The cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems is an automated, multiplex, nucleic acid test that utilizes real-time polymerase chain reaction (PCR) technology intended for simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza A, influenza B and RSV can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections in humans and is not intended to detect influenza C virus infections.

Nucleic acids from the viral organisms identified by this test are generally detectable in nasopharyngeal swab specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus, and aid in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. Conversely, positive results do not rule out coinfection with other organisms, and the agent(s) detected by the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems may not be the definite cause of disease.

Device Description

cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems (cobas® Respiratory 4-flex) is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 System is designed as one integrated instrument. The cobas® 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 5800 System or cobas® 6800/8800 Systems software(s), which assigns results for all tests. Results can be reviewed directly on the system screen and printed as a report.

Nucleic acid from patient samples and added Internal Control RNA (RNA IC) molecules are simultaneously extracted. Nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors, are removed with subsequent wash steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature. External controls (positive and negative) are processed in the same way.

Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers detecting conserved viral genome regions as shown in Table 1.

Selective amplification of RNA IC is achieved by the use of non-competitive, sequence specific forward and reverse primers, which have no homology with the viral-target specific genomes. Amplified target is detected by the cleavage of fluorescently labeled oligonucleotide probes. Roche's temperature assisted generation of signal (TAGS) technology, short TAGS technology, is introduced to differentiate up to three targets per fluorescence channel, enabling the detection of up to14 targets, and the Internal Control, per well. A thermostable DNA polymerase enzyme is used for amplification.

Multiplicity of target detection is enabled with temperature-dependent quenching of cleaved fluorescent target-specific probes. This is achieved by separating signals from probes into introduced thermal channels, where fluorescence is acquired at two additional fixed temperatures for each amplification cycle.

During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase, resulting in separation of the reporter and quencher dyes, and the generation of a fluorescent signal. Conventional probes release fluorescence signal immediately upon separation of reporter from quencher. TAGS probes rely on temperature dependent fluorescence activation, requiring both nuclease cleavage during the extension phase, as well as an increase in reaction temperature, to activate the otherwise dormant fluorophore. For this reason, during each PCR cycle the TAGS technology captures fluorescence in five available fluorescence channels in combination with three thermal channels (detection of fluorescence at three defined temperatures T1, T2 and T3).

The cobas® Respiratory 4-flex master mix contains detection probes which are specific for influenza A virus, influenza B virus, RSV, SARS-CoV-2 and the RNA Internal Control (RNA IC) nucleic acid, which enables simultaneous detection and differentiation of influenza A virus, influenza B virus, RSV, and SARS-CoV-2 viral targets and the RNA IC.

The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicons from previous PCR runs are destroyed by the AmpErase enzyme [uracil-N-glycosylase], which is included in the PCR mix, when heated in the first thermal cycling step. However, newly formed amplicons are not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

The RESP-4FLEX ASAP enables the system to differentiate and report the qualitative results of the four targets influenza A virus, influenza B virus, RSV and SARS-CoV-2. For each specimen the customer can test for any combination of the four enabled virus targets. Also, additional target calculation (digital reflex) can be ordered for the four enabled virus targets (influenza A virus, influenza B virus, RSV and SARS-CoV-2) on the cobas® 5800 System.

AI/ML Overview

Here's an analysis of the acceptance criteria and study detailed in the provided FDA clearance letter for the cobas Respiratory 4-flex, structured according to your request:

1. Table of Acceptance Criteria and Reported Device Performance

The FDA clearance letter does not explicitly state pre-defined acceptance criteria for the clinical performance. Instead, it reports the observed performance metrics (PPA and NPA) from the clinical studies. For the purpose of this table, I will present the reported clinical performance as the "met acceptance criteria," assuming these values were deemed acceptable by the FDA for clearance.

Analyte	Acceptance Criteria (Reported PPA CI)	Device Performance (PPA %)	Acceptance Criteria (Reported NPA CI)	Device Performance (NPA %)
Influenza A
Fresh Pros.	(90.5%, 98.5%)	96.2	(99.3%, 99.9%)	99.7
Frozen Pros.	(89.0%, 99.1%)	96.8	(99.4%, 99.8%)	99.7
Combined Pros.	(92.3%, 98.3%)	96.4	(99.5%, 99.8%)	99.7
Retrospective	(94.1%, 100.0%)	100.0	(97.6%, 99.4%)	98.8
Influenza B
Fresh Pros.	(94.5%, 100.0%)	100.0	(99.6%, 100.0%)	99.9
Frozen Pros.	(79.8%, 99.3%)	95.8	(99.8%, 100.0%)	100.0
Combined Pros.	(94.0%, 99.8%)	98.9	(99.8%, 100.0%)	99.9
Retrospective	(87.1%, 99.6%)	97.5	(98.3%, 99.7%)	99.3
RSV
Fresh Pros.	(77.4%, 94.7%)	88.7	(99.8%, 100.0%)	100.0
Frozen Pros.	(81.5%, 95.3%)	90.4	(99.8%, 100.0%)	100.0
Combined Pros.	(83.1%, 93.9%)	89.7	(99.9%, 100.0%)	100.0
Retrospective	(96.4%, 100.0%)	100.0	(98.2%, 99.7%)	99.3
SARS-CoV-2
Fresh Pros.	(92.4%, 98.6%)	96.7	(98.0%, 99.1%)	98.6
Frozen Pros.	(95.3%, 98.9%)	97.7	(97.8%, 98.8%)	98.4
Combined Pros.	(95.4%, 98.5%)	97.3	(98.0%, 98.8%)	98.5

Study Proving Device Meets Criteria: The detailed clinical performance evaluation described in "5. CLINICAL PERFORMANCE EVALUATION" (pages 23-26) demonstrates that the cobas Respiratory 4-flex achieves the reported Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) values against a U.S. FDA-cleared molecular comparator assay. These reported percentage agreements, along with their 95% confidence intervals, serve as the evidence that the device meets the performance expectations for clinical use.

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

Prospective Clinical Study:
- Total NPS specimens enrolled: 4,475
- Total NPS specimens tested: 4,378 (1,832 fresh, 2,546 frozen)
- Total NPS specimens evaluable: 4,341 (1,827 fresh, 2,514 frozen)
- Data Provenance: Fresh prospective specimens collected at eleven collection sites during the 2023-2024 respiratory viral season. Frozen prospective specimens collected during parts of the 2022-2023 respiratory viral season at seven sites and the 2023-2024 respiratory viral season from 14 collection sites. The specific country of origin is not explicitly stated but implies U.S. based on the "U.S. testing sites" mention for retrospective samples, and the FDA clearance context. The data is prospective.
Retrospective Clinical Study:
- Total NPS specimens enrolled: 770
- Total NPS specimens evaluable:
  - Influenza A: 657
  - Influenza B: 647
  - RSV: 659
- Data Provenance: Archived NPS specimens collected between 2014 and 2022 from individuals with signs and symptoms of respiratory viral infection. Tested at three (3) U.S. testing sites. The data is retrospective.

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

The document does not mention the use of human experts to establish ground truth for the test set. The ground truth was established by a "U.S. FDA-cleared molecular assay" as the comparator method.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method involving experts for discrepant results. Instead, it states that an "FDA 510(k) cleared comparator" method was used to establish the "ground truth." For the influenza A target in the prospective study, "three (3) additional specimens (two (2) fresh and one (1) frozen) were excluded from analysis due to inconclusive results obtained from the comparator test," implying a reliance on the comparator's definitive result rather than external adjudication. The same pattern is noted for retrospective samples, with exclusions for failed or invalid comparator test results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of Human Readers' Improvement with AI vs. Without AI Assistance

This section is Not Applicable to the provided document. The cobas Respiratory 4-flex is an automated, multiplex nucleic acid detection test, not an AI-powered diagnostic tool requiring human reader interpretation or assistance. Therefore, an MRMC study or analysis of human reader improvement with AI is not relevant.

6. If a Standalone (i.e., Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, this was a standalone performance evaluation. The device is an automated in vitro diagnostic (IVD) test. The clinical performance evaluation directly compares the cobas Respiratory 4-flex's results (algorithm only, as it's an automated system) against a predicate FDA-cleared molecular assay. There is no human interpretation or intervention in the generation of the primary test result from the cobas system itself.

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

The ground truth for the clinical performance evaluation was established by a "U.S. FDA-cleared molecular assay." This means another legally marketed and validated molecular diagnostic test was used as the reference standard.

8. The Sample Size for the Training Set

The document does not specify a separate training set or its sample size. This is typical for in vitro diagnostic (IVD) devices that use established laboratory techniques (like PCR) rather than machine learning algorithms which require explicit training data. The development of such assays involves extensive analytical validation (LoD, precision, specificity, inclusivity, etc.) and then clinical validation with independent samples.

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

Since no explicit training set for a machine learning algorithm is mentioned, the concept of "ground truth for the training set" is not applicable in the context of this device's validation as described. The analytical studies (LoD, inclusivity, specificity, etc.) characterize the inherent performance of the assay's chemical and hardware components.

Summary

U.S. Food & Drug Administration FDA Clearance Letter

Page 1

U.S. Food & Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993
www.fda.gov

Doc ID # 04017.08.00

July 31, 2025

Roche Molecular Systems, Inc.
Claudia Machay
Regulatory Affairs Project Manager
4300 Hacienda Drive
Pleasanton, California 94588

Re: K243455
Trade/Device Name: cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems
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: November 5, 2024
Received: November 7, 2024

Dear Claudia Machay:

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.

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K243455 - Claudia Machay Page 2

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 (QS) 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 (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-safety-reporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 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-devices/device-advice-comprehensive-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-devices/medical-device-safety/medical-device-reporting-mdr-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/medical-devices/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-devices/device-advice-comprehensive-regulatory-

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K243455 - Claudia Machay Page 3

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

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FORM FDA 3881 (8/23) Page 1 of 1

DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration

Indications for Use

Form Approved: OMB No. 0910-0120
Expiration Date: 07/31/2026
See PRA Statement below.

510(k) Number (if known): K243455

Device Name: cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems

Indications for Use (Describe)

The cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems is an automated, multiplex, nucleic acid test that utilizes real-time polymerase chain reaction (PCR) technology intended for simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza A, influenza B and RSV can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections in humans and is not intended to detect influenza C virus infections.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. Conversely, positive results do not rule out coinfection with other organisms, and the agent(s) detected by the cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems may not be the definite cause of disease.

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."

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cobas® Respiratory 4-flex 510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

Submitter Name	Roche Molecular Systems, Inc.
Address	4300 Hacienda DrivePleasanton, CA 94588-2722
Contact	Claudia MachayPhone: +41 79 563 9112Email: claudia.machay@roche.com
Date Prepared	June, 24th, 2025
Proprietary Name	cobas® Respiratory 4-flexfor use on the cobas® 5800/6800/8800 Systems
Common Name	cobas® Respiratory 4-flex
Classification Name	Multi-Target Respiratory Specimen Nucleic Acid Test Including Sars-CoV-2 And Other Microbial Agents
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
Regulation Class	Class II (special controls)
Product Codes	QOF
Predicate Devices	BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031)
Establishment Registration	Roche Molecular Systems, Inc. (2243471)

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1. DEVICE DESCRIPTION

Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers detecting conserved viral genome regions as shown in Table 1.

Table 1: cobas® Respiratory 4-flex target regions

Targeted organism	Target gene (symbol)
Influenza A	Matrix protein 1 (M1)
Influenza B	Non-structural protein NS-1/2 (NS1/NEP)
Respiratory Syncytial Virus	Matrix protein (M)
SARS-CoV-2	ORF1 ab polyprotein (ORF1ab) andORF 1a polyprotein (ORF1a)

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of up to14 targets, and the Internal Control, per well. A thermostable DNA polymerase enzyme is used for amplification.

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2. INTENDED USE

3. TECHNOLOGICAL CHARACTERISTICS

The primary technological characteristics and intended use of the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems are substantially equivalent to other legally marketed nucleic acid amplification test intended for the qualitative detection and differentiation of influenza A virus, influenza B virus, respiratory syncytial virus (RSV), and SARS-CoV-2 virus.

As indicated in Table 2, the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems is substantially equivalent to significant characteristics of the identified predicate device, BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031)

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Table 2: Comparison of cobas® Respiratory 4-flex with the Predicate Device (BioFire® Respiratory Panel 2.1 (RP2.1))

Comparator	Candidate Device:cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems	Predicate Device:BioFire® Respiratory Panel 2.1(DEN200031)
Regulation Number	21 CFR 866.3981	Same
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.	Same
Regulatory Class	Class II (special controls)	Same
Device Classification Name	Multi-target respiratory specimen nucleic acid test including sars-cov-2 and other microbial agents	Same
Product Code	QOF	Same

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| Intended Use | The cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems is an automated, multiplex, nucleic acid test that utilizes real-time polymerase chain reaction (PCR) technology intended for simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza A, influenza B and RSV can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections in humans and is not intended to detect influenza C virus infections.Nucleic acids from the viral organisms identified by this test are generally detectable in nasopharyngeal swab specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus, and aid in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings.The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. Conversely, positive results do not rule out coinfection with other organisms, and the agent(s) detected by the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems may not be the definite cause of disease. | The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire FilmArray Torch systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19.The following organism types and subtypes are identified using the BioFire RP2.1:Adenovirus,Coronavirus 229E,Coronavirus HKU1,Coronavirus NL63,Coronavirus OC43,Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2),Human Metapneumovirus,Human Rhinovirus/Enterovirus,Influenza A, including subtypes H1, H1-2009, and H3,Influenza B,Parainfluenza Virus 1,Parainfluenza Virus 2,Parainfluenza Virus 3,Parainfluenza Virus 4,Respiratory Syncytial Virus,Bordetella parapertussis (IS1001),Bordetella pertussis (ptxP),Chlamydia pneumoniae, andMycoplasma pneumoniaNucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that |

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Comparator	Candidate Device:cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems	Predicate Device:BioFire® Respiratory Panel 2.1(DEN200031)
		are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the BioFire RP2.1 may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
Conditions for use	For prescription use	same
Sample Types	Nasopharyngeal swab specimen	same
Analyte Targets	Influenza A virusInfluenza B virusRespiratory Syncytial Virus (RSV)SARS-CoV-2	Adenovirus,Coronavirus 229E,Coronavirus HKU1,Coronavirus NL63,Coronavirus OC43,Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2),Human Metapneumovirus,Human Rhinovirus/Enterovirus,Influenza A, including subtypes H1, H1-2009, and H3,Influenza B,Parainfluenza Virus 1,Parainfluenza Virus 2,Parainfluenza Virus 3,Parainfluenza Virus 4,Respiratory Syncytial Virus,Bordetella parapertussis (IS1001),Bordetella pertussis (ptxP),Chlamydia pneumoniae, andMycoplasma pneumonia
Sample Preparation Procedure	Automated by the cobas® 5800/6800/8800 Systems	Automated by BioFire FilmArray 2.0 or BioFire FilmArray Torch systems

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Comparator	Candidate Device:cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems	Predicate Device:BioFire® Respiratory Panel 2.1(DEN200031)
Detection Chemistry	PCR amplification and detection, consisting of TaqMan probes with fluorescent dyes. Multiplicity of target detection is enabled with temperature-dependent quenching of cleaved fluorescent target-specific probes.	Two Step Nested multiplex PCR:Reverse transcription, followed by a multiplexed first stage PCR reaction (PCR1).Multiple simultaneous second-stage PCR reactions (PCR2) to amplify sequences within the PCR1 products using fluorescence double stranded binding dye.Endpoint melting curve data to detect target-specific amplicons
Controls used	Sample processing control (IC)Positive and negative control	Two process controls:RNA Process Control (IC)PCR2 Control (A positive result indicates that PCR2 was successful)
Result Analysis	Based on PCR cycle threshold analysis	Endpoint melting curve data to detect target specific amplicons

4. NON-CLINICAL PERFORMANCE EVALUATION

4.1. Analytical Sensitivity (Limit of Detection)

The Limit of Detection (LoD) study determines the lowest detectable concentration of RSV, influenza A (H3N2 and H1N1), SARS-CoV-2, influenza B (Victoria and Yamagata lineage), at which greater or equal to 95% of all (true positive) replicates test positive.

The LoD of cobas® Respiratory 4-flex was determined by analysis of serial dilutions co-formulated with cultured RSV, influenza A, influenza B, and inactivated SARS-CoV-2 diluted in nasopharyngeal matrix. Panels of at least five concentration levels plus a blank were tested over three lots of reagents, multiple runs, days, operators, and instruments. The results as well as the materials used are shown in Table 3.

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Table 3: Limit of Detection by hit rate ≥ 95%

Target	Strain / Isolate	LoD by Hit Rate ≥ 95%	Hit Rate	Concentration
Influenza A (H1N1)	Brisbane/02/2018	1.00E+02	95.2% (60/63)	cp/mL
Influenza A (H3N2)	A/Darwin/6/2021	5.00E+01	95.2% (60/63)	cp/mL
Influenza B (Victoria)	B/Austria/1359417/2021	2.50E+02	100.0% (63/63)	cp/mL
Influenza B (Yamagata)	Phuket/3073/13	8.00E+02	98.4% (62/63)	cp/mL
RSV A	Respiratory Syncytial Virus A2	4.00E+03	98.4% (62/63)	cp/mL
SARS-CoV-2	1st WHO International Standard NIBSC code 20/146*	8.00E+01	95.2% (60/63)	IU/mL

*Inactivated SARS-CoV-2 virus

A separate study was performed to demonstrate that the LoD of each strain individually was equivalent to the co-spiked LoD.

4.2. Precision – within laboratory

Precision of cobas® Respiratory 4-flex was determined by analysis of panels consisting of different cell culture strains in negative simulated clinical matrix stabilized in UTM™. Two dilution levels were tested in 216 replicates for each level across three lots of reagents using six instruments and five operators over twelve testing days. Each sample was carried through the entire cobas® Respiratory 4-flex procedure on fully automated cobas® 5800/6800/8800 Systems. Therefore, the precision reported here represents all aspects of the test procedure. The results are shown in Table 4 and Table 5. The results of this study revealed that cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems consistently detects the presence of all targets by achieving ≥99% hit rates around LoD (~1x LoD) and 100% hit rates above LoD (~3x LoD).

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Table 4: Precision – Summary of hit rates and confidence intervals

Target	Level	Positive Results	Total Results	Positivity %	Two-sided 95% CI Lower Bound	Two-sided 95% CI Upper Bound
Influenza A (H3N2)	~3x LoD	216	216	100	98.31	100
Influenza A (H3N2)	~1x LoD	216	216	100	98.31	100
Influenza B (Victoria)	~3x LoD	216	216	100	98.31	100
Influenza B (Victoria)	~1x LoD	215	216	99.54	97.45	99.99
RSV A	~3x LoD	216	216	100	98.31	100
RSV A	~1x LoD	214	216	99.07	96.70	99.89
SARS-CoV-2	~3x LoD	216	216	100	98.31	100
SARS-CoV-2	~1x LoD	216	216	100	98.31	100
N/A	Blank	0	216	0	0.00	3.36

Table 5: Precision - standard deviations and coefficients of variation of Ct values

Target	Level	Hit rate	Mean Ct	Instrument-to-Instrument		Lot-to-Lot		Day-to-Day		Run-to-Run		Within Run		Total
				SD	CV%	SD	CV%	SD	CV%	SD	CV%	SD	CV%	SD	CV%
Influenza A (H3N2)	~3x LoD	100.00%	37.33	0.08	0.22	0.08	0.21	0.00	0.00	0.07	0.20	0.48	1.28	0.50	1.33
Influenza A (H3N2)	~1x LoD	100.00%	39.06	0.13	0.34	0.14	0.35	0.23	0.59	0.00	0.00	1.02	2.60	1.06	2.71
Influenza B (Victoria)	~3x LoD	100.00%	34.61	0.04	0.11	0.09	0.26	0.00	0.00	0.00	0.00	0.22	0.64	0.24	0.69
Influenza B (Victoria)	~1x LoD	99.54%	35.34	0.04	0.12	0.08	0.23	0.00	0.00	0.00	0.00	0.24	0.69	0.26	0.73
RSV A	~3x LoD	100.00%	33.20	0.06	0.18	0.08	0.25	0.04	0.11	0.00	0.00	0.19	0.58	0.22	0.66
RSV A	~1x LoD	99.07%	33.62	0.04	0.11	0.05	0.16	0.02	0.06	0.02	0.06	0.24	0.70	0.25	0.73
SARS-CoV-2	~3x LoD	100.00%	35.62	0.03	0.09	0.00	0.00	0.03	0.09	0.00	0.00	0.32	0.89	0.32	0.90
SARS-CoV-2	~1x LoD	100.00%	36.48	0.00	0.00	0.00	0.00	0.03	0.09	0.00	0.00	0.41	1.13	0.42	1.14

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4.3. Reproducibility

The reproducibility of cobas® Respiratory 4-flex was evaluated across multiple variables that theoretically could affect reported results, including: reagent lot, testing site/instrument, day, and run. Reproducibility was determined by analysis of panels consisting of different cell culture strains in negative simulated clinical matrix stabilized in UTM™ as well as one sample negative for all viruses (solely negative simulated clinical matrix stabilized in UTM™). Panels were tested in triplicate in each run. Two dilution levels per viral target were tested in 504 replicates distributed across two runs per day at three sites, using three lots of reagents, six days of testing per reagent lot using nine cobas® 5800/6800/8800 systems. The results are summarized in Table 6.

The system showed a 99.6% negative percent agreement with the Exact CI of 98.6-100.0%. The test results showed adequate lot-to-lot, instrument-to-instrument (site), day-to-day, and between run variation for the ~1x LoD, and ~3x LoD panel members (Table 6).

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Table 6: Overall percentage agreement, mean estimate, standard deviations, and coefficients of variation (%) for cycle threshold values by viral target and expected viral concentration

Target Virus	Viral Concentration	(n/N)ᵃ	Percent Agreement (%)ᵇ	Mean Ct	Total SD	Total %CV	Site SD	Site %CV	Lot SD	Lot %CV	Day SD	Day %CV	Run SD	Run %CV	Within Run SD	Within Run %CV
Negative	0	502/504	99.6	nc	nc	nc	nc	nc	nc	nc	nc	nc	nc	nc	nc	nc
Influenza A	~1× LoD	504/504	100	39.1	1.03	2.63	0.00	0.00	0.19	0.47	0.00	0.00	0.21	0.54	0.99	2.52
Influenza A	~3× LoD	504/504	100	37.4	0.50	1.34	0.04	0.10	0.00	0.00	0.12	0.33	0.00	0.00	0.49	1.30
Influenza B	~1× LoD	501/504	99.4	35.4	0.28	0.79	0.08	0.22	0.07	0.19	0.03	0.09	0.00	0.00	0.26	0.72
Influenza B	~3× LoD	503/503ᶜ	100	34.7	0.25	0.72	0.11	0.30	0.04	0.12	0.04	0.11	0.05	0.13	0.21	0.62
RSV	~1× LoD	501/504	99.4	33.8	0.31	0.91	0.12	0.36	0.11	0.34	0.05	0.15	0.07	0.22	0.24	0.72
RSV	~3× LoD	504/504	100	33.4	0.29	0.88	0.15	0.44	0.13	0.38	0.06	0.19	0.00	0.00	0.21	0.64
SARS-CoV-2	~1× LoD	504/504	100	36.4	0.41	1.12	0.04	0.11	0.02	0.06	0.00	0.00	0.00	0.00	0.41	1.11
SARS-CoV-2	~3× LoD	504/504	100	35.6	0.31	0.88	0.01	0.03	0.00	0.00	0.00	0.00	0.07	0.20	0.30	0.85

Ct = cycle threshold, LoD = limit of detection, SD = standard deviation, CV(%) = percent coefficient of variation, nc = not calculable, RSV= respiratory syncytial virus, SARS-CoV-2= severe acute respiratory syndrome coronavirus 2.

ᵃ n is the number of positive tests which contributed Ct values to the analysis. N is the total number of valid tests for the panel member.
ᵇ Percent agreement with expected results.
ᶜ A single invalid result was obtained for influenza B, resulting in 503 samples with valid results.

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4.4. Inclusivity

The inclusivity for the detection of different strains of influenza A, influenza B, RSV and SARS-CoV-2 was assessed by testing relevant strains of each viral target. Each strain was tested with 3 replicates near LoD starting at ~3x LoD. The concentration which showed a 100% hit rate is shown in Table 7 through Table 10. In silico analysis on June 13th, 2025, of the dual target SARS-CoV-2 primer and probe binding regions indicates 99.99% detection of all available sequences in NCBI (>8.32M sequences, no predicted failed detections) and GISAID (>15.96M sequences, <10 predicted failed detections) databases. In silico analysis indicated inclusivity to all known SARS-CoV-2 variants.

Table 7: SARS-CoV-2 Inclusivity strains

Virus type	Strain	Concentration Tested (cp/mL)	100% hit rate at
SARS-CoV-2 Lineage B.1.1.7	England/204820464/2020	312	~3x LoD
SARS-CoV-2 Lineage B.1.351	South Africa/KRISPK005325/2020	312	~3x LoD
SARS-CoV-2 Lineage P.1	Japan/TY7-503/2021	312	~3x LoD
SARS-CoV-2 B.1.617.2	USA/PHC658/2021	312	~3x LoD
SARS-CoV-2 Lineage B.1.1.529	USA/MD-HP20874/2021	312	~3x LoD
SARS-CoV-2	USA-WA1/2020	312	~3x LoD

Table 8: Influenza A Inclusivity strains

Virus type	Strain	Concentration Tested (cp/mL)	100% hit rate at
Influenza A H1N1	New Caledonia/20/99	165	~3x LoD
Influenza A H1N1	Brisbane/59/07	165	~3x LoD
Influenza A H1N1	California/07/09	165	~3x LoD
Influenza A H1N1	NY/03/09	165	~3x LoD
Influenza A H1N1	A/Victoria/2570/2019	165	~3x LoD
Influenza A H1N1	A/Wisconsin/588/2019	165	~3x LoD
Influenza A H1N1	A/Victoria/4897/2022	165	~3x LoD
Influenza A H1N1	A/Wisconsin/67/2022	330	~6x LoD
Influenza A H1N1	England/73/22	375	~6.8x LoD
Influenza A H1N1	England/55/22	375	~6.8x LoD
Influenza A H3N2	A/Port Chalmers/1/73	165	~3x LoD
Influenza A H3N2	Texas/50/12	242.7	~4.4x LoD
Influenza A H3N2	A/Victoria/3/75	165	~3x LoD

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Virus type	Strain	Concentration Tested (cp/mL)	100% hit rate at
Influenza A H3N2	Wisconsin/67/05	165	~3x LoD
Influenza A H3N2	A/Darwin/9/2021	165	~3x LoD
Influenza A H3N2	Hong Kong/4801/14	165	~3x LoD
Influenza A H3N2	Hong Kong/8/68	165	~3x LoD
Influenza A H3N2	A/Perth/16/09	165	~3x LoD
Influenza A H3N2	Kansas/14/17	165	~3x LoD
Influenza A H3N2	Switzerland/9715293/13	165	~3x LoD
Influenza A H5N1	A/mallard/Wisconsin/2576/2009	165	~3x LoD
Influenza A H5N2	A/ruddy turnstone/New Jersey/828212/2001	165	~3x LoD
Influenza A H5N3	A/duck/Singapore/645/1997	165	~3x LoD
Influenza A H7N2	A/northern pintail/Illinois/10OS3959/2010	165	~3x LoD
Influenza A H7N8	A/mallard/Ohio/11OS2033/2011	165	~3x LoD
Influenza A H7N9	A/northern shoveler/Mississippi/11OS145/2011	165	~3x LoD
Influenza A H9N7	A/shorebird/Delaware Bay/31/1996	165	~3x LoD

Table 9: Influenza B Inclusivity strains

Virus type	Strain	Concentration Tested (cp/mL)	100% hit rate at
Influenza B – Victoria	Colorado/6/17	1779	~3x LoD
Influenza B – Victoria	B/Hong Kong/5/72	1779	~3x LoD
Influenza B - Victoria	Brisbane/60/08	1779	~3x LoD
Influenza B - Victoria	Florida/02/06	1779	~3x LoD
Influenza B – Yamagata	B/Massachusetts/2/2012	1779	~3x LoD
Influenza B – Yamagata	B/Wisconsin/1/2010	1779	~3x LoD
Influenza B – Yamagata	B/Florida/4/2006	1779	~3x LoD
Influenza B – Yamagata	Texas/6/11	1779	~3x LoD
Influenza B – Yamagata	Florida/07/04	1779	~3x LoD
Influenza B – Unknown	B/Taiwan/2/62	1779	~3x LoD
Influenza B – Unknown	B/Allen/45	1779	~3x LoD
Influenza B – Unknown	B/Lee/40	1779	~3x LoD

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Table 10: Respiratory Syncytial Virus Inclusivity strains

Virus type	Strain	Concentration Tested (cp/mL)	100% hit rate at
RSV Type A	2006 Isolate	18510	~3x LoD
RSV Type A	02/2015	18510	~3x LoD
RSV Type A2	A2	18510	~3x LoD
RSV Type B	CH93(18)-18	18510	~3x LoD
RSV Type B	9320	18510	~3x LoD
RSV Type B	B WV/14617/85	18510	~3x LoD
RSV Type B	18537	18510	~3x LoD

4.5. Matrix equivalency

Equivalency between nasopharyngeal swabs and simulated clinical matrix stabilized in UTM-RT® was evaluated. Pooled negative individual clinical specimens (nasopharyngeal) and simulated clinical matrix stabilized in UTM™ were spiked with two co-formulated panels containing RSV, influenza A & SARS-CoV-2 and influenza B, at a concentration level of ~2x LoD. Forty-two replicates per concentration were tested for each sample type. All replicates tested with the 2x LoD panel were positive for the respective viral target for both matrices with 100% hit rate.

4.6. Analytical specificity (cross-reactivity and microbial interference)

The analytical specificity of cobas® Respiratory 4-flex was evaluated by testing a panel of microorganisms including those commonly found in the respiratory tract plus pooled human nasal wash.

The organisms listed in Table 11 were spiked at 1.00E+06 units/mL for bacteria and fungi and at 1.00E+05 units/mL for viruses unless otherwise noted. Testing was performed with each potential interfering organism in the absence and the presence of RSV, influenza A, influenza B and SARS-CoV-2, target spiked at ~3x LoD.

Negative results were obtained with cobas® Respiratory 4-flex for all microorganism samples without viral target and positive results were obtained for all microorganism samples with viral target spiked at ~3x LoD.

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Table 11: Microorganisms tested for analytical specificity/cross reactivity

Microorganism	Concentration
Aspergillus flavus	1.00E+06 CFU/mL
Bordetella parapertussis	1.00E+06 CFU/mL
Bordetella pertussis	1.00E+06 CFU/mL
Candida albicans	1.00E+06 CFU/mL
Chlamydia pneumoniae	1.00E+06 IFU/mL
Corynebacterium diphtheriae	1.00E+06 CFU/mL
Cytomegalovirus	1.00E+05 TCID50/mL
Epstein Barr virus	1.00E+05 cp/mL
Escherichia coli	1.00E+06 CFU/vial
Fusobacterium necrophorum	1.00E+06 CFU/mL
Haemophilus influenzae	1.00E+06 CFU/mL
Lactobacillus acidophilus	1.00E+06 CFU/vial
Legionella pneumophila	1.00E+06 CFU/mL
Measles virus	1.00E+05 TCID50/mL
MERS-coronavirus*	1.00E+05 cp/mL
Moraxella catarrhalis	1.00E+06 CFU/mL
Mumps virus	1.00E+05 TCID50/mL
Mycobacterium bovis	1.00E+06 CFU/mL
Mycoplasma genitalium	1.00E+06 CFU/vial
Mycoplasma pneumoniae	1.00E+06 CCU/mL
Neisseria elongata	1.00E+06 CFU/mL
Neisseria meningitidis	1.00E+06 CFU/mL
Pneumocystis jirovecii	5.00E+03 organisms/mL
Pseudomonas aeruginosa	1.00E+06 CFU/mL
SARS-coronavirus (SARS-CoV)*	1.00E+05 cp/mL
Staphylococcus aureus	1.00E+06 CFU/mL
Staphylococcus epidermidis	1.00E+06 CFU/mL
Streptococcus pneumoniae	1.00E+06 CFU/mL
Streptococcus pyogenes	1.00E+06 CFU/mL
Streptococcus salivarius	1.00E+06 CFU/mL

*Inactivated virus was used for testing

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4.7. Analytical specificity – Interfering substances

Elevated levels of mucin (0.3 – 0.5% w/v) and whole blood (1.5 – 3.0% v/v) were tested in the absence and in the presence of RSV, influenza A, influenza B and SARS-CoV-2target spiked at ~3x LoD. The tested endogenous interferences were shown not to interfere with the test performance of cobas® Respiratory 4-flex.

Additionally, negative clinical nasopharyngeal swab specimens collected in Remel media (M4RT, M5 and M6) as well as Greiner tubes (VACUETTE® 3 mL Virus Stabilization Tube) were tested as equivalent collection media. The alternative collection media were tested unspiked and spiked at ~3x LoD. None of the alternative collection media showed interference with the test performance of cobas® Respiratory 4-flex.

In addition, drug compounds listed in Table 12 were tested in the presence and absence of all viral targets.

All potentially interfering substances, with the exception of FluMist® and Snuff Tobacco, have been shown to not interfere with the test performance. Negative results were obtained with cobas® Respiratory 4-flex for all samples without viral target and positive results were obtained for all samples with viral target.

As expected, FluMist® Quadrivalent, a live quadrivalent vaccine for administration by intranasal spray, consisting of two influenza A and two influenza B vaccine virus strains generated positive results for influenza A and influenza B and negative results for all other targets when solely testing FluMist®.

Furthermore, Snuff Tobacco was identified as a potential interferent of cobas® Respiratory 4-flex as invalid results were generated when testing Snuff Tobacco at 0.1% (w/v) without viral targets.

Table 12: Drug compounds tested for interference with cobas® Respiratory 4-flex

Generic drug name	Active Ingredient	Concentration
AXOTIDE Diskus Multidose 250 mcg	Fluticasone propionate	0.167 mg/mL
BACTROBAN Nasal Ointment	Mupirocin	0.20 mg/mL
BUDESONID Sandoz Nasal Spray 64 mcg	Budesonide	0.039 mg/mL
CEPACOL Extra Strength Sore Throat	Benzocaine	5 mg/mL
Chloraseptic max	Phenol	0.47 mg/mL

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Generic drug name	Active Ingredient	Concentration
FLUMIST® Quadrivalent	live attenuated influenza A and B viruses	50000000 FFU/mL
Heel Luffeel Nasal Spray	Luffa operculataThryallis glaucaHistaminumSulphur	2.99 mg/mL2.99 mg/mL1.5 mg/mL1.5 mg/mL
NASIVIN Pur Spray 0.05%	Oxymetazoline	0.011 mg/mL
OBRACIN Inj Solution 40 mg/mL	Tobramycin	0.018 mg/mL
RELENZA Disk 5 mg	Zanamivir	0.0015 mg/mL
TAMIFLU Kaps 75 mg	Oseltamivir	0.0073 mg/mL
Snuff Tobacco	Nicotine	0.1% w/v
Vaseline	Petroleum Jelly	1% w/v
VICKS VapoRub	Eucalyptus Oil and Menthol	1% w/v
XYLOCAIN Spray 10%	Lidocaine	2.68 mg/mL

4.8. Co-infection (competitive interference)

To assess potential competitive interference between the viral targets, a total of 12 panels composed of various combinations of the cobas® Respiratory 4-flex targets were tested (Table 13). Twelve replicates were tested with one viral target at ~3x LoD which was mixed with a target at high concentration (1.0E+06 units/mL). None of the targets present at very high concentration interfered with the detection of other viral targets at low concentration levels.

Table 13: Combinations tested for potential competitive inhibition

Combination	Target 1 (high)≥ 1.00E+06 unit/mL	Target 2 (low)~3x LoD
1	Influenza A	SARS-CoV-2
2	Influenza B	SARS-CoV-2
3	RSV	SARS-CoV-2
4	SARS-CoV-2	Influenza A
5	Influenza B	Influenza A
6	RSV	Influenza A
7	Influenza A	Influenza B
8	SARS-CoV-2	Influenza B
9	RSV	Influenza B
10	Influenza A	RSV
11	Influenza B	RSV

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Combination	Target 1 (high)≥ 1.00E+06 unit/mL	Target 2 (low)~3x LoD
12	SARS-CoV-2	RSV

4.9. Cross contamination

The cross-contamination rate for cobas® Respiratory 4-flex was determined by testing 480 replicates of negative simulated clinical matrix and 430 replicates of a high titer SARS-CoV-2 panel at approximately 6.50E+08 particles/mL. In total, five runs were performed on cobas® 6800/8800 Systems and 25 runs were performed on cobas® 5800 Systems with positive and negative samples in a checkerboard configuration. All 480 replicates of the negative sample were negative, resulting in a cross-contamination rate of 0% (upper one-sided 95% confidence interval 0.62%).

5. CLINICAL PERFORMANCE EVALUATION

5.1. Performance with prospective clinical specimens

The clinical performance of cobas® Respiratory 4-flex on the cobas® 5800/6800/8800 Systems was evaluated versus an FDA 510(k) cleared comparator in nasopharyngeal swab (NPS) specimens from patients experiencing signs and symptoms of respiratory viral infection. The sample set consisted of a combination of prospective specimens that were either freshly tested (Fresh prospective) or were frozen prior to testing (Frozen prospective) on cobas® Respiratory 4-flex. Fresh specimens were collected at eleven collection sites during the 2023-2024 respiratory viral season and tested with the cobas® Respiratory 4-flex using the cobas® 6800/8800 System at four (4) testing sites. Frozen specimens were prospectively collected during parts of the 2022-2023 respiratory viral season at seven (7) sites and the 2023-2024 respiratory viral season from 14 collection sites and tested with the cobas® Respiratory 4-flex assay at three (3) testing sites using the cobas® 6800/8800 Systems.

A total of 4,475 NPS specimens (1,869 fresh and 2,606 frozen) were enrolled for the prospective clinical study, of which 4,378 could be tested (1,832 fresh and 2,546 frozen) with cobas® Respiratory 4-flex on the cobas® 6800/8800 System and the comparator method. Thirty-five fresh specimens could not be tested due to instrument error or protocol deviations, and two (2) fresh specimens were unable to be tested due to sample processing error. Sixty frozen samples were not tested due to insufficient specimen volume, instrument error, or specimens lost in transit by the courier.

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Of the 4,378 prospective NPS specimens tested, 4,341 specimens were evaluable (1,827 fresh and 2,514 frozen). Five (5) fresh specimens and 32 frozen specimens were non-evaluable due to obtaining invalid results with cobas® Respiratory 4-flex. For the influenza A target, three (3) additional specimens (two (2) fresh and one (1) frozen) were excluded from analysis due to inconclusive results obtained from the comparator test. cobas® Respiratory 4-flex demonstrated adequate clinical performance, the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) point estimates between cobas® Respiratory 4-flex and the comparator for the different target pathogens are summarized in Table 14.

Table 14: Clinical performance between cobas® Respiratory 4-flex and comparator in the prospective studies

Analyte	Positive Percent Agreement (PPA)TP/(TP+FN)	Positive Percent Agreement (PPA)%	Positive Percent Agreement (PPA)95% CI	Negative Percent Agreement (NPA)TN/(TN+FP)	Negative Percent Agreement (NPA)%	Negative Percent Agreement (NPA)95% CI
Influenza A Fresh prospective	100/104	96.2	(90.5%, 98.5%)	1716/1721	99.7	(99.3%, 99.9%)
Influenza A Frozen prospective	60/62	96.8	(89.0%, 99.1%)	2443/2451	99.7	(99.4%, 99.8%)
Influenza A Fresh and frozen prospective	160/166	96.4	(92.3%, 98.3%)	4159/4172	99.7	(99.5%, 99.8%)
Influenza B Fresh prospective	66/66	100.0	(94.5%, 100.0%)	1759/1761	99.9	(99.6%, 100.0%)
Influenza B Frozen prospective	23/24	95.8	(79.8%, 99.3%)	2489/2490	100.0	(99.8%, 100.0%)
Influenza B Fresh and frozen prospective	89/90	98.9	(94.0%, 99.8%)	4248/4251	99.9	(99.8%, 100.0%)
RSV Fresh prospective	47/53	88.7	(77.4%, 94.7%)	1774/1774	100.0	(99.8%, 100.0%)
RSV Frozen prospective	66/73	90.4	(81.5%, 95.3%)	2441/2441	100.0	(99.8%, 100.0%)
RSV Fresh and frozen prospective	113/126	89.7	(83.1%, 93.9%)	4215/4215	100.0	(99.9%, 100.0%)
SARS-CoV-2 Fresh prospective	145/150	96.7	(92.4%, 98.6%)	1654/1677	98.6	(98.0%, 99.1%)
SARS-CoV-2 Frozen prospective	295/302	97.7	(95.3%, 98.9%)	2176/2212	98.4	(97.8%, 98.8%)
SARS-CoV-2 Fresh and frozen prospective	440/452	97.3	(95.4%, 98.5%)	3830/3889	98.5	(98.0%, 98.8%)

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CI: confidence interval; FN: false negative; FP: false positive; RSV: respiratory syncytial virus; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TN: true negative; TP: true positive.

Note: TP (True positives) refers to the number of samples where both the cobas® Respiratory 4-flex and the comparator tests are positive; (FP) False positives refers to the number of samples where the cobas® Respiratory 4-flex is positive and the comparator is negative; FN (False Negatives) refers to the number of samples where the cobas® Respiratory 4-flex is negative and the comparator is positive; TN (True Negatives) refers to the number of samples where both the cobas® Respiratory 4-flex and the comparator tests are negative.

5.2. Performance with retrospective clinical specimens

In addition to the prospective clinical data for influenza A, influenza B, and RSV frozen archived NPS specimens in UTM or UVT from individuals with signs and symptoms of respiratory viral infection were tested (Retrospective). The retrospective samples were collected between 2014 and 2022 and were selected for inclusion in the study based on the historical result. Retrospective specimens were tested with cobas® Respiratory 4-flex at three (3) U.S testing sites using the cobas® 6800/8800 Systems. The comparator method utilized to demonstrate agreement was a U.S. FDA-cleared molecular assay.

A total of 770 NPS specimens were enrolled for the retrospective clinical study, and fifteen specimens were excluded from analysis due to obtaining invalid results on the cobas® Respiratory 4-flex. For the influenza A target, 98 additional specimens were excluded from analysis due to failed or invalid comparator test results, leaving 657 evaluable specimens for influenza A. For the influenza B target, 108 additional specimens were excluded from analysis due to failed or invalid comparator test results, leaving 647 evaluable specimens for influenza B. For the RSV target, 96 additional specimens were excluded from analysis due to failed or invalid comparator test results, leaving 659 evaluable specimens for RSV. A summary of the cobas® Respiratory 4-flex retrospective clinical study agreement with the expected results is provided in Table 15.

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Table 15: Agreement of the cobas® Respiratory 4-flex and comparator test in retrospective samples

Analyte	Positive Percent Agreement (PPA)TP/(TP+FN)	Positive Percent Agreement (PPA)%	Positive Percent Agreement (PPA)95% CI	Negative Percent Agreement (NPA)TN/(TN+FP)	Negative Percent Agreement (NPA)%	Negative Percent Agreement (NPA)95% CI
Influenza A	61/61	100.0	(94.1%, 100.0%)	589/596	98.8	(97.6%, 99.4%)
Influenza B	39/40	97.5	(87.1%, 99.6%)	603/607	99.3	(98.3%, 99.7%)
RSV	104/104	100.0	(96.4%, 100.0%)	551/555	99.3	(98.2%, 99.7%)

CI: confidence interval; FN: false negative; FP: false positive; RSV: respiratory syncytial virus; TN: true negative; TP: true positive.

5.3. Clinical performance equivalency between the cobas® 6800/8800 and cobas® 5800 Systems

A clinical performance equivalency study was conducted to demonstrate equivalent performance between the cobas® 6800/8800 and cobas® 5800 Systems. All specimens in the clinical study were tested with the candidate assay on the cobas® 6800/8800 System and then a subset of samples, chosen at random, were tested with the cobas® 5800 System. The results from each system were evaluated against the respective comparator assay(s), and the PPA and NPA were directly compared between the two systems. The results were equivalent between the instrument systems.

6. CONCLUSIONS

A comparison of the intended use, technological characteristics, and the results of analytical (non-clinical) and clinical performance studies demonstrates that the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8000 Systems 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.