cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems
Device Facts
| Record ID | K243455 |
|---|---|
| Device Name | cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems |
| Applicant | Roche Molecular Systems, Inc. |
| Product Code | QOF · Microbiology |
| Decision Date | Jul 31, 2025 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3981 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K243455 · Jul 31, 2025 | cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems | Roche Molecular Systems, Inc. | Archived nasopharyngeal swab specimens from routine clinical care (2014-2022) | Retrospective clinical study used to evaluate the performance of the cobas Respiratory 4-flex assay against an FDA-cleared molecular comparator. | Retrospective study; Archived specimens; Clinical performance |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Retrospective clinical study; Retrospective evaluation of archived clinical specimens; Follow-up/Duration: Samples collected between 2014 and 2022; Study Period: 2014-2022 | Patients with signs and symptoms of respiratory viral infection; Sample Size: 770 NPS specimens enrolled; Number of Sites: 3 U.S. testing sites | FDA-cleared molecular assay | Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) |
Indications for 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.
Device Story
Automated, multiplex, real-time PCR test for simultaneous qualitative detection/differentiation of SARS-CoV-2, influenza A, influenza B, and RSV. Input: nasopharyngeal swab specimens. Process: automated nucleic acid extraction/purification using magnetic glass particles; selective amplification of target viral genome regions (M1, NS1/NEP, M, ORF1ab/ORF1a) and RNA internal control; detection via fluorescently labeled oligonucleotide probes using TAGS (temperature-assisted generation of signal) technology. System: cobas 5800/6800/8800. Used in clinical laboratory settings by trained personnel. Output: qualitative results reviewed on system screen or printed report. Aids clinicians in differential diagnosis when used with other clinical/epidemiological data. Benefits: rapid, simultaneous identification of common respiratory pathogens to inform patient management.
Clinical Evidence
Prospective clinical study of 4,341 evaluable nasopharyngeal swab specimens collected during 2022-2024. Performance compared to FDA-cleared molecular assay. Results: Influenza A (PPA 96.4%, NPA 99.7%), Influenza B (PPA 98.9%, NPA 99.9%), RSV (PPA 89.7%, NPA 100%), SARS-CoV-2 (PPA 97.3%, NPA 98.5%).
Technological Characteristics
Automated nucleic acid extraction/purification using magnetic glass particles. Real-time PCR amplification using thermostable DNA polymerase. Detection via TAGS (temperature-assisted generation of signal) technology using fluorescently labeled oligonucleotide probes. Employs dUTP/AmpErase (uracil-N-glycosylase) for carryover contamination control. System: cobas 5800/6800/8800. Connectivity: integrated system software for data management.
Indications for Use
Indicated for individuals with signs and symptoms of respiratory tract infection to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections using nasopharyngeal swab specimens.
Regulatory Classification
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.
Special Controls
*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.
Predicate Devices
- BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031)
Submission Summary (Full Text)
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**FDA** U.S. FOOD & DRUG
ADMINISTRATION
# **510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY AND INSTRUMENT**
# **I Background Information:**
# **A 510(k) Number**
K243455
# **B Applicant**
Roche Molecular Systems, Inc.
# **C Proprietary and Established Names**
cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems
# **D Regulatory Information**
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 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 | MI - Microbiology |
# **II Submission/Device Overview:**
# **A Purpose for Submission:**
The purpose of this submission is to show that the cobas Respiratory 4-flex is substantially equivalent to the BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) and to obtain clearance for the cobas Respiratory 4-flex.
# **B Measurand:**
The cobas Respiratory 4-flex detects and identifies nucleic acids from influenza A, influenza B, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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### C Type of Test:
A multiplexed, reverse transcription polymerase chain reaction (RT-PCR) test, which utilizes temperature assisted generation of signal (TAGS) technology, intended for use with the automated cobas 5800/6800/8800 Systems for the qualitative in vitro detection and identification of influenza A, influenza B, RSV, and SARS-CoV-2 nucleic acids in nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of respiratory tract infections.
### III Intended Use/Indications for Use:
### A Intended Use(s):
See Indications for Use below.
### B Indication(s) for 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.
### Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For in vitro diagnostic Use Only
### C Special Instrument Requirements:
For use with the cobas 5800/6800/8800 Systems, only.
### IV Device/System Characteristics:
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# A Device Description:
The 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 reverse transcription, PCR amplification, and detection. The cobas Respiratory 4-flex is for use with the fully automated cobas 5800 System, cobas 6800 System, or cobas 8800 System. 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 analytical module.
The cobas Respiratory 4-flex has four (4) different reportable targets: influenza A, influenza B, RSV, and SARS-CoV-2. Reporting of these targets is based on detection of one or more of the nucleic acid targets.
Prior to initiating a test on the cobas 5800/6800/8800 Systems, NPS specimens are processed using the cobas omni Secondary Tube. To process specimens, first 0.8 mL of cobas Microbial Inactivation Solution (MIS) is added to the secondary tube. The user then transfers 0.4 mL of NPS specimen (in UTM/UVT) to the secondary tube. Next, the specimen is added to the cobas System and the specimen type “Diluted in cobas MIS” is selected to run the cobas Respiratory 4-flex.
# Flexible Result Reporting
To provide flexible reporting strategies, the cobas Respiratory 4-flex has several ordering/result reporting options, which are described independently for each cobas System below.
# Cobas 5800
Configuring Target Viral Groups. Each NPS specimen can be tested for any combination of viral targets (i.e., influenza A, influenza B, RSV, and SARS-CoV-2).
Digital Reflex Result Reporting Option. Based on the initial test results, additional targets can be ordered in a predefined timeframe. Results for the additional ordered targets will then be calculated and reported based on the already measured raw data. The sample is not re-processed to obtain additional test results.
# Cobas 6800/8800
Configuring Target Viral Groups. Each NPS specimen can be tested for all four panel targets or a predefined subgroup of panel targets using one of the three available Assay Specific Analysis Packages (ASAPs). Reflex testing options are not available when running the cobas Respiratory 4-flex on the cobas 6800/8800 Systems with software version 1.4.
The available ASAPs for these systems include:
- RESP-4FLEX ASAP: Result calculation of all four targets influenza A, influenza B, RSV and SARS-CoV-2.
- RESP-4FLEX-CAB ASAP: Result calculation for targets influenza A, influenza B and SARS-CoV-2.
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- RESP-FLEX-FLUR ASAP: Result calculation for targets influenza A, influenza B and RSV.
Regardless of the result reporting strategy implemented, automated data management is performed by the cobas 5800 System and cobas 6800/8800 System software, which assigns results for all tests. Results can be reviewed directly on the System screen and printed as a report.
### Interpretation of Results for cobas 5800 and cobas 6800/8800 Systems
To report a patient NPS specimen result, all external positive control targets must be positive and all external negative control targets must be negative. When the external controls yield the expected results, patient specimen results are reported for each of the four (4) assay targets. For each target, a negative, positive, or invalid result is reported according to the interpretative criteria outlined in **Table 1**. A patient specimen can have one more target combinations reported as invalid if at least one target is positive, *and* the RNA IC is not detected.
**Table 1.** Individual Target Result Interpretation for Patient Specimens
| Target Result* | Interpretation |
| --- | --- |
| Negative | No target signal detected for the corresponding viral target and RNA IC signal detected. |
| Positive | Target signal detected for the corresponding viral target and the RNA IC signal may or may not be detected. |
| Invalid | No target signal detected for the corresponding viral target and the RNA IC signal is not detected. If clinically indicated for the corresponding viral target, repeat assay with same sample. If the result is still invalid, a new specimen should be obtained. |
\*Shown for each of the four (4) viral targets (influenza A, influenza B, RSV, SARS-CoV-2) individually.
### B Principle of Operation:
cobas Respiratory 4-flex is based on a fully automated sample preparation process (nucleic acid extraction and purification) followed by reverse transcription, PCR amplification, and detection. Nucleic acid from patient NPS specimens and added Internal Control RNA (RNA-IC) molecules are simultaneously extracted. Nucleic acid is released by addition of proteinase and lysis reagent to the specimen. 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 specimen is achieved using target-specific forward and reverse primers detecting conserved viral genome regions, as shown in **Table 2**.
**Table 2.** cobas Respiratory 4-flex Target Regions
| Targeted Organism | Targeted Gene (Symbol) |
| --- | --- |
| Influenza A | Matrix protein 1 (M1) |
| Influenza B | Non-structural protein NS-1/2 (NS1/NEP) |
| RSV | Matrix protein (M) |
| SARS-CoV-2 | ORF1 ab polyprotein (ORF1ab) and ORF 1a polyprotein (ORF1a) |
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Selective amplification of RNA-IC is achieved using 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. Temperature assisted generation of signal (TAGS) technology is introduced to differentiate up to three targets per fluorescence channel and the RNA-IC, in each 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 reported 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 test captures fluorescence in five available fluorescence channels in combination with three thermal channels (detection of fluorescence at three defined temperatures: T1, T2, and T3), which enables simultaneous detection and differentiation of the amplified 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.
### C Instrument Description Information:
1. Instrument Name:
cobas 5800 System with software 1.0.5 or higher using cobas 5800 RESP-4FLEX ASAP v1.1.0, and cobas 6800/8800 Systems with software 1.4.7 or higher using cobas RESP-4FLEX, RESP-4FLEX-CAB and RESP-4FLEX-FLUR ASAP v12.1.0.
2. Specimen Identification:
Specimen identification information can be configured in an automated fashion or manually entered.
3. Specimen Sampling and Handling:
Nasopharyngeal swab (NPS) specimens collected in Copan UTM or BD UVT.
4. Calibration:
The user does not perform calibration of the cobas 5800/6800/8800 Systems.
5. Quality Control:
The RNA Internal Control (RNA-IC) consists of MS2 bacteriophage harboring a non-target RNA sequence. The RNA-IC, which is introduced into each NPS specimen during sample
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processing, is used to monitor the entire sample preparation and reverse transcription/PCR amplification process. In addition, the test utilizes external controls – cobas Respiratory flex Positive Control (low titer positive control consisting of non-infectious plasmid DNA) and cobas Buffer Negative Control, provided separately.
## V Substantial Equivalence Information:
### A Predicate Device Name(s):
BioFire Respiratory Panel 2.1 (RP2.1)
### B Predicate 510(k) Number(s):
DEN200031
### C Comparison with Predicate(s):
| Device & Predicate Device(s): | K243455 | DEN200031 |
| --- | --- | --- |
| Device Trade Name | cobas Respiratory 4-flex for use on the cobas 5800/6800/8800 Systems | BioFire Respiratory Panel 2.1 (RP2.1) |
| **Similarities** | | |
| Regulation Number | Same | 21 CFR 866.3981 |
| Regulation Name | Same | 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. |
| Primary Product Code | Same | QOF |
| Intended Use/Indications For 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 | 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, |
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| | 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. | 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*, and *Mycoplasma pneumonia* Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral 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 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 |
| --- | --- | --- |
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| | | evaluating a patient with possible respiratory tract infection. |
| --- | --- | --- |
| Differences | | |
| Analyte Targets | Influenza A, Influenza B, Respiratory Syncytial Virus, and Severe Acute Respiratory Syndrome Coronavirus (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, and Mycoplasma pneumonia |
| Sample Preparation Procedure | Automated by cobas 5800/6800/8800 Systems | Automated by BioFire FilmArray 2.0 or BioFire FilmArray Torch Systems |
| 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 | Sample processing internal control (IC) and external positive and external negative controls. | 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. |
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# **VI Standards/Guidance Documents Referenced:**
# Standards
- CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures.
- CLSI EP07-A3. Interference Testing in Clinical Chemistry.
- ISO 14971:2019. Medical devices – Application of risk management to medical devices.
- IEC 62366-1:2015+AMD1:2020 CSV. Medical devices – Application of usability engineering to medical devices.
- IEC 62304:2006. Medical device software – Software life-cycle processes
- ISO 15223-1:2021. Medical devices – Symbols to be used with information to be supplied by the manufacturer – Part 1: General requirements.
- ISO 13485:2016. Medical devices – Quality management systems – Requirements for regulatory purposes.
- ISO 20916:2019. In vitro diagnostic medical devices – Clinical performance studies using specimens from human subjects – Good study practice.
# Special Controls
- Class II Special Controls as per 21 CFR 866.3981
# Guidance Documents
- Respiratory Viral Panel Multiplex Nucleic Acid Assay – Class II Special Controls Guidance for Industry and FDA Staff (October 2009).
- Instrumentation for Clinical Multiplex Test Systems – Class II Special Controls Guidance for Industry and FDA Staff (March 2005).
- Cybersecurity in Medical Devices: Quality System Considerations and Content of Premarket Submissions Guidance for Industry and Food and Drug Administration Staff (September 2023).
- Off-The-Shelf Software Use in Medical Devices Guidance for Industry and Food and Drug Administration Staff (August 2023).
- Content of Premarket Submissions for Device Software Functions Guidance for Industry and Food and Drug Administration Staff (June 2023).
# **VII Performance Characteristics (if/when applicable):**
# **A Analytical Performance:**
# 1. Precision/Reproducibility:
# a. Within-Laboratory Precision
Within-laboratory precision was evaluated at a single site using the cobas Respiratory 4-flex run on the cobas 5800 System and cobas 6800/8800 System. Contrived panels containing known quantities of representative target analytes were prepared in simulated NPS matrix. The cultured influenza A, influenza B, RSV and inactivated SARS-CoV-2 material that was used to generate the positive panel members are denoted in **Table 3**. The contrived positive panels were co-formulated with target analytes at a low positive
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concentration (~1x LoD) and moderate concentration (~3x LoD). A negative panel was also included in the study. The study was conducted by five (5) operators using three test cassette lots over the course of 12 non-consecutive days, on three cobas 5800 Systems and three cobas 6800/8800 Systems. Each panel member was tested in triplicate twice per day over 12 different days. In total, 216 replicates were collected per panel member.
**Table 3.** Viral Strains Used to Evaluate Within-Laboratory Precision
| Target Channel | Organism | Viral Strain | Vendor: Catalog No. |
| --- | --- | --- | --- |
| RSV | RSV A | Respiratory Syncytial Virus A2 | Microbiologics: Lot: D2126D |
| Influenza A | Influenza A (H3N2) | A/Darwin/6/2021 | Microbiologics: SD-DRW0621A |
| SARS-CoV-2 | SARS-CoV-2^{1} | England/02/2020 | NIBSC^{2}: 20/146 |
| Influenza B | Influenza B (Victoria lineage) | B/Austria/1359417/2021 | Microbiologics: SD-AT1721A |
$^{1}$Inactivated virus
$^{2}$NIBSC - National Institute for Biological Standards and Control
The qualitative results (i.e., % agreement with expected results) from the study are illustrated in **Table 4**.
**Table 4.** Within-Laboratory Precision Study – Qualitative Results
| 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 |
| | ~1x LoD | 216 | 216 | 100 | 98.31 | 100 |
| Influenza B (Victoria) | ~3x LoD | 216 | 216 | 100 | 98.31 | 100 |
| | ~1x LoD | 215 | 216 | 99.54 | 97.45 | 99.99 |
| RSV A | ~3x LoD | 216 | 216 | 100 | 98.31 | 100 |
| | ~1x LoD | 214 | 216 | 99.07 | 96.70 | 99.89 |
| SARS-CoV-2 | ~3x LoD | 216 | 216 | 100 | 98.31 | 100 |
| | ~1x LoD | 216 | 216 | 100 | 98.31 | 100 |
| N/A | Blank | 0 | 216 | 0 | 0.00 | 3.36 |
All low positive panel members exhibited a detection rate >99% and all moderate positive panel members exhibited a detection rate of 100%. The negative panel was negative 100% of the time. The results of the study demonstrate acceptable assay variability.
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The means, standard deviations, and coefficients of variation (%) for cycle threshold (Ct) values by target analyte and expected concentration (Positive Panel Members) are shown in **Table 5**.
**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 | 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 |
| | ~1x LoD | 100 | 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 | 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 |
| | ~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 | 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 |
| | ~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 | 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 |
| | ~1x LoD | 100 | 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 |
Note: SD= standard deviation, CV= coefficient of variation, Ct= cycle threshold. LoD limit of detection
# **b. Reproducibility**
A reproducibility study was conducted at three testing sites (one internal) using the cobas Respiratory 4-flex run on the cobas 5800 System and cobas 6800/8800 System. The study incorporated potential sources of variation introduced by site (three (3) testing sites), day (six (6) different days), operator (nine (9) operators), test cassette lot (three (3) total, >2 per site), instrument (five (5) cobas 6800/8800 Systems and four (4) cobas 5800 Systems, with at least one (1) cobas 5800 or cobas 6800/8800 System per site).
The same contrived co-formulated panels used to evaluate precision (see **Table 3**) were included in the reproducibility study. Each panel member was tested in triplicate, twice per day. In total, 504 replicates were collected per panel member (72 at site 1 and 216 at both sites 2 and 3). The qualitative results of the study are illustrated in **Table 6**.
**Table 6.** Reproducibility Study- Qualitative Results
| Target Channel | Level | % Agreement with Expected Results (n Detected/ N Tested) | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | Overall (95% CI) |
| NA | Negative | 100% (72/72) | 99.1% (214/216) | 100% (216/216) | 99.6% (502/504) (98.6-99.9%) |
| Influenza B | | 100% (72/72) | 99.1% (214/216) | 99.5% (215/216) | 99.4% (501/504) (98.3-100%) |
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| Target Channel | Level | % Agreement with Expected Results (n Detected/ N Tested) | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | Overall (95% CI) |
| RSV | ~1x LoD | 100% (72/72) | 99.5% (215/216) | 99.1% (214/216) | 99.4% (501/504) (98.3-99.8%) |
| Influenza A | | 100% (72/72) | 100% (216/216) | 100% (216/216) | 100% (504/504) (99.3-100%) |
| SARS-CoV-2 | | 100% (72/72) | 100% (216/216) | 100% (216/216) | 100% (504/504) (99.3-100%) |
| Influenza B | ~3x LoD | 100% (72/72) | 100% (215/215) | 100% (216/216) | 100% (503/503)^{a} (99.3-100%) |
| RSV | | 100% (72/72) | 100% (216/216) | 100% (216/216) | 100% (504/504) (99.3-100%) |
| Influenza A | | 100% (72/72) | 100% (216/216) | 100% (216/216) | 100% (504/504) (99.3-100%) |
| SARS-CoV-2 | | 100% (72/72) | 100% (216/216) | 100% (216/216) | 100% (504/504) (99.3-100%) |
$^{a}$At site 2, a single invalid result was obtained for influenza B, resulting in 503 samples with valid results.
The cobas Respiratory 4-flex results showed acceptable lot-to-lot, instrument-to-instrument (site), day-to-day, and between run variation for the ~1x LoD, and ~3x LoD panel members (Table 7). Additionally, the cobas 5800 and cobas 6800/8800 Systems demonstrated equivalent reproducibility.
Table 7. Reproducibility - Standard Deviations and Coefficients of Variation of Ct Values
| Target Virus | Level | (n/N)^{a} | Mean Ct | Total | | Site | | Lot | | Day | | Run | | Within Run | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % |
| Negative | 0 | 502/504 | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc |
| Influenza A | ~1× LoD | 504/504 | 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 |
| | ~3× LoD | 504/504 | 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 | 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 |
| | ~3× LoD | 503/503^{b} | 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 | 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 |
| | ~3× LoD | 504/504 | 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 | 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 |
| | ~3× LoD | 504/504 | 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 tests which matched the expected results. N is the total number of valid tests for the panel member.
$^{b}$ A single invalid result was obtained for influenza B, resulting in 503 samples with valid results.
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# 2. Linearity:
Not applicable; this is a qualitative assay.
# 3. Analytical Specificity/Interference:
# Analytical Reactivity (Inclusivity)
The inclusivity of the cobas Respiratory 4-flex was evaluated using a combination of in silico analysis of publicly available sequence information and laboratory testing of contrived specimens containing viral isolates that were selected to represent phylogenetic, geographic, and temporal diversity.
# a. Wet-Testing
This study was performed to determine the analytical reactivity of the cobas Respiratory 4-flex with clinically relevant strains, serotypes, or subtypes of the target species. The inclusivity panel was prepared by spiking various target microorganism strains/serotypes/subtypes encompassing temporal and geographical diversity into simulated NPS matrix at a concentration of ~3x LoD and were tested in triplicate. Strains that did not yield 100% reactivity at ~3x LoD were prepared at higher concentrations and tested in triplicate until 100% reactivity was observed. The strains evaluated and the results of inclusivity testing are shown in Tables 8-11, below.
Table 8. Strains of Influenza A Evaluated for Inclusivity
| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| H1N1 | A/New Caledonia/20/99 | Zeptometrix | 0810036CF | 165 | ~3x | 100% (3/3) |
| | A/Brisbane/59/07 | Zeptometrix | 0810244CF | 165 | ~3x | 100% (3/3) |
| H1N1 pdm09 | A/California/07/09 | Zeptometrix | 0810165CF | 165 | ~3x | 100% (3/3) |
| | A/NY/03/09 | Zeptometrix | 0810249CF | 165 | ~3x | 100% (3/3) |
| | A/Victoria/2570/2019 | Microbiologics | SD-VIC219A-7 | 165 | ~3x | 100% (3/3) |
| | A/Wisconsin/588/2019 | Microbiologics | SD-WA519A-8 | 165 | ~3x | 100% (3/3) |
| | A/Victoria/4897/2022 | Microbiologics | SD-VIC9722B | 165 | ~3x | 100% (3/3) |
| | A/Wisconsin/67/2022 | Microbiologics | SD-WI6722MS 1B | 330 | ~6x | 100% (3/3) |
| | A/England/73/2022 | UHSA | N/A | 357 | ~6.8x | 100% (3/3) |
| | A/England/55/2022 | UHSA | N/A | 357 | ~6.8x | 100% (3/3) |
| H3 | A/Port Chalmers/1/73 | ATCC | VR-810 | 165 | ~3x | 100% (3/3) |
| | A/Texas/50/12 | Zeptometrix | 0810238CF | 242.7 | ~4.4x | 100% (3/3) |
| | A/Victoria/3/75 | ATCC | VR-822 | 165 | ~3x | 100% (3/3) |
| | A/Wisconsin/67/05 | Zeptometrix | 810252CF | 165 | ~3x | 100% (3/3) |
| | A/Darwin/9/2021 | Microbiologics | SD-DRW921-6 | 165 | ~3x | 100% (3/3) |
| | A/Hong Kong/8/68 | Zeptometrix | 0810250CF | 165 | ~3x | 100% (3/3) |
| | A/Perth/16/09 | Zeptometrix | 0810251CF | 165 | ~3x | 100% (3/3) |
| | A/Hong Kong/4801/14 | Zeptometrix | 0810526CF | 165 | ~3x | 100% (3/3) |
| | A/Kansas/14/17 | Zeptometrix | 0810586CF | 165 | ~3x | 100% (3/3) |
| | A/Switzerland/9715293/13 | Zeptometrix | 0810511CF | 165 | ~3x | 100% (3/3) |
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| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| H5 | A/mallard/Wisconsin/2576/2009 (H5N1) | BEI | NR-31131 | 165 | ~3x | 100% (3/3) |
| | A/duck/Singapore/645/1997 (H5N3) | BEI | NR-3558 | 165 | ~3x | 100% (3/3) |
| | A/ruddy turnstone/New Jersey/828212/2001 (N5N2) | BEI | NR-44298 | 165 | ~3x | 100% (3/3) |
| H7 | A/northern pintail/Illinois/10OS3959/2010 (H7N3) | BEI | NR-35979 | 165 | ~3x | 100% (3/3) |
| | H7N9, A/northern shoveler/Mississippi/11OS145/2011 | BEI | NR-36001 | 165 | ~3x | 100% (3/3) |
| | H7N8, A/mallard/Ohio/11OS2033/2011 | BEI | NR-36008 | 165 | ~3x | 100% (3/3) |
| H9 | H9N7, A/shorebird/Delaware Bay/31/1996 | BEI | NR-45171 | 165 | ~3x | 100% (3/3) |
UHSA-UK Health Security Agency
**Table 9.** Strains of Influenza B Evaluated for Inclusivity
| Lineage | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| Victoria | B/Colorado/6/17 | Zeptometrix | 0810573CF | 1779 | ~3x | 100% (3/3) |
| | B/Hong Kong/5/72 | ATCC | VR-823 | 1779 | ~3x | 100% (3/3) |
| | B/Brisbane/60/08 | Zeptometrix | 0810254CF | 1779 | ~3x | 100% (3/3) |
| | B/Florida/02/06 | Zeptometrix | 0810037CF | 1779 | ~3x | 100% (3/3) |
| Yamagata | B/Florida/07/04 | Zeptometrix | 0810256CF | 1779 | ~3x | 100% (3/3) |
| | B/Massachusetts/2/2012 | ATCC | VR-1813 | 1779 | ~3x | 100% (3/3) |
| | B/Wisconsin/1/2010 | ATCC | VR-1883 | 1779 | ~3x | 100% (3/3) |
| | B/Florida/4/2006 | ATCC | VR-1804 | 1779 | ~3x | 100% (3/3) |
| | B/Texas/6/11 | Zeptometrix | 0810242CF | 1779 | ~3x | 100% (3/3) |
| Unknown | B/Allen/45 | ATCC | VR-102 | 1779 | ~3x | 100% (3/3) |
| | B/Lee/40 | ATCC | VR-101 | 1779 | ~3x | 100% (3/3) |
| | B/Taiwan/2/62 | ATCC | VR-295 | 1779 | ~3x | 100% (3/3) |
**Table 10.** Strains of RSV Evaluated for Inclusivity
| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| A | 2006 Isolate | Zeptometrix | 0810040ACF-CL | 18510 | ~3x | 100% (3/3) |
| | 02/2015 | Zeptometrix | 0810475CF | 18510 | ~3x | 100% (3/3) |
| | A2 | ATCC | VR-1540 | 18510 | ~3x | 100% (3/3) |
| B | CH93(18)-18 | Zeptometrix | 0810040CF-CL | 18510 | ~3x | 100% (3/3) |
| | 9320 | Zeptometrix | VPL-030 | 18510 | ~3x | 100% (3/3) |
| | WV/14617/85 | ATCC | VR-1400 | 18510 | ~3x | 100% (3/3) |
| | 18537 | ATCC | VR-1580 | 18510 | ~3x | 100% (3/3) |
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Table 11. Strains of SARS-CoV-2 Evaluated for Inclusivity
| Lineage | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| B.1.1.7 | England/204820464/2020 | Zeptometrix | 0810614CFHI-CL | 312 | ~3x | 100% (3/3) |
| B.1.351 | South Africa/KRISPK005325/2020 | Zeptometrix | 0810613CFHI-CL | 312 | ~3x | 100% (3/3) |
| B.1.617.2 | USA/PHC658/2021 | Zeptometrix | 0810624CFHI-CL | 312 | ~3x | 100% (3/3) |
| B.1.1.529 | USA/MDHP20874/2021 | Zeptometrix | 0810642CFHI-CL | 312 | ~3x | 100% (3/3) |
| P.1 | Japan/TY7-503/2021 | Zeptometrix | 0810616CFHI-CL | 312 | ~3x | 100% (3/3) |
| - | USA-WA1/2020 | Zeptometrix | 0810587CFHI | 312 | ~3x | 100% (3/3) |
# b. In silico
# SARS-CoV-2 In Silico Analysis
The inclusivity of the cobas Respiratory 4-flex was evaluated using in silico analysis of the forward primers, reverse primers, and probes for the SARS-CoV-2 target in relation to all sequences available in the NCBI and GISAID gene databases. For both SARS-CoV-2 gene targets taken together, in silico analysis on June 13, 2025 (>8.32M sequences in NCBI and >15.96M sequences in GISAID) indicates that the majority of sequence variants are predicted to be detected, with only <0.07% of sequences in NCBI and <0.06% of sequences in GISAID, with any mismatch in primer/probe binding sites for both targets. In silico analysis indicates that the cobas Respiratory 4-flex SARS-CoV-2 test detects all known variant strains.
No sequences in the NCBI database, and seven (7) unique sequences in GISAID (<0.00005%) had mismatches that were predicted to affect detection and performance of the test. Due to the low percentage representation in the databases of mismatches, performance of the cobas Respiratory 4-flex is not expected to be impacted.
# Cross-Reactivity and Microbial Interference
Cross-reactivity and microbial interference of the cobas Respiratory 4-flex was evaluated by testing a panel of bacteria, fungi, and viruses commonly found in the respiratory tract for cross-reactivity and interference. The effect of non-target microorganisms on the performance of the cobas Respiratory 4-flex was tested by introducing non-target microorganisms into simulated NPS matrix spiked with and without co-formulated SARS-CoV-2, influenza A, influenza B and RSV viruses (Table 3) at ~3x LoD. Three (3) replicates in target positive background and three (3) replicates in target negative background were tested for each non-target microorganism. Bacteria and fungi were spiked at 1.0e+6 units/mL and viruses at 1.0e+5 units/mL, or the highest concentration possible. As summarized in Table 12, no cross-reactivity or microbial interference was observed at the concentrations tested, and no invalid results were obtained.
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Table 12. Cross-reactivity and Microbial Interference Results for Non-Target Microorganisms
| Microorganism | Concentration | Negative Sample (Specificity Test) | | | | Positive Sample (Interference Test) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Target Result: n Detected/N Tested | | | | Target Result: n Detected/N Tested | | | |
| | | SARS-CoV-2 | Influenza A | Influenza B | RSV | SARS-CoV-2 | Influenza A | Influenza B | RSV |
| *Aspergillus flavus* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Bordetella parapertussis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Bordetella pertussis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Candida albicans* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Chlamydia pneumoniae* | 1.00E+06 IFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Corynebacterium diphtheriae* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Cytomegalovirus Epstein Barr virus | 1.00E+05 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| | 1.00E+05 cp/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Escherichia coli* *Fusobacterium necrophorum* | 1.00E+06 CFU/vial | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Haemophilus influenzae* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Lactobacillus acidophilus* *Legionella pneumophila* | 1.00E+06 CFU/vial | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Measles virus | 1.00E+05 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| MERS-coronavirus* | 1.00E+05 cp/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Moraxella catarrhalis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mumps virus | 1.00E+05 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Mycobacterium bovis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Mycoplasma genitalium* | 1.00E+06 CFU/vial | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Mycoplasma pneumoniae* | 1.00E+06 CCU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Neisseria elongata* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Neisseria meningitidis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Pneumocystis jirovecii* | 5.00E+03 organisms/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Pooled nasal wash | NA | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
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| Microorganism | Concentration | Negative Sample (Specificity Test) | | | | Positive Sample (Interference Test) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Target Result: n Detected/N Tested | | | | Target Result: n Detected/N Tested | | | |
| | | SARS-CoV-2 | Influenza A | Influenza B | RSV | SARS-CoV-2 | Influenza A | Influenza B | RSV |
| *Pseudomonas aeruginosa* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| SARS-coronavirus (SARS-CoV)* | 1.00E+05 cp/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Staphylococcus aureus* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Staphylococcus epidermidis* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Streptococcus pneumoniae* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Streptococcus pyogenes* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| *Streptococcus salivarius* | 1.00E+06 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
CFU = Colony Forming Units; CCU = Colony Changing Units; IFU= Inclusion Forming Units; TCID₅₀ = Median Tissue Culture Infectious Dose.
*Inactivated virus was used for testing
### Interfering Substances
An analytical study was performed to assess the potential inhibitory effects of endogenous and exogenous substances that may be commonly found in NPS specimens. All substances in Table 13 were evaluated in the presence and absence of target organisms (Table 3) co-formulated at ~3x LoD. Testing for each condition was performed in replicates of ten.
None of the evaluated substances, at the concentrations tested, interfered with detection of the candidate assay targets, except for FluMist and snuff tobacco. Specifically, in the absence of target organisms, FluMist caused positive influenza A and influenza B results and snuff tobacco caused invalid results for all target organisms. The FluMist results were anticipated, as FluMist contains both influenza A and influenza B viruses.
Table 13. Substances Evaluated for Interference
| Substance | Active Ingredient | Concentration Tested | SARS-CoV-2, Influenza A, Influenza B, RSV Absent | SARS-CoV-2, Influenza A, Influenza B, RSV Present |
| --- | --- | --- | --- | --- |
| | | | Valid Negative Results/ Total # of Valid Results | Valid Positive Results/ Total # of Valid Results |
| Mucin | NA | 0.3% w/v | 10/10 | 10/10 |
| | | 0.5% w/v | 10/10 | 10/10 |
| Human Whole Blood | NA | 1.5% (v/v) | 10/10 | 10/10 |
| | | 3.0% (v/v) | 10/10 | 10/10 |
| NNSC (Target Negative No Substance Control) | NA | NA | 10/10 | 0/10 |
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| Substance | Active Ingredient | Concentration Tested | SARS-CoV-2, Influenza A, Influenza B, RSV Absent | SARS-CoV-2, Influenza A, Influenza B, RSV Present |
| --- | --- | --- | --- | --- |
| | | | Valid Negative Results/ Total # of Valid Results | Valid Positive Results/ Total # of Valid Results |
| AXOTIDE Diskus Multidose 250 mcg | Fluticasone propionate | 0.167 mg/mL | 10/10 | 10/10 |
| BACTROBAN Nasal Ointment | Mupirocin | 0.20 mg/mL | 10/10 | 10/10 |
| BUDESONID Sandoz Nasal Spray 64 mcg | Budesonide | 0.039 mg/mL | 10/10 | 10/10 |
| CEPACOL Extra Strength Sore Throat | Benzocaine | 5 mg/mL | 10/10 | 10/10 |
| Chloraseptic max | Phenol | 0.47 mg/mL | 10/10 | 10/10 |
| FLUMIST Quadrivalent | live attenuated influenza A and B viruses | 50000000 FFU/mL | 10/10^{a} | 10/10 |
| Heel Luffeel Nasal Spray | Luffa operculata Thryallis glauca Histaminum Sulphur | 2.99 mg/mL 2.99 mg/mL 1.5 mg/mL 1.5 mg/mL | 10/10 | 10/10 |
| NASIVIN Pur Spray 0.05% | Oxymetazoline | 0.011 mg/mL | 10/10 | 10/10 |
| OBRACIN Inj Solution 40 mg/mL | Tobramycin | 0.018 mg/mL | 10/10 | 10/10 |
| RELENZA Disk 5 mg | Zanamivir | 0.0015 mg/mL | 10/10 | 10/10 |
| TAMIFLU Kaps 75 mg | Oseltamivir | 0.0073 mg/mL | 10/10 | 10/10 |
| Snuff Tobacco | Nicotine | 0.1% w/v | 0/0^{b} | 10/10 |
| Vaseline | Petroleum Jelly | 1% w/v | 10/10 | 10/10 |
| VICKS VapoRub | Eucalyptus Oil and Menthol | 1% w/v | 10/10 | 10/10 |
| XYLOCAIN Spray 10% | Lidocaine | 2.68 mg/mL | 10/10 | 10/10 |
NA-Not Applicable
$^{a}$ FluMist is expected to give positive (detected) results for influenza A and influenza B targets. 10/10 represents SARS-CoV-2 and RSV targets.
$^{b}$ All replicates had a negative RNA-IC result and were therefore invalid.
### Competitive Interference (Co-Infection)
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 (Table 3) were tested. Samples were prepared with one viral target at ~3x LoD mixed with another target at a high concentration (1.0E+06 units/mL). Twelve replicates per condition were tested. As shown in Table 14, none of the targets present at very high concentration interfered with the detection of other viral targets at low concentration levels.
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**Table 14.** Competitive Interference (Co-infection) Study Results
| Combination | Target 1 (high) ≥ 1.00E+06 unit/mL | Target 2 (low) ~3x LoD | % Detected (# Detected / # Tested) | | |
| --- | --- | --- | --- | --- | --- |
| | | | Target 1 | Target 2 | Target 3 |
| 1 | Influenza A | SARS-CoV-2 | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 2 | Influenza B | SARS-CoV-2 | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 3 | RSV | SARS-CoV-2 | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 4 | SARS-CoV-2 | Influenza A | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 5 | Influenza B | Influenza A | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 6 | RSV | Influenza A | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 7 | Influenza A | Influenza B | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 8 | SARS-CoV-2 | Influenza B | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 9 | RSV | Influenza B | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 10 | Influenza A | RSV | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 11 | Influenza B | RSV | 100% (12/12) | 100% (12/12) | 100% (12/12) |
| 12 | SARS-CoV-2 | RSV | 100% (12/12) | 100% (12/12) | 100% (12/12) |
# 4. Assay Reportable Range:
Not applicable; this is a qualitative assay.
# 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
# a. Controls
The assay contains an Internal Control (RNA-IC) added to each test specimen and external positive and negative controls. For more information, see section **IV.C.5.Quality Control**, above.
# b. Sample Stability
Stability studies have been performed to support the following sample stability claims:
Primary Sample (NP swab in UTM)
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- Up to 12-hours at 2-25°C, followed by up to 3-days at 2-8°C, followed by up to 30-days at ≤ -20°C. Frozen samples may undergo up to three freeze/thaw cycles.
# 6. Detection Limit:
Limit of detection (LoD) studies determine the lowest detectable concentration of SARS-CoV-2, influenza A, influenza B, and RSV at which equal to or greater than 95% of all replicates test positive. To determine the LoD for each target, co-spiked panels were formulated using cultured viral material or inactivated virus diluted in nasopharyngeal (NP) matrix. Twenty-one replicates per reagent lot per dilution were tested for five (5) 2-fold dilutions using three reagent lots distributed across six (6) cobas 5800 Systems, three (3) cobas 6800 Systems and one (1) cobas 8800 System. The strains evaluated, as well as their corresponding LoD values are shown in **Table 15**.
**Table 15.** LoD Study Results
| Target | Subtype/Strain/Isolate | LoD Concentration (By Hit Rate) | n Detected/ N Tested (% Positive) |
| --- | --- | --- | --- |
| Influenza A | H3N2, A/Darwin/6/2021 | 50 cp/mL | 60/63 (95.24%) |
| | H1N1 pdm09, Brisbane/02/2018 | 100 cp/mL | 60/63 (95.24%) |
| Influenza B | Yamagata, Phuket/3073/13 | 800 cp/mL | 62/63 (98.41%) |
| | Victoria, B/Austria/1359417/2021 | 250 cp/mL | 63/63 (100%) |
| RSV | Respiratory Syncytial Virus A2 | 4000 cp/mL | 62/63 (95.41%) |
| SARS-CoV-2* | England/02/2020 NIBSC code: 20/146 | 80 IU/mL | 60/63 (95.24%) |
\*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.
# 7. Assay Cut-Off:
Data from several analytical studies were used to analyze the Ct distribution and set cutoffs for all organisms to maximize sensitivity and specificity.
# 8. Accuracy (Instrument):
Not applicable.
# 9. Carry-Over:
A total of 430 SARS-CoV-2 high positive samples prepared at approximately 6.50E+08 particles/mL in negative simulated NPS matrix and 480 negative samples consisting of negative simulated NPS matrix were tested in 25 checkerboard configuration runs, using both cobas 5800 and 6800/8800 Systems. The cross-contamination rate calculated per system (cobas 5800 and cobas 6800/8800) and for all systems combined was in both cases 0.0%.
# **B Comparison Studies:**
# 1. Method Comparison with Predicate Device:
Not applicable.
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# 2. Matrix Comparison:
A subset of analytical studies were conducted using simulated matrix, therefore equivalency between clinical NPS matrix and simulated NPS matrix was evaluated. For this study, pooled clinical NPS matrix and simulated matrix were spiked with co-formulated panels containing RSV, influenza A, influenza B and SARS-CoV-2 at a concentration level of ~2x LoD. Forty-two replicates were tested per co-formulated panel and matrix type combination. All replicates tested with the ~2x LoD panels were positive for the respective viral targets. The results demonstrate equivalent performance of the cobas Respiratory 4-flex when testing samples contrived in clinical NPS matrix and simulated matrix and support the use of simulated matrix in a subset of analytical studies.
# 3. Media Equivalency Study
An analytical study was performed to assess the performance of cobas Respiratory 4-flex with common transport medias that may be utilized to collect NPS specimens for testing. Negative clinical NPS matrix in each of the media types illustrated in **Table 16** were evaluated in the presence and absence of target organisms (**Table 3**) co-formulated at ~3x LoD. Testing for each condition was performed in replicates of ten. None of the media types evaluated interfered with detection of the candidate assay targets at the concentration tested.
**Table 16.** Media Equivalency Study Results
| Media Type | SARS-CoV-2, Influenza A, Influenza B, RSV Absent | SARS-CoV-2, Influenza A, Influenza B, RSV Present |
| --- | --- | --- |
| | Valid Negative Results/ Total # of Valid Results | Valid Positive Results/ Total # of Valid Results |
| Remel M4RT (Transport Media) | 10/10 | 10/10 |
| Remel M5 (Transport Media) | 10/10 | 10/10 |
| Remel M6 (Transport Media) | 10/10 | 10/10 |
| Greiner VACUETTE Virus Stabilization Tube (Transport Media) | 10/10 | 10/10 |
# **C Clinical Studies:**
# 1. Clinical Sensitivity:
# **Prospective Clinical Studies**
The clinical performance of the cobas Respiratory 4-flex was established in multi-center prospective studies conducted with nasopharyngeal swab (NPS) specimens in VTM or UVT from individuals exhibiting signs and symptoms of respiratory viral infection. Prospective NPS specimens were collected at eleven collection sites (eight (8) in the U.S. and three (3) OUS) during the 2023-2024 respiratory viral season and tested fresh (Category I specimens).
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Category I specimens were tested with the cobas Respiratory 4-flex using the cobas 6800/8800 System at four (4) testing sites (three (3) in the U.S and one (1) OUS). Additionally, residual (leftover) and de-identified NPS specimens (Category II) were prospectively collected during parts of the 2022-2023 respiratory viral seasons at seven (7) U.S. sites and the 2023-2024 respiratory viral season from 14 U.S. collection sites. Category II specimens were tested with the cobas Respiratory 4-flex at three (3) U.S testing sites using the cobas 6800/8800 Systems. The comparator method utilized to establish performance was a U.S. FDA-cleared molecular assay. Comparator testing was performed in accordance with the package insert.
A total of 4,475 NPS specimens (1,869 Category I and 2,606 Category II) were enrolled for the prospective clinical study, of which 4,378 could be tested (1,832 Category I and 2,546 Category II) with cobas Respiratory 4-flex on the cobas 6800/8800 System and the comparator method. Thirty-five Category I specimens could not be tested due to instrument error or protocol deviations, and two (2) Category I specimens were unable to be tested due to sample processing error. Sixty Category II samples were not tested due to insufficient specimen volume, instrument error, or specimens lost in transit by the courier.
Of the 4,378 prospective NPS specimens tested, 4,341 specimens were evaluable (1,827 Category I and 2,514 Category II). Five (5) Category I specimens and 32 Category II 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) Category I and one (1) Category II) were excluded from analysis due to inconclusive results obtained from the comparator test. The initial prospective clinical study invalid rate was 1.3% (57/4,378), and 0.9% (40/4,378) following retesting. **Table 17** below provides a summary of demographic information for the 4,341 specimens included in the prospective clinical study.
**Table 17.** Demographics of Subjects from Prospective Population that were Included in the Performance Analysis
| Age group (years) | Total Number (%) | | |
| --- | --- | --- | --- |
| | Category I | Category II | Overall |
| **Total** | 1,827 | 2,514 | 4,341 |
| <6 | 49 (2.7%) | 183 (7.3%) | 232 (5.3%) |
| 6 - <14 | 215 (11.8%) | 225 (8.9%) | 440 (10.1%) |
| 14 - <18 | 84 (4.6%) | 85 (3.4%) | 169 (3.9%) |
| 18 - <65 | 1,249 (68.4%) | 1,721 (68.5%) | 2,970 (68.4%) |
| >=65 | 230 (12.6%) | 300 (11.9%) | 530 (12.2%) |
| Missing | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
A summary of the cobas Respiratory 4-flex prospective clinical study performance is provided in **Table 18**.
Positive Percent Agreement (PPA) for the NPS specimen tested on the candidate and comparator device was calculated as $$100\% \times (TP/(TP+FN))$$. True positive (TP) indicates that both the cobas Respiratory 4-flex and the comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the cobas Respiratory 4-flex was
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negative while the comparator result was positive. Negative Percent Agreement (NPA) for the NPS specimen tested on the candidate and comparator device was calculated as $100\% \times (\text{TN} / (\text{TN}+\text{FP}))$. True negative (TN) indicates that both the cobas Respiratory 4-flex and the comparator method had negative results, and false positive (FP) indicates that the cobas Respiratory 4-flex was positive while the comparator result was negative.
**Table 18.** Clinical Performance of the cobas Respiratory 4-flex in the Prospective Studies
| Analyte | Positive Percent Agreement | | | Negative Percent Agreement | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | TP/ (TP+FN) | % | 95% CI | TN/ (TN+FP) | % | 95% CI |
| **Influenza A** | Cat I | 100/104 | 96.2 | 90.5-98.5 | 1716/1721 | 99.7 | 99.3-99.9 |
| | Cat II | 60/62 | 96.8 | 89.0-99.1 | 2443/2451 | 99.7 | 99.4-99.8 |
| | **All** | **160/166** | **96.4** | **92.3-98.3** | **4159/4172** | **99.7** | **99.5-99.8** |
| **Influenza B** | Cat I | 66/66 | 100 | 94.5-100 | 1759/1761 | 99.9 | 99.6-100 |
| | Cat II | 23/24 | 95.8 | 79.8-99.3 | 2489/2490 | 100.0 | 99.8-100 |
| | **All** | **89/90** | **98.9** | **94.0-99.8** | **4248/4251** | **99.9** | **99.8-100** |
| **RSV** | Cat I | 47/53 | 88.7 | 77.4-94.7 | 1774/1774 | 100 | 99.8-100 |
| | Cat II | 66/73 | 90.4 | 81.5-95.3 | 2441/2441 | 100.0 | 99.8-100 |
| | **All** | **113/126** | **89.7** | **83.1-93.9** | **4215/4215** | **100.0** | **99.9-100** |
| **SARS-CoV-2** | Cat I | 145/150 | 96.7 | 92.4-98.6 | 1654/1677 | 98.6 | 98.0-99.1 |
| | Cat II | 295/302 | 97.7 | 95.3-98.9 | 2176/2212 | 98.4 | 97.8-98.8 |
| | **All** | **440/452** | **97.3** | **95.4-98.5** | **3830/3889** | **98.5** | **98.0-98.8** |
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.
Number of samples with positive results for more than one target observed in the prospective cohort as detected by the cobas Respiratory 4-flex and comparator method are listed in **Table 19**.
**Table 19.** Multiple Detection Combination by cobas Respiratory 4-flex in the Prospective Studies
| Sample Category | Analyte 1 | Analyte 2 | Total Multiple Detections | Number of Specimen with False Positive Detections | False Positive Analyte(s) |
| --- | --- | --- | --- | --- | --- |
| Category I | Influenza A | SARS-CoV-2 | 5 | 3 | SARS-CoV-2 (3) |
| Category I | Influenza B | RSV | 1 | 1 | Influenza B (1) |
| Category II | Influenza A | SARS-CoV-2 | 3 | 0 | None |
| Category II | Influenza B | RSV | 1 | 0 | None |
| Category II | Influenza B | SARS-CoV-2 | 1 | 1 | SARS-CoV-2 (1) |
| Category II | RSV | SARS-CoV-2 | 4 | 4 | SARS-CoV-2 (4) |
Note: False positive is when a sample is detected by cobas Respiratory 4-flex but not detected by comparator method.
## 2. Clinical Specificity:
See section **VII. Performance Characteristics. C. Clinical Studies 1. Clinical Sensitivity**, above.
## 3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
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## Clinical Equivalency Study
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, and the PPA and NPA were directly compared between the two systems. The results were equivalent between the instrument systems.
### D Clinical Cut-Off:
Not applicable.
### E Expected Values/Reference Range:
The cobas Respiratory 4-flex prospective clinical studies included a total of 4,378 prospectively collected NPS specimens, of which 4,341 NPS specimens were evaluable. Of these evaluable specimens, 1,827 NPS specimens were collected during the 2023-2024 respiratory viral season and tested fresh (Category I) with the cobas Respiratory 4-flex, 785 specimens were collected during the 2022-2023 respiratory viral season and frozen prior to testing (Category II) and 1,729 were collected during the 2023-2024 respiratory viral season and frozen prior to testing (Category II). The number and percentage of cases positive for SARS-CoV-2, influenza A, influenza B, and RSV, as determined by the cobas Respiratory 4-flex, are presented in **Tables 20-22**, stratified by collection site. **Tables 23** below shows the expected values, as determined by the cobas Respiratory 4-flex, stratified by the patient's age.
**Table 20.** cobas Respiratory 4-flex – Expected Values by Specimen Collection Site for Category I NPS Specimens Collected in 2023-2024 in the Prospective Study
| Sample Category | Collection Period | Collection site | Target Virus | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Influenza A | Influenza B | Respiratory Syncytial Virus | SARS-CoV-2 |
| Category I | 2023-2024 | 1 | 0.0% (0/139) | 2.9% (4/139) | 0.0% (0/139) | 5.0% (7/139) |
| | | 2 | 0.6% (1/170) | 0.0% (0/171) | 2.3% (4/171) | 2.9% (5/171) |
| | | 3 | 0.8% (3/389) | 0.5% (2/389) | 0.5% (2/389) | 2.3% (9/389) |
| | | 4 | 0.0% (0/61) | 1.6% (1/61) | 1.6% (1/61) | 8.2% (5/61) |
| | | 5 | 17.5% (25/143) | 5.6% (8/144) | 9.0% (13/144) | 13.2% (19/144) |
| | | 6 | 14.7% (11/75) | 4.0% (3/75) | 4.0% (3/75) | 12.0% (9/75) |
| | | 7 | 0.0% (0/6) | 0.0% (0/6) | 0.0% (0/6) | 16.7% (1/6) |
| | | 8 | 6.3% (7/112) | 1.8% (2/112) | 0.9% (1/112) | 15.2% (17/112) |
| | | 9 | 2.7% (7/258) | 2.3% (6/258) | 1.9% (5/258) | 4.7% (12/258) |
| | | 10 | 0.0% (0/21) | 0.0% (0/21) | 0.0% (0/21) | 0.0% (0/21) |
| | | 11 | 11.3% (51/451) | 9.3% (42/451) | 4.0% (18/451) | 18.6% (84/451) |
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| Sample Category | Collection Period | Collection site | Target Virus | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Influenza A | Influenza B | Respiratory Syncytial Virus | SARS-CoV-2 |
| | | Overall | 5.8% (105/1825) | 3.7% (68/1827) | 2.6% (47/1827) | 9.2% (168/1827) |
**Table 21.** cobas Respiratory 4-flex – Expected Values by Specimen Collection Site for Category II NPS Specimens Collected in 2022-2023 in the Prospective Study
| Sample Category | Collection Period | Collection site | Target Virus | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Influenza A | Influenza B | Respiratory Syncytial Virus | SARS-CoV-2 |
| Category II | 2022-2023 | 1 | 0.4% (1/223) | 0.0% (0/223) | 0.0% (0/223) | 2.7% (6/223) |
| | | 2 | 0.0% (0/18) | 0.0% (0/18) | 0.0% (0/18) | 0.0% (0/18) |
| | | 3 | 0.0% (0/51) | 0.0% (0/51) | 0.0% (0/51) | 62.7% (32/51) |
| | | 4 | 2.7% (1/37) | 0.0% (0/38) | 0.0% (0/38) | 5.3% (2/38) |
| | | 5 | 0.0% (0/36) | 2.8% (1/36) | 0.0% (0/36) | 5.6% (2/36) |
| | | 6 | 0.0% (0/12) | 0.0% (0/12) | 0.0% (0/12) | 16.7% (2/12) |
| | | 7 | 2.5% (10/407) | 0.0% (0/407) | 0.2% (1/407) | 11.1% (45/407) |
| | | Overall | 1.5% (12/784) | 0.1% (1/785) | 0.1% (1/785) | 11.3% (89/785) |
**Table 22.** cobas Respiratory 4-flex – Expected Values by Specimen Collection Site for Category II NPS Specimens Collected in 2023-2024 in the Prospective Study
| Sample Category | Collection Period | Collection site | Target Virus | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Influenza A | Influenza B | Respiratory Syncytial Virus | SARS-CoV-2 |
| Category I | 2023-2024 | 1 | 4.5% (3/67) | 0.0% (0/67) | 0.0% (0/67) | 6.0% (4/67) |
| | | 2 | 2.4% (1/41) | 2.4% (1/41) | 4.9% (2/41) | 12.2% (5/41) |
| | | 3 | 3.8% (7/185) | 5.9% (11/185) | 13.0% (24/185) | 7.6% (14/185) |
| | | 4 | 0.0% (0/33) | 0.0% (0/33) | 12.1% (4/33) | 6.1% (2/33) |
| | | 5 | 7.1% (3/42) | 0.0% (0/42) | 16.7% (7/42) | 0.0% (0/42) |
| | | 6 | 0.0% (0/326) | 0.6% (2/326) | 1.2% (4/326) | 17.8% (58/326) |
| | | 7 | 9.2% (16/174) | 0.0% (0/174) | 3.4% (6/174) | 17.8% (31/174) |
| | | 8 | 3.1% (5/161) | 0.6% (1/161) | 3.1% (5/161) | 23.6% (38/161) |
| | | 9 | 0.0% (0/66) | 1.5% (1/66) | 1.5% (1/66) | 4.5% (3/66) |
| | | 10 | 0.6% (1/168) | 0.0% (0/168) | 1.2% (2/168) | 17.3% (29/168) |
| | | 11 | 1.0% (2/201) | 0.0% (0/201) | 0.0% (0/201) | 12.9% (26/201) |
| | | 12 | 7.7% (12/155) | 1.3% (2/155) | 5.8% (9/155) | 6.5% (10/155) |
| | | 13 | 0.0% (0/56) | 8.9% (5/56) | 1.8% (1/56) | 14.3% (8/56) |
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| Sample Category | Collection Period | Collection site | Target Virus | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Influenza A | Influenza B | Respiratory Syncytial Virus | SARS-CoV-2 |
| | | 14 | 11.1% (6/54) | 0.0% (0/54) | 0.0% (0/54) | 25.9% (14/54) |
| | | Overall | 3.2% (56/1729) | 1.3% (23/1729) | 3.8% (65/1729) | 14.0% (242/1729) |
**Table 23.** cobas Respiratory 4-flex – Expected Values by Patient Age for the Prospective Studies
| Study Phase | Analyte | Positive Results Stratified by Age (Years) % Positive (n Detected/ N Tested) | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | All Ages | ≤5 | >5 - 21 | >21 - 65 | >65 |
| Category I: 2023-2024 | Influenza A | 5.8% (105/1825) | 15.4% (6/39) | 9.1% (32/352) | 4.8% (58/1205) | 3.9% (9/229) |
| | Influenza B | 3.7% (68/1827) | 7.7% (3/39) | 7.9% (28/353) | 3.0% (36/1206) | 0.4% (1/229) |
| | RSV | 2.6% (47/1827) | 25.6% (10/39) | 3.4% (12/353) | 1.7% (21/1206) | 1.7% (4/229) |
| | SARS-CoV-2 | 9.2% (168/1827) | 7.7% (3/39) | 7.9% (28/353) | 9.7% (117/1206) | 8.7% (20/229) |
| Category II: 2022-2023 | Influenza A | 1.5% (12/784) | NC (0/0) | 0.0% (0/47) | 1.8% (11/598) | 0.7% (1/139) |
| | Influenza B | 0.1% (1/785) | NC (0/0) | 0.0% (0/47) | 0.2% (1/599) | 0.0% (0/139) |
| | RSV | 0.1% (1/785) | NC (0/0) | 0.0% (0/47) | 0.2% (1/599) | 0.0% (0/139) |
| | SARS-CoV-2 | 11.3% (89/785) | NC (0/0) | 2.1% (1/47) | 11.0% (66/599) | 15.8% (22/139) |
| Category II: 2023-2024 | Influenza A | 3.2% (56/1729) | 4.9% (9/183) | 4.1% (18/434) | 2.9% (28/967) | 0.7% (1/145) |
| | Influenza B | 1.3% (23/1729) | 1.1% (2/183) | 4.4% (19/434) | 0.2% (2/967) | 0.0% (0/145) |
| | RSV | 3.8% (65/1729) | 17.5% (32/183) | 3.7% (16/434) | 1.3% (13/967) | 2.8% (4/145) |
| | SARS-CoV-2 | 14.0% (242/1729) | 5.5% (10/183) | 6.7% (29/434) | 15.0% (145/967) | 40.0% (58/145) |
Note: NC = Not Calculable due to no samples from the age group.
#### F Other Supportive Instrument Performance Characteristics Data:
Not applicable.
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# **VIII Proposed Labeling:**
The labeling supports the finding of substantial equivalence for this device.
# **IX Conclusion:**
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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