant results were investigated using a different U.S. FDA EUA SARS-CoV-2 molecular test or U.S. FDA-cleared molecular respiratory panel. This suggests an adjudication process for discrepancies, where an additional, independent test confirms or refutes the initial finding of the comparator or investigational device. The exact "X+Y" method (e.g., 2+1) is not explicitly stated, but the process of re-testing discrepant samples with a "tie-breaker" method is described.
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This document describes the validation of an in vitro diagnostic (IVD) PCR test kit, not an AI-assisted diagnostic imaging or interpretation system that would involve human "readers" or "interpreters." The device is largely automated.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- This is a standalone diagnostic test in the sense that the GeneXpert Xpress System performs the sample preparation, nucleic acid amplification, and real-time fluorescent signal detection to produce a result (qualitative positive/negative). Human input is for specimen collection, loading the cartridge, and reviewing the auto-generated results. The performance metrics presented (PPA, NPA, LoD, etc.) are of the "device only" system. So, yes, a standalone performance was done for the device.
The type of ground truth used (expert consensus, pathology, outcomes data, etc):
- For analytical studies: Contrived samples with known concentrations of viral strains or microorganisms, often verified against internal standards or external reference materials.
- For clinical studies: Results from U.S. FDA-cleared and U.S. FDA EUA molecular diagnostic tests served as the ground truth (comparator method). Discrepancies were resolved using additional molecular tests.
The sample size for the training set:
- The document does not provide information on a training set size. This is common for traditional IVD device submissions, where analytical and clinical validation data (test set) are collected after the assay design (analogous to "training") is complete. The "training" in this context would likely refer to the internal assay development and optimization, for which specific sample sizes might not be publicly disclosed or directly relevant to the regulatory submission for substantial equivalence.
How the ground truth for the training set was established:
- As no explicit "training set" is mentioned in the context of device performance validation, the method for establishing its ground truth is not described. For assay development, ground truth would be established through a combination of highly characterized samples, pure cultures, and perhaps clinical samples with confirmed results from gold-standard methods (e.g., viral culture, sequencing, or highly sensitive and specific lab-developed tests).

Summary

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left, there is a symbol representing the Department of Health & Human Services - USA. To the right of this symbol, there is the FDA logo in blue, with the words "U.S. FOOD & DRUG" stacked on top of the word "ADMINISTRATION".

January 10, 2025

Cepheid Yen Nguven Director, Regulatory Affairs 904 East Caribbean Drive Sunnyvale, California 94089

Re: K242071

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

Dated: July 15, 2024 Received: July 16, 2024

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 (the 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 available 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.

{1}------------------------------------------------

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (OS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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-regulatory

{2}------------------------------------------------

assistance/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,

Anna M. Mielech -S

Anna Mielech, PhD. Deputy Branch Chief (Acting) Viral Respiratory and HPV Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

{3}------------------------------------------------

Indications for Use

510(k) Number (if known) K242071

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.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{4}------------------------------------------------

Image /page/4/Picture/0 description: The image shows the Cepheid logo, which is a blue abstract shape. Below the logo is the text "Cepheid." in a serif font. Underneath that is the text "Xpert® Xpress CoV-2/Flu/RSV plus" in a serif font.

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

{5}------------------------------------------------

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 of three curved lines. The text is in a serif font, with "Xpert" in bold and the registered trademark symbol after it.

1. 510(k) SUMMARY	3
1.1. DEVICE DESCRIPTION	4
1.2. DEVICE INTENDED USE	5
1.3. SUBSTANTIAL EQUIVALENCE	6
1.4. PERFORMANCE STUDIES	8
1.4.1. ANALYTICAL PERFORMANCE	8
1.4.2. CLINICAL PERFORMANCE	26
1.5. CONCLUSIONS	31

{6}------------------------------------------------

1. 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:	January 7, 2025
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 numberClassification nameProduct code	21 CFR 866.3981, Multi-Target Respiratory SpecimenNucleic Acid Test Including SARS-CoV-2 and OtherMicrobial Agents, QOF.21 CFR 862.2570, Real Time Nucleic Acid AmplificationSystem, OOI.
ClassificationAdvisory Panel	Microbiology (83)
Prescription Use	Yes
Predicate DeviceAssay:	Xpert Xpress CoV-2/Flu/RSV plus (K231481)

{7}------------------------------------------------

1.1. Device Description

Nasopharyngeal Sample Collection Kit for Viruses
- Copan UTM® 3C057N (Flexible Minitip Flocked Swab with UTM® Medium O without Beads)
- Copan eNAT® Molecular Collection and Preservation Medium P/N 6U074S01 о (Flexible Minitip Flocked Swab with eNAT® Medium)

{8}------------------------------------------------

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 6U073S01 O (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.

1.2. Device Intended Use

The Xpert Xpress CoV-2/Flu/RSV plus test. performed on the GeneXpert Xpress System, 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 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 A, 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 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 the disease.

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

{9}------------------------------------------------

1.3. Substantial Equivalence

Table 1 shows the similarities and differences between the subject device and the predicate device.

			Table 1: Comparison of Similarities and Differences Between Subject and Predicate Device
--	--	--	------------------------------------------------------------------------------------------	--	--	--

	Subject Device	Predicate Device
Attribute	Xpert® Xpress CoV-2/Flu/RSV pluson the GeneXpert Xpress System	Xpert® Xpress CoV-2/Flu/RSV pluson the GeneXpert Instrument Systems[K231481]
Regulation	Same	21 CFR 866.3981Devices to detect and identify nucleic acidtargets in respiratory samples frommicrobial agents that cause the SARS-CoV-2 respiratory infection and other microbialagents when in a multi-analyte test
Product Code	Same	QOFMulti-target respiratory specimen nucleicacid test including SARS-CoV-2 and othermicrobial agents
Device Class	Same	II (Special Controls)
Technology/Detection	Same	Real-time reverse transcription polymerasechain reaction (RT-qPCR)
Intended Use	The Xpert® Xpress CoV-2/Flu/RSV plustest, performed on the GeneXpert® XpressSystem, is an automated multiplexed real-time reverse transcriptase polymerasechain reaction (RT-PCR) test intended foruse in the simultaneous in vitro qualitativedetection and differentiation of severeacute respiratory syndrome coronavirus(SARS-CoV-2), influenza A, influenza B,and/or respiratory syncytial virus (RSV)viral RNA in nasopharyngeal swab andanterior nasal swab specimens collectedfrom individuals with signs and symptomsof respiratory tract infection. Clinical signsand symptoms of respiratory tractinfection 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	The Xpert® Xpress CoV-2/Flu/RSV plustest, performed on the GeneXpert® Dx andGeneXpert® Infinity Systems, is anautomated multiplexed real-time reversetranscriptase polymerase chain reaction(RT-PCR) test intended for use in thesimultaneous in vitro qualitative detectionand differentiation of severe acuterespiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/orrespiratory syncytial virus (RSV) viral RNAin nasopharyngeal swab and anterior nasalswab specimens collected from individualswith signs and symptoms of respiratory tractinfection. Clinical signs and symptoms ofrespiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSVcan be similar.The Xpert Xpress CoV-2/Flu/RSV plus isintended for use in the differential detectionof SARS-CoV-2, influenza A, influenza Band/or RSV RNA and aids in the diagnosisof COVID-19, influenza and/or RSVinfections if used in conjunction with otherclinical and epidemiological information,and laboratory findings. SARS-CoV-2,influenza A, influenza B, and RSV
	Subject Device	Predicate Device
Attribute	Xpert® Xpress CoV-2/Flu/RSV pluson the GeneXpert Xpress System	Xpert® Xpress CoV-2/Flu/RSV pluson the GeneXpert Instrument Systems[K231481]
	RNA are generally detectable innasopharyngeal swab and anterior nasalswab specimens during the acute phase ofinfection.	RNA 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.	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 not bethe 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.	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.
Assay Targets	Same	SARS-CoV-2, Influenza A, Influenza B,RSV viral RNA
Specimen Type	Same	Nasopharyngeal swab (NPS) Anterior nasal swab (NS)
TransportMedia	Same	Universal Transport Medium (UTM) /Viral Transport Medium (VTM) eNAT
Test Format	Same	Single Use
Automation	Same	Automated Nucleic Acid Extraction,Detection and Results Interpretation
Assay Results	Same	Qualitative
InternalControl	Same	Sample Processing Control (SPC)Probe Check Control (PCC)
InstrumentSystems	Cepheid GeneXpert® Xpress System	Cepheid GeneXpert® Instrument Systems
Time to Result	Same	36 min or less for sample preparation andRT-PCR

{10}------------------------------------------------

Image /page/10/Picture/0 description: The image contains the logo for Cepheid. The logo consists of a blue graphic element resembling stylized wings or feathers on the left. To the right of the graphic is the company name, "Cepheid," in a simple, sans-serif font, with a small trademark symbol next to it.

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

{11}------------------------------------------------

1.4. Performance Studies

1.4.1. Analytical Performance

Analytical Sensitivity (Limit of Detection) - 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 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 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 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

{12}------------------------------------------------

Image /page/12/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 black font and appears to be the name of a product.

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 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 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 4 summarizes the effective predicted inclusivity for E, N2 and RdRP amplicons for the variants of interests and concern.

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

Amplicon	Exact Match	1 Mismatcha	2 or MoreMismatches	% Total <2Mismatches
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%

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

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

{13}------------------------------------------------

Image /page/13/Picture/0 description: The image shows the Cepheid logo, which consists of a blue graphic and the word "Cepheid." in black font. Below the logo is the text "Xpert® Xpress CoV-2/Flu/RSV plus" in black font. The text appears to be advertising a diagnostic test.

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.

			Result
Virus	Strain	Target Conc.	SARS-CoV-2	Flu A	Flu B	RSV
SARS-CoV-2	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 (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-EHC-2811C/20221) B.1.1.529 (Omicron)	100 (genomeequivalents/mL)	POS	NEG	NEG	NEG
	SARS-CoV-2 RNA, USA/WA2/2020(C09)b	100 copies/mL	POS	NEG	NEG	NEG

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

{14}------------------------------------------------

Image /page/14/Picture/0 description: The image shows the Cepheid logo. The logo consists of a blue graphic element resembling stylized wings or feathers on the left. To the right of the graphic is the word "Cepheid" in a simple, sans-serif font, with a small circle or dot following the word.

Virus	Strain	Target Conc.	Result
			SARS-CoV-2	Flu A	Flu B	RSV
	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
Flu AH3N2	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
			Result
Virus	Strain	Target Conc.	SARS-CoV-2	Flu A	Flu B	RSV
	A/Singapore/INFIMH-16-0019/2016	2.5 CEID50/mL	NEG	POS	NEG	NEG
	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
	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
	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
AvianFlu Ae	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)	NAf	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
Flu B (VictoriaLineage)	B/Florida/02/06	0.004 TCID50/mL	NEG	NEG	POS	NEG
	B/Brisbane/60/2008	0.005 TCID50/mL	NEG	NEG	POS	NEG
	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
Virus	Strain	Target Conc.	Result
			SARS-CoV-2	Flu A	Flu B	RSV
Flu B(YamagataLineage)	B/Florida/07/2004	0.03 TCID50/mL	NEG	NEG	POS	NEG
	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	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 (2000/3-4)	0.0015 TCID50/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)	0.0089 TCID50/mL	NEG	NEG	NEG	POS
RSV B	RSV-B/WV14617/85	0.04 TCID50/mL	NEG	NEG	NEG	POS
	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
	RSV-B (cp23 Clone 1A2)	4200 copies/mL	NEG	NEG	NEG	POS

{15}------------------------------------------------

Image /page/15/Picture/0 description: The image shows the Cepheid logo. The logo consists of a blue graphic element resembling stylized wings or feathers on the left. To the right of the graphic is the word "Cepheid" in a simple, sans-serif font, with a small circle or dot following the word.

{16}------------------------------------------------

Image /page/16/Picture/0 description: The image shows the logo for Cepheid. The logo consists of a blue graphic on the left and the word "Cepheid" in black on the right. The graphic is made up of three curved lines that resemble a stylized wing or feather.

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

b. In vitro transcripts from Twist Biosciences.

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

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

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 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.

{17}------------------------------------------------

Image /page/17/Picture/0 description: The image shows the Cepheid logo, which includes a blue graphic element resembling feathers or wings, along with the text "Cepheid.". Below the logo, the text "Xpert® Xpress CoV-2/Flu/RSV plus" is displayed, indicating a product or test related to CoV-2, Flu, and RSV.

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 non-target 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 6 is expected based on the in silico analysis.

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)

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

{18}------------------------------------------------

Image /page/18/Picture/0 description: The image shows the Cepheid company logo, which includes a blue wing-like graphic and the company name in a serif font. Below the logo is the text "Xpert® Xpress CoV-2/Flu/RSV plus", indicating a product or service offered by Cepheid. The text is in a similar serif font and suggests a diagnostic test related to COVID-19, influenza, and respiratory syncytial virus.

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

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 10 copies/mL of genomic DNA. Viruses were tested at concentrations of ≥ 1 x 105 TCIDso/mL. The analytical specificity was 100%. Results are shown in Table 7.

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

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
Count	Strain	Tested Concentration	SARS-CoV-2	Flu A	Flu B	RSV
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
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
Count	Strain	Tested Concentration	SARS-CoV-2	Flu A	Flu B	RSV
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

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

Image /page/19/Picture/0 description: The image shows the logo for Cepheid. The logo consists of a stylized blue wing-like design on the left, followed by the company name "Cepheid." The font is simple and modern, with a small registered trademark symbol next to the "d" in Cepheid.

{20}------------------------------------------------

Image /page/20/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 element with the word "Cepheid." next to it. The text "Xpert® Xpress CoV-2/Flu/RSV plus" is written in a smaller font size than the word "Cepheid."

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 1x10> TCID50/mL), Hemophilus influenzae (seeded at 1x10° CFU/mL), Staphylococcus aureus or Staphylococcus epidermidis (each seeded at 1x107 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.

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 CEID50/mL for Flu B/Washington/2/2019; 0.99 TCID50/mL for RSV A/2/Australia/61), and 1.11 TCIDso/mL for RSV B/9320/MA/77. The competitive strains were evaluated at >10 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

{21}------------------------------------------------

Tables 8 through 12 for high concentration of Flu A, Flu B, RSV A, RSV B and SARS-CoV-2, respectively.

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

			Table 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 10. Summary of Competitive Interference Study with RSV A at High Concentration

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

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

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 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		1/3	3/3
RSV A		3/3	Not tested
RSV B		3/3	Not tested

{22}------------------------------------------------

Image /page/22/Picture/0 description: The image shows the Cepheid logo, which includes a blue feather-like design 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.

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 8). In addition, SARS-CoV-2 at concentrations above 1e5 RNA copies/mL inhibited detection of Flu B at 3x LoD (Table 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 H1N1, 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 13.

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

Table 13. Potentially Interfering Substances Tested

{23}------------------------------------------------

Image /page/23/Picture/0 description: The image shows the logo for Cepheid. The logo consists of a blue graphic element resembling stylized wings or feathers, positioned to the left of the company name. The word "Cepheid" is written in a dark, sans-serif font, with a small trademark symbol next to it.

Substance ID	Substance/Class	Substance/Active Ingredient	Concentrations Tested
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

The results from the study (Table 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 106 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).

{24}------------------------------------------------

Image /page/24/Picture/0 description: The image shows the Cepheid logo. The logo consists of a blue graphic on the left and the word "Cepheid" in blue on the right. The graphic is a stylized representation of three curved lines, resembling feathers or wings.

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

		Number of Correct Results/Number Tested for Each Virus and the No Virus Control
Substance	ConcentrationTested	No Virus Control	SARS-CoV-2/USA-WA-1	Flu A/California/7/2009	Flu A/Idaho/07/2018	Flu A/ Hong 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
Albuterol Sulfate	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	8/8a	8/8	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
FluticasonePropionate NasalSpray	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
Human peripheralblood	1e6 cells/mL	8/8	8/8	8/8b	8/8	8/8	8/8	8/8	6/8	8/8b	8/8	8/8b	8/8b
mononuclearcells	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
	0.25e6 cells/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
Menthol	1.7 mg/mL	16/16a	8/8	8/8	8/8	8/8	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
Substance	ConcentrationTested	No Virus Control	SARS-CoV-2/USA-WA-1	Flu A/California/7/2009	Flu A/Idaho/07/2018	Flu A/Hong 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
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 µg/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
Zinc	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
	0.1µg/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

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

Image /page/25/Picture/0 description: The image shows the Cepheid logo. The logo consists of three blue curved lines that resemble wings or feathers on the left side. To the right of the wing-like symbol is the word "Cepheid" in a sans-serif font, also in blue. A small circle is present to the right of the word.

Dual 510(k) and CLIA Waiver by Application

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 valicates.

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 bead. 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.

{26}------------------------------------------------

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 TCIDs0/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.

Reproducibility

The reproducibility of the Xpert Xpress CoV-2/Flu/RSV plus test was established at 3 external sites using a 10-member panel including 2 negative, 4 low positive (~1.5x LoD) and 4 moderate positive (~3x LoD) samples. The negative samples 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 5 days, using 1 lot of Xpert Xpress CoV-2/Flu/RSV plus cartridges at 3 participating sites, each with 3 operators to yield a total of 90 observations per panel member (3 Sites x 3 Operators x 1 Lot x 5 Days x 1 Run x 2 Replicates = 90 observations/panel member). The results from the study are summarized in Table 15.

The percent agreement of the correct results compared to the expected results analyzed by each of the 3 operators and each site is shown in Table 15. 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.

{27}------------------------------------------------

Image /page/27/Picture/0 description: The image shows the Cepheid logo. The logo consists of a stylized blue wing-like design on the left and the word "Cepheid" in a sans-serif font on the right. The wing design is made up of three curved lines that resemble feathers or wings.

Sample	Site 1				Site 2				Site 3
	Op 1	Op 2	Op 3	Site	Op 1	Op 2	Op 3	Site	Op 1	Op 2	Op 3	Site
Negative - 1	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
Negative - 2	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
SARS-CoV-2Low Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
SARS-CoV-2Mod Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%9/9	100%10/10	100%10/10	100%29/29	100%(89/89) a[95.9-100.0]
Flu ALow Pos	100%10/10	100%10/10	100%10/10	100%30/30	90%9/10	90%9/10	100%10/10	93.3%28/30	100%10/10	100%10/10	100%10/10	100%30/30	97.8%(88/90)[92.3-99.4]
Flu AMod Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
Flu BLow Pos	100%10/10	90%9/10	100%10/10	96.7%29/30	100%10/10	100%10/10	90%9/10	96.7%29/30	100%10/10	100%10/10	100%10/10	100%30/30	97.8%(88/90)[92.3-99.4]
Flu BMod Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
RSVLow Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]
RSVMod Pos	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%10/10	100%10/10	100%10/10	100%30/30	100%(90/90)[95.9-100.0]

Table 15. Summary of Reproducibility Results - % Agreement

a. One replicate was excluded due to a repeat non-determinate test result.

The evaluation of reproducibility 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; %) betweensites, between-operators, between-days, and within-run for each panel member are presented in Table 16.

{28}------------------------------------------------

Image /page/28/Picture/0 description: The image shows the Cepheid logo. The logo consists of three blue curved lines that resemble a wing or a stylized letter "C". To the right of the wing-like symbol is the word "Cepheid" in a simple, sans-serif font, with a period at the end.

	Analyte	N		Variance Source
Sample			Mean Ct	Site		Operator		Day		Within-run		Total
				SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)
Negative - 1	SPC	90	28.54	0.00	0.0	0.10	0.3	0.23	0.8	0.87	3.0	0.90	3.2
Negative - 2	SPC	90	28.51	0.10	0.3	0.00	0.0	0.00	0.0	0.36	1.3	0.37	1.3
SARS-CoV-2Low Pos	SARS-CoV-2	90	36.86	0.09	0.2	0.00	0.0	0.00	0.0	0.60	1.6	0.61	1.7
SARS-CoV-2Mod Pos	SARS-CoV-2	89a	35.74	0.00	0.0	0.12	0.3	0.00	0.0	0.30	0.8	0.33	0.9
Flu A	Flu A1	90	35.88	0.24	0.7	0.00	0.0	0.23	0.6	1.11	3.1	1.16	3.2
Low Pos	Flu A2	72b	38.34	0.00	0.0	0.32	0.8	0.00	0.0	1.34	3.5	1.38	3.6
Flu A	Flu A1	90	34.64	0.00	0.0	0.1	0.3	0.00	0.0	0.40	1.2	0.41	1.2
Mod Pos	Flu A2	90	36.75	0.26	0.7	0.34	0.9	0.00	0.0	0.90	2.4	1.00	2.7
Flu BLow Pos	Flu B	90	36.05	0.00	0.0	0.00	0.0	0.48	1.3	1.12	3.1	1.21	3.4
Flu BMod Pos	Flu B	90	35.12	0.09	0.2	0.08	0.2	0.00	0.0	0.61	1.7	0.62	1.8
RSVLow Pos	RSV	90	35.92	0.00	0.0	0.16	0.5	0.00	0.0	0.75	2.1	0.77	2.1
RSVMod Pos	RSV	90	35.07	0.00	0.0	0.2	0.6	0.00	0.0	0.57	1.6	0.60	1.7

Table 16. Summary of Nested ANOVA by Coefficient Variation

One replicate was excluded due to a repeat non-determinate test result. a.

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

{29}------------------------------------------------

1.4.2. 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 23 geographically diverse sites in the United States (US) using specimens collected from individuals showing signs and symptoms of respiratory infection. Of the 23 sites, 22 performed Xpert testing and specimen collection, and 1 site performed 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 (3333/3334) and frozen (1/3334) 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 II specimens were collected are presented in Table 17.

Prospectively Collected Clinical Specimens	NPS(N=2300)	NS(N=2261)	Overall(N=4561)
Gender
Female	1296 (56.3%)	1374 (60.8%)	2670 (58.5%)
Male	1004 (43.7%)	887 (39.2%)	1891 (41.5%)
Age Group (Years)
<5	273 (11.9%)	402 (17.8%)	675 (14.8%)
6-21	600 (26.1%)	550 (24.3%)	1150 (25.2%)
22-59	1129 (49.1%)	1015 (44.9%)	2144 (47.0%)
≥60	298 (13.0%)	294 (13.0%)	592 (13.0%)
Specimen Testing
Fresh	1654 (71.9%)	1679 (74.3%)	3333 (73.1%)
Frozen	646 (28.1%)	582 (25.7%)	1228 (26.9%)
Race
Prospectively Collected in 2022
American Indian or Alaska Native	1 (0.0%)	3 (0.1%)	4 (0.1%)
Asian	53 (2.3%)	58 (2.6%)	111 (2.4%)
Asian, White	6 (0.3%)	2 (0.1%)	8 (0.2%)
Black or African American	403 (17.5%)	389 (17.2%)	792 (17.4%)
Black or African American, White	6 (0.3%)	7 (0.3%)	13 (0.3%)
Native Hawaiian or Other Pacific Islander	1 (0.0%)	1 (0.0%)	2 (0.0%)
White	1042 (45.3%)	1035 (45.8%)	2077 (45.5%)

Table 17. Demographic Summary for Category I and II Specimens

{30}------------------------------------------------

Image /page/30/Picture/0 description: The image shows the logo for Cepheid. The logo consists of a blue graphic element resembling stylized wings or feathers, positioned above the company name. The text "Cepheid" is written in a modern, sans-serif font, with a small circle or dot placed after the name.

	NPS(N=2300)	NS(N=2261)	Overall(N=4561)
Prospectively Collected Clinical Specimens
Other Mixed	2 (0.1%)	4 (0.2%)	6 (0.1%)
Missing, Declined to Answer or unknown	141 (6.1%)	180 (8.0%)	321 (7.0%)
Prospectively Collected Pre- Pandemic
Not Available	645 (28.0%)	582 (25.7%)	1227 (26.9%)
Ethnicity
Prospectively Collected in 2022
Hispanic	136 (5.9%)	146 (6.5%)	282 (6.2%)
Non-Hispanic	1411 (61.3%)	1413 (62.5%)	2824 (61.9%)
Missing, Declined to Answer or Unknown	108 (4.7%)	120 (5.3%)	228 (5.0%)
Prospectively Collected Pre-Pandemic
Not Available	645 (28.0%)	582 (25.7%)	1227 (26.9%)
COVID-19 Vaccination Status
Prospectively Collected in 2022
Vaccinated	1293 (56.2%)	1184 (52.4%)	2477 (54.3%)
Not Vaccinated	328 (14.3%)	468 (20.7%)	796 (17.5%)
Unknown	34 (1.5%)	27 (1.2%)	61 (1.3%)
Prospectively Collected Pre-Pandemic
Not Available (Not Vaccinated)	645 (28.0%)	582 (25.7%)	1227 (26.9%)

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

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 3147 specimens, including 1565 NPS and 1582 NS specimens that yielded 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 4310 prospective (Category I and II) specimens, including 2175 NPS and 2135 NS specimens that yielded valid results by both the Xpert Xpress CoV2/Flu/RSV plus and the U.S. FDAcleared molecular Flu A/B/RSV assay were included in the performance evaluation for Flu A, Flu B, and RSV targets.

{31}------------------------------------------------

For the NPS specimens (both fresh and frozen specimens, combined), Xpert Xpress CoV-2/Flu/RSV plus demonstrated a PPA and NPA of 98.7% and 98.5% for SARS-CoV-2. respectively; 99.1% and 98.5% for Flu A, respectively; 96.6% and 99.9% for Flu B, respectively; 97.8% and 100.0% for RSV, respectively (Table 18). The initial non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test using NPS specimens was 2.5% (58/2300). On repeat testing, 51 specimens yielded valid results. The final non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 0.3% (7/2300).

Analyte	SpecimenCollection	Numbers ofSpecimens	TP	FN	TN	FP	PPA(%)	95%CI	NPA(%)	95%CI
SARS-CoV-2	Fresh	1564	294	4a	1247	19b	98.7	96.6 - 99.5	98.5	97.7 - 99.0
SARS-CoV-2	Frozen	1	1	0	0	0	100	20.7 - 100.0	NA	NA
SARS-CoV-2	Overall	1565	295	4	1247	19	98.7	96.6 - 99.5	98.5	97.7 - 99.0
Flu A	Fresh	1582	79	0	1495	8c	100	95.4 - 100.0	99.5	99.0 - 99.7
Flu A	Frozen	593	130	2d	440	21e	98.5	94.6 - 99.6	95.4	93.1 - 97.0
Flu A	Overall	2175	209	2	1935	29	99.1	96.6 - 99.7	98.5	97.9 - 99.0
Flu B	Fresh	1582	0	0	1582	0	NA	NA	100	99.8 - 100.0
Flu B	Frozen	593	57	2f	532	2g	96.6	88.5 - 99.1	99.6	98.6 - 99.9
Flu B	Overall	2175	57	2	2114	2	96.6	88.5 - 99.1	99.9	99.7 - 100.0
RSV	Fresh	1582	6	0	1576	0	100	61.0 - 100.0	100	99.8 - 100.0
RSV	Frozen	593	84	2h	506	1i	97.7	91.9 - 99.4	99.8	98.9 - 100.0
RSV	Overall	2175	90	2	2082	1	97.8	92.4 - 99.4	100	99.7 - 100.0

Table 18. Xpert Xpress CoV-2/Flu/RSV plus Performance Results for NPS Specimens

TP: True Positive; FP: False Positive; TN: False Negative; CI: 95% two-sided Confidence Interval NA: Not Applicable

a. Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 1/4 SARS-CoV-2 positive; 3/4 SARS-CoV-2 negative

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 6/19 SARS-CoV-2 positive; b. 13/19 SARS-CoV-2 negative

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 6/8 Flu A positive; 2/8 Flu A C. negative
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/2 tests not performed due to d. the specimens being stored for a longer duration than allowed per the package insert
e. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 21/21 tests not performed due to the specimens being stored for a longer duration than allowed 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: 2/2 test not performed due to g. 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: 2/2 test 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 test not performed due to specimens being stored for a longer duration than recommended per the package insert

{32}------------------------------------------------

For the NS specimens (both fresh and frozen specimens, combined), Xpert Xpress CoV-2/Flu/RSV plus demonstrated a PPA and NPA of 98.4% and 99.3% for SARS CoV-2, respectively; 97.6% and 98.9% for Flu A, respectively; 100.0% and 99.9% for Flu B, respectively; 97.0% and 99.9% for RSV, respectively (Table 19). The initial non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test using NS specimens was 2.9% (65/2261). On repeat testing, 46 specimens gave valid results upon retest. The final non-determinate rate for the Xpert Xpress CoV-2/Flu/RSV plus test was 0.8% (19/2261).

Analyte	SpecimenCollection	Numbers ofSpecimens	TP	FN	TN	FP	PPA(%)	95%CI	NPA(%)	95%CI
SARS-CoV-2	Fresh	1582	302	5a	1266	9b	98.4	96.2 - 99.3	99.3	98.7 - 99.6
SARS-CoV-2	Frozen	0	0	0	0	0	NA	NA	NA	NA
SARS-CoV-2	Overall	1582	302	5	1266	9	98.4	96.2 - 99.3	99.3	98.7 - 99.6
Flu A	Fresh	1603	107	4c	1489	3d	96.4	91.1 - 98.6	99.8	99.4 - 99.9
Flu A	Frozen	532	99	1e	413	19f	99.0	94.6 - 99.8	95.6	93.2 - 97.2
Flu A	Overall	2135	206	5	1902	22	97.6	94.6 - 99.0	98.9	98.3 - 99.2
Flu B	Fresh	1603	0	0	1603	0	NA	NA	100	99.8 - 100.0
Flu B	Frozen	532	34	0	495	3g	100	89.9 - 100.0	99.4	98.2 - 99.8
Flu B	Overall	2135	34	0	2098	3	100	89.9 - 100.0	99.9	99.6 - 100.0
RSV	Fresh	1603	6	1h	1596	0	85.7	48.7 - 97.4	100	99.8 - 100.0
RSV	Frozen	532	91	2i	437	2j	97.8	92.5 - 99.4	99.5	98.4 - 99.9
RSV	Overall	2135	97	3	2033	2	97.0	91.6 - 99.0	99.9	99.6 - 100.0

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

TP: True Positive; FP: False Positive; TN: False Negative; CI: 95% two-sided Confidence Interval NA: Not Applicable

a. Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 1/5 SARS-CoV-2 positive; 4/5 SARS-CoV-2 negative

Discrepant test results based on a U.S. FDA EUA SARS-CoV-2 molecular test: 2/9 SARS-CoV-2 positive; 4/9 b. SARS-CoV-2 negative; 2/9 invalid results; 1/9 discrepant testing was inadvertently not performed

Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/4 Flu A C. negative
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/3 Flu A d. negative
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 1/1 tests not performed due to e. the specimens being stored for a longer duration than allowed per the package insert.
f. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 19/19 tests not performed due to the specimens being stored for a longer duration than allowed per the package insert.
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 3/3 tests not performed due to g. the specimens being stored for a longer duration than allowed per the package insert.
Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 1/1 RSV positive h.
1. Discrepant test results based on a U.S. FDA-cleared molecular respiratory panel: 2/2 test not performed due to the specimens being stored for a longer duration than allowed per the package insert.

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

{33}------------------------------------------------

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 21, where bolded values indicate concordant results.

Table 20. Multi-Target Detection by Xpert Xpress CoV-2/Flu/RSV plus for
Specimens Collected in 2022

Infection		SARS-CoV-2 only	Flu A only	RSV Only	SARS-CoV-2 and Flu A	Negative	Total	Co-Infection Rate (%)
Xpert Xpress CoV-2/Flu/RSV plus	SARS-CoV-2 only	568	0	0	1	25	594
	Flu A only	0	179	0	0	11	190
	RSV only	0	0	12	0	0	12
	SARS-CoV-2 and Flu A	0	0	0	1	0	1	0.1
	Negative	9	3	1	0	2218	2231
Total		577	182	13	2	2254	3028
	Co-Infection Rate (%)	0.3

As presented in Table 20, a total of 3028 Category I and II 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 0.1% (1/797) and the rate of co-infection by the comparator was 0.3% (2/774).

	Table 21. 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

		Hologic Panther Fusion Flu A/B/RSV
	Infection	Flu Aonly	Flu Bonly	RSVonly	Flu Aand FluB	Flu AandRSV	Flu BandRSV	Negative	Total	Co-infectionRate (%)
The Xpert Xpress CoV-2/Flu/RSV plusTest	Flu A only	406	0	0	1	1	0	44	452	2.0
	Flu B only	0	85	0	0	0	0	3	88
	RSV only	0	0	179	0	0	0	3	182
	Flu A and Flu B	1	4	0	1	0	0	1	7
	Flu A and RSV	0	0	2	0	5	0	0	7
	Flu B and RSV	0	0	0	0	0	1	0	1
	Negative	7	1	4	0	0	0	3561	3573
	Total	414	90	185	2	6	1	3612	4310
	Co-infectionRate (%) for theComparator	1.3

As presented in Table 21, of the 4310 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 2.0%

{34}------------------------------------------------

Image /page/34/Picture/0 description: The image shows the Cepheid logo, which includes a stylized blue wing-like design above the word "Cepheid." Below the logo is the text "Xpert® Xpress CoV-2/Flu/RSV plus." The text indicates that the image is related to a diagnostic test for COVID-19, influenza, and respiratory syncytial virus.

(15/737) and the rate of co-infection by the comparator was 1.3% (9/698).

1.5. Conclusions

The results of the analytical and clinical performance studies summarized above demonstrate 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.