cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems

K213804 · Roche Molecular Systems, Inc. · QQX · Oct 22, 2022 · Microbiology

Device Facts

• Record ID: K213804
• Device Name: cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems
• Applicant: Roche Molecular Systems, Inc.
• Product Code: QQX · Microbiology
• Decision Date: Oct 22, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3981
• Device Class: Class 2
• Attributes: AI/ML

AI Performance

Output	Acceptance	Observed	Dev DS	Test DS
SARS-CoV-2 RNA Detection (Nasopharyngeal Swab)	Not specified	PPA: 98.7% (93.1%, 99.8%); NPA: 99.7% (99.0%, 99.9%)	Algorithm trained using selected data from testing of clinical specimens or cultures spiked into clinical background to confirm adequate separation between Ct values and assay cut-offs.	Multi-center clinical study: 938 evaluable nasopharyngeal swab specimens from 968 participants.
SARS-CoV-2 RNA Detection (Nasal Swab)	Not specified	PPA: 97.4% (91.1%, 99.3%); NPA: 99.9% (99.3%, 100%)	Algorithm trained using selected data from testing of clinical specimens or cultures spiked into clinical background to confirm adequate separation between Ct values and assay cut-offs.	Multi-center clinical study: 934 evaluable nasal swab specimens from 968 participants.

Indications for Use

cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems is a real-time RT-PCR test intended for the qualitative detection of nucleic acids from SARS-CoV-2 in nasal and nasopharyngeal specimens collected from symptomatic individuals suspected of COVID-19 by their healthcare provider. Results are for the detection of SARS-CoV-2 RNA. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection with other pathogens. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Results are meant to be used in conjunction with clinical observations, patient history, recent exposures and epidemiological information, and laboratory data, in accordance with the guided by the relevant public health authorities. cobas SARS-CoV-2 is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and on the use of the cobas 6800/8800 Systems.

Device Story

The cobas SARS-CoV-2 Qualitative is a fully automated, real-time RT-PCR assay for use on the cobas 6800/8800 Systems. It processes nasal and nasopharyngeal specimens through automated nucleic acid extraction and purification using magnetic glass particles. The system performs selective amplification of SARS-CoV-2 ORF1 a/b and pan-Sarbecovirus E-gene targets, alongside an RNA internal control. Detection utilizes TaqMan probe-based fluorescence chemistry; reporter and quencher dyes are separated via 5' to 3' exonuclease activity during PCR cycles. The system software manages data, assigns results based on cycle threshold (Ct) analysis, and displays reports. Used in clinical laboratory settings by trained personnel, the device provides qualitative results to assist healthcare providers in diagnosing COVID-19. Clinical correlation with patient history and other diagnostic data is required for final infection status determination.

Clinical Evidence

Multi-center study of 968 symptomatic participants. Evaluated nasopharyngeal (NPS) and nasal swab (NS) specimens against a composite comparator (concordant results from up to three EUA molecular assays). For NPS (n=938), PPA was 98.7% (95% CI: 93.1-99.8%) and NPA was 99.7% (95% CI: 99.0-99.9%). For NS (n=934), PPA was 97.4% (95% CI: 91.1-99.3%) and NPA was 99.9% (95% CI: 99.3-100.0%).

Technological Characteristics

Real-time RT-PCR; TaqMan FRET chemistry. Automated extraction via magnetic glass particles. Targets: ORF1 a/b and E-gene. Includes AmpErase (uracil-N-glycosylase) for carry-over prevention. System: cobas 6800/8800 (sample supply, transfer, processing, analytic modules). Connectivity: LIS interface. Reagent monitoring via RFID tags.

Indications for Use

Indicated for symptomatic individuals suspected of COVID-19. Specimens: nasal and nasopharyngeal swabs. For use by qualified clinical laboratory personnel.

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 COVID-19 Test 2 (K211079)

Submission Summary (Full Text)

{0}

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number
K213804

B Applicant
Roche Molecular Systems, Inc.

C Proprietary and Established Names
cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QQX | 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:
New Device

B Measurand:
SARS-CoV-2 RNA

C Type of Test:

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 1 of 20

{1}

Nucleic acid amplification test with paired reporter and quencher fluorescence labeled probes (TaqMan Technology) using fluorescence resonance energy transfer (FRET)

## III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) for Use:

cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems is a real-time RT-PCR test intended for the qualitative detection of nucleic acids from SARS-CoV-2 in nasal and nasopharyngeal specimens collected from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the detection of SARS-CoV-2 RNA. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other pathogens.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Results are meant to be used in conjunction with clinical observations, patient history, recent exposures and epidemiological information, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. cobas SARS-CoV-2 is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and on the use of the cobas 6800/8800 Systems.

### C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

### D Special Instrument Requirements:

This test is to be used with the Roche cobas 6800/8800 system only.

## IV Device/System Characteristics:

### A Device Description:

cobas SARS-CoV-2 Qualitative is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas 6800/8800 Systems consists of two separate instruments, the cobas 6800 System and the cobas 8800 System, which both are fully integrated and allow users to perform multiple PCR-based in vitro nucleic acid amplification tests. 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 6800/8800 Systems software(s), which assigns test results for all tests. Results can be reviewed directly on the system screen and printed as a report.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 2 of 20

{2}

cobas 6600 instrument

cobas 8800 instrument

The main system functionality is provided by two Software components: the cobas 6800/8800 System Software and Assay Specific Analysis Packages (ASAPs). The cobas 6800/8800 System Software provides basic functionality such as a Graphical User Interface (GUI), instrument management, database functionality, report engines, and LIS interfaces. ASAPs are built using a common Software framework and provides the assay test run conditions (sample preparation and PCR parameters), result analysis functionality (result calculation and algorithms), and result report formatting.

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.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 3 of 20

{3}

Selective amplification of target nucleic acid from the sample is achieved using target specific forward and reverse primers for ORF1 a/b non-structural region that is unique to SARS-CoV-2. Additionally, a conserved region in the structural protein envelope E-gene were chosen for pan-Sarbecovirus detection. The pan-Sarbecovirus detection sets will also detect SARS-CoV-2 virus. Selective amplification of RNA IC is achieved using non-competitive sequence specific forward and reverse primers which have no homology with the coronavirus genome. A thermostable DNA polymerase enzyme is used for amplification.

# B Principle of Operation:

The cobas SARS-CoV-2 Qualitative master mix contains detection probes which are specific for the coronavirus type SARS-CoV-2, members of the Sarbecovirus subgenus, and the RNA IC nucleic acid. The coronavirus and RNA IC detection probes are each labeled with unique fluorescent dyes that act as a reporter. Each probe also has a second dye which acts as a quencher. When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by the quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the $5^{\prime}$ to $3^{\prime}$ exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Each reporter dye is measured at defined wavelengths, which enables simultaneous detection and discrimination of the amplified coronavirus target and the RNA IC. The master mix includes deoxy uridine 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 $55^{\circ}\mathrm{C}$ .

Reagents loaded onto the cobas 6800/8800 Systems are stored at appropriate temperatures and their expiration is monitored by the system. The cobas 6800/8800 Systems allow reagents to be used only if all the conditions are met. The system automatically prevents use of expired reagents via encoded radio frequency identification (RFID) tags that are placed on the reagent cassettes, RMC cassettes and reagent bottles. RFID Labels are used to apply information on test specific and generic reagent cassettes, control tube containers and generic reagent bottles utilized on the cobas 6800/8800 Systems.

Components of the cobas SARS-CoV-2 Qualitative Kit

| Components Name | Formulation |
| --- | --- |
| Proteinase Solution (PASE) | Tris buffer, < 0.05% EDTA, calcium chloride, calcium acetate, 8% proteinase, glycerol EUH210: Safety data sheet available on request. EUH208: Contains Subtilisin. May produce an allergic reaction. |
| RNA Internal Control (RNA IC) | Tris buffer, < 0.05% EDTA, < 0.001% non-Sarbecovirus related armored RNA construct containing primer and probe specific primer sequence regions (non-infectious RNA in MS2 bacteriophage), < 0.1% sodium azide |

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 4 of 20

{4}

| Elution Buffer (EB) | Tris buffer, 0.2% methyl-4 hydroxybenzoate |
| --- | --- |
| Master Mix Reagent 1 (MMX-R1) | Manganese acetate, potassium hydroxide, <0.1% sodium azide |
| SARS-CoV-2 Master Mix Reagent 2 (SARS-CoV-2 MMX-R2) | Tricine buffer, potassium acetate, <18% dimethyl sulfoxide, glycerol, <0.1% Tween 20, EDTA, <0.12% dATP, dCTP, dGTP, dUTPs, <0.01% upstream and downstream SARS-CoV-2 and Sarbecovirus primers, <0.01% Internal Control forward and reverse primers, <0.01% fluorescent-labeled oligonucleotide probes specific for SARS-CoV-2, Sarbecovirus, and the RNA Internal Control, <0.01% oligonucleotide aptamer, <0.1% Z05D DNA polymerase, <0.10% AmpErase (uracil-N-glycosylase) enzyme (microbial), <0.1% sodium azide |

## Components of the cobas SARS-CoV-2 Qualitative Control Kit

| Components Name | Formulation |
| --- | --- |
| SARS-CoV-2 Positive Control (SARS-CoV-2 (+) C) | Tris buffer, < 0.05% Sodium azide, < 0.005% EDTA, < 0.003% Poly rA, < 0.01% Non-infectious plasmid DNA (microbial) containing SARS-CoV-2 sequence, <0.01% Non-infectious plasmid DNA (microbial) containing pan-Sarbecovirus sequence |

## Components of the cobas Buffer Negative Control Kit

| Components Name | Formulation |
| --- | --- |
| cobas Buffer Negative Control (BUF (-) C) | Tris buffer, < 0.1% sodium azide, EDTA, < 0.002% Poly rA RNA (synthetic) |

Automated data management is performed by the Assay Specific Analysis Package software which assigns test results for all tests. Results can be review directly on the system screen, exported, or printed as a report.

The results interpretation for detecting SARS-CoV-2 is summarized below.

## cobas SARS-CoV-2 Qualitative Results Interpretation

| Target 1 (SCoV2) | Target 2 (PanSarb) | Interpretation |
| --- | --- | --- |
| Positive | PanSarb Positive | All Target Results were valid. Result for SARS-CoV-2 RNA is Detected. |
| Positive | Negative | All Target Results were valid. Result for SARS-CoV-2 RNA is Detected. A positive Target 1 result and a negative Target 2 result is suggestive of 1) a sample at concentrations near or below the limit of detection of the test, 2) a mutation in the Target 2, target region, or 3) other factors. |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 5 of 20

{5}

| Negative | Positive | All Target Results were valid.
Result for SARS-CoV-2 RNA is Presumptive Positive. A negative Target 1 result and a positive Target 2 result is suggestive of 1) a sample at concentrations near or below the limit of detection of the test, 2) a mutation in the Target 1 target region in the oligo binding sites, or 3) infection with some other Sarbecovirus (e.g., SARS-CoV or some other Sarbecovirus previously unknown to infect humans), or 4) other factors. For samples with a Presumptive Positive result, additional confirmatory testing may be conducted, if it is necessary to differentiate between SARS-CoV-2 and SARS-CoV-1 or other Sarbecovirus currently unknown to infect humans, for epidemiological purposes or clinical management. |
| --- | --- | --- |
| Negative | Negative | All Target Results were valid.
Result for SARS-CoV-2 RNA is Not Detected. |
| Positive | Invalid | Not all Target Results were valid.
Result for SARS-CoV-2 RNA is Detected. |
| Invalid | Positive | Not all Target Results were valid.
Result for SARS-CoV-2 RNA is Presumptive Positive. For samples with a Presumptive Positive result, additional confirmatory testing may be conducted, if it is necessary to differentiate between SARS-CoV-2 and SARS-CoV-1 or other Sarbecovirus currently unknown to infect humans, for epidemiological purposes or clinical management. |
| Negative | Invalid | Not all Target Results were valid.
Sample should be retested. If the result is still invalid, a new specimen should be obtained. |
| Invalid | Negative | Not all Target Results were valid.
Sample should be retested. If the result is still invalid, a new specimen should be obtained. |
| Invalid | Invalid | All Target Results were invalid.
Sample should be retested. If the result is still invalid, a new specimen should be obtained. |

C Instrument Description Information:

1. Instrument Name: cobas 6800/8800 Systems and software version 1.4.7; cobas SCoV2-QL ASAP Software version 12.1.0
2. Specimen Identification: Specimen identification can be configured in an automated fashion or entered manually. All corresponding user interactions are recorded in the audit trail on the Instrument Gateway
3. Specimen Sampling and Handling: Nasopharyngeal swab (NPS) or Anterior Nasal swab (ANS) specimens collected in transport media.
4. Calibration: Not Applicable

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 6 of 20

{6}

5. Quality Control: The RNA Internal Control (RNA-IC), used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. In addition, the test utilizes external controls- cobas SARS-CoV-2 Qualitative Control Kit (low titer positive control) and cobas Buffer Negative Control Kit (negative control), provided separately.

V Substantial Equivalence Information:

A Predicate Device Name(s):

BioFire COVID-19 Test 2

B Predicate 510(k) Number(s):

K211079

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K213804 | K211079 |
| --- | --- | --- |
| Device Trade Name | cobas SARS-CoV-2 Qualitative | BioFire COVID-19 Test 2 |
| 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 |
| Product Code | QQX | same |
| Intended Use | cobas SARS-CoV-2 Qualitative for use on the cobas 6800/8800 Systems is a real-time RT-PCR test intended for the qualitative detection of nucleic acids from SARS-CoV-2 in nasal and nasopharyngeal specimens collected from symptomatic individuals suspected of COVID-19 by their healthcare provider. Results are for the detection of SARS-CoV-2 RNA. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other pathogens. Negative results do not preclude SARS-CoV-2 infection and should | The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider. Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 |

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 7 of 20

{7}

| | not be used as the sole basis for patient management decisions. Results are meant to be used in conjunction with clinical observations, patient history, recent exposures and epidemiological information, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. cobas SARS-CoV-2 is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and on the use of the cobas 6800/8800 Systems. | RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional. |
| --- | --- | --- |
| Conditions for use | For prescription use | same |
| Sample Types | Nasopharyngeal swab specimen Nasal swab specimen | Nasopharyngeal swab specimen |
| Analyte Targets | SARS-CoV-2 | SARS-CoV-2 |
| Sample Preparation Procedure | Automated by cobas 6800/8800 Systems | Automated by BioFire FilmArray 2.0 or BioFire FilmArray Torch systems |
| Amplification Technology | Real-time PCR | Nested multiplex RT-PCR |
| Analyte | RNA | RNA |
| Detection Chemistry | Paired reporter and quencher fluorescence labeled probes (TaqMan Technology) using fluorescence resonance energy transfer (FRET) | 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 | 1. The RNA Internal Control (RNA-IC)
2. External Positive and negative controls | Two process controls:
1. RNA Process Control (IC)
2. 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 |
| Test Interpretation | Automated test interpretation and report generation. User cannot access raw data. | same |

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 8 of 20

{8}

VI Standards/Guidance Documents Referenced:

Class II Special Controls as per 21 CFR 866.3981.

VII Performance Characteristics:

A Analytical Performance:

1. Precision/Reproducibility:

a) Within-laboratory Precision:

Within-laboratory precision was examined using a panel of heat-inactivated SARS-CoV-2 (USA-WA1/2020) cultures diluted in simulated clinical matrix stabilized in UTM. Sources of variability were examined with a panel consisting of three concentration levels (0.3x, 1x and 3x LoD), using three lots of cobas SARS-CoV-2 Qualitative reagents and three instruments over a course of 15 testing days (2 runs/day x 3 instruments x 5 days/instrument) for a total of 30 runs. A total of 90 replicates per concentration level were tested over the course of the study. A negative panel was tested along with the positive panel members. The tables below summarize the results from the inter-lot precision study, showing the mean Ct values and calculated SD and % CV for each panel member.

Qualitative Summary of Within Laboratory Precision

| Target | Panel Member | Level (x LoD) | Total Results (N) | Positive Results | Positivity % | Two-sided 95% CI Lower Bound | Two-sided 95% CI Upper Bound |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Target 1 (SARS-CoV-2) | High negative | ~0.3x | 90 | 9 | 10% | 5% | 18% |
| | Low positive | ~1.0x | 90 | 82 | 91% | 83% | 96% |
| | Moderate positive | ~3.0x | 90 | 90 | 100% | 96% | 100% |
| Target 2 (pan-Sarbecovirus) | High negative | ~0.3x | 90 | 31 | 34% | 25% | 45% |
| | Low positive | ~1.0x | 90 | 84 | 93% | 86% | 97% |
| | Moderate positive | ~3.0x | 90 | 90 | 100% | 96% | 100% |
| N/A | Negative | Blank | 90 | 0 | 0% | 0% | 4% |

Summary of the Precision Studies based on Ct values

| Target | Level (x LoD) | Hit rate | Mean Ct | Within Run | | Run-to-Run | | Day-to-Day | | Instrument -to- Instrument | | Lot-to-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | SD | SD | CV% | CV% | CV% | SD | CV% | SD | CV% | SD | CV% |
| | ~0.3x | 10.0% | 32.51 | 0.5 | 0.0 | 0.0 | 0.0 | 0.0 | 1.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.5 | 1.4 |
| | ~1.0x | 91.1% | 32.1 | 0.6 | 0.0 | 0.1 | 0.3 | 0.0 | 1.8 | 0.0 | 0.0 | 0.2 | 0.6 | 0.6 | 1.9 |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 9 of 20

{9}

b) Reproducibility:

The reproducibility of cobas SARS-CoV-2 Qualitative was evaluated at three testing sites, using three reagent lots, with a four-member panel of positive and negative samples resulting in a total number 216 tests per concentration (not including controls). The positive panel members contained the SARS-CoV-2 target in negative simulated clinical matrix stabilized in UTM at three different concentrations (0.3x, 1x, 3x LoD). Each site tested two reagent lots for six days. Two runs were performed each day and three replicates of each panel member were performed for each run. An overall SARS-CoV-2 positive result was determined by a positive detection in either or both SARS-CoV-2 or/and pan-Sarbecovirus channels. The study results are summarized in Table below.

Summary of Reproducibility Variance Components based on Ct values

| Viral Target | Panel Member Concentration | nᵃ/N | Percent Agreementᵃ (%)ᵇ | Mean Ct | Between Site | | Between Lot | | Between Day | | Between Runs | | Within Run | | Total SD | Total CV(%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | | |
| Negative | 0 | 214/216ᶜ | 99.1 | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc | nc |
| SARS-CoV-2 | ~0.3xLoD | 45/216 | 20.8 | 33.6 | 0.00 | 0.0 | 0.00 | 0.0 | 0.11 | 0.3 | 0.00 | 0.0 | 0.35 | 1.1 | 0.37 | 1.1 |
| SARS-CoV-2 | ~1xLoD | 196/216 | 90.7 | 33.2 | 0.00 | 0.0 | 0.09 | 0.3 | 0.00 | 0.0 | 0.17 | 0.5 | 0.37 | 1.1 | 0.42 | 1.3 |
| SARS-CoV-2 | ~3xLoD | 216/216 | 100.0 | 32.2 | 0.05 | 0.2 | 0.02 | 0.1 | 0.00 | 0.0 | 0.03 | 0.1 | 0.24 | 0.8 | 0.25 | 0.8 |
| pan-Sarbecovirus | ~0.3xLoD | 158/216 | 73.1 | 36.5 | 0.18 | 0.5 | 0.00 | 0.0 | 0.00 | 0.0 | 0.00 | 0.0 | 0.71 | 2.0 | 0.74 | 2.0 |
| pan-Sarbecovirus | ~1xLoD | 214/216 | 99.1 | 35.4 | 0.00 | 0.0 | 0.00 | 0.0 | 0.00 | 0.0 | 0.00 | 0.0 | 0.67 | 1.9 | 0.67 | 1.9 |
| pan-Sarbecovirus | ~3xLoD | 216/216 | 100.0 | 34.1 | 0.11 | 0.3 | 0.05 | 0.2 | 0.00 | 0.0 | 0.00 | 0.0 | 0.32 | 0.9 | 0.34 | 1.0 |

Ct = cycle threshold, LoD = limit of detection, SD = standard deviation, CV(%) = percent coefficient of variation, SARS CoV 2 = severe acute respiratory syndrome coronavirus 2, nc = not calculable
Note: SARS-CoV-2 is a dual target assay. Inactivated viral culture material was diluted to ~0.3/1/3x LoD based on the target 2 (SARS-CoV-2) LoD.
ᵃ n is the number of positive tests which contribute Ct values to the analysis. N is the total number of valid tests for the panel member.
ᵇ Percent agreement with expected results.
ᶜ Two negative panel members were tested positive (one for each target). Sequencing showed that one of these samples was positive and the other was negative. The Ct values and the curve analysis of these samples suggest a low level of contamination during specimen handling.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 10 of 20

{10}

2. Linearity:

Not applicable; this is a qualitative assay.

3. Analytical Specificity/Interference:

a) Cross-Reactivity:

The potential for cross-reactivity (analytical specificity) for cobas SARS-CoV-2 Qualitative assay was evaluated in a study testing samples containing organisms that are commonly found in the respiratory tract and might cause similar clinical symptoms as SARS-CoV-2. A panel of 47 viruses, bacteria, and fungi were included in the study, with each organism tested in three replicates. The organisms were spiked at concentrations of 1×10⁵ units/mL for viruses and 1×10⁶ units/mL for bacteria and fungi. Testing was performed with each potential interfering organism in the absence and presence of SARS-CoV-2 target (spiked at ~3xLoD). No cross reactivity or microbial interference was observed with the organisms at the concentrations tested, as shown below. Additional in-silico analysis confirmed no homology was found between any primers or probes for Bordetella parapertussis, Aspergillus sp, and Fusobacterium necrophorum, however no wet testing with these organisms was performed.

Summary of Analytical Cross-Reactivity for cobas SARS-CoV-2 Qualitative

| Microorganism | Concentration | Positivity Rate | Negativity rate |
| --- | --- | --- | --- |
| Human coronavirus 229E | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Human coronavirus OC43 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Human coronavirus HKU1 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Human coronavirus NL63 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| MERS coronavirus | 1.0E+05 genomic equivalent/mL | 100% | 100% |
| SARS coronavirus | 1.0E+05 PFU/mL | 100% | 100%* |
| Adenovirus B (Type 34) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Bocavirus | 1.0E+05 cp/mL | 100% | 100% |
| Cytomegalovirus | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Epstein Barr virus | 1.0E+05 cp/mL | 100% | 100% |
| Human Metapneumovirus (hMPV) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Measles virus | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Mumps virus | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Parainfluenza virus Type 1 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Parainfluenza virus Type 2 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Parainfluenza virus Type 3 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Parainfluenza virus Type 4 | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Influenza A (H1N1) | 1.0E+05 TCID_{50}/mL | 100% | 100% |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 11 of 20

{11}

| Microorganism | Concentration | Positivity Rate | Negativity rate |
| --- | --- | --- | --- |
| Influenza A virus (H1N1-2009) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Influenza A virus (H1N3) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Influenza A virus (H3N2) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Influenza B | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Enterovirus E (Type 1) | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Parechovirus | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Respiratory syncytial virus | 1.0E+05 PFU/mL | 100% | 100% |
| Rhinovirus | 1.0E+05 TCID_{50}/mL | 100% | 100% |
| Candida albicans | 1.0E+06 CFU/mL | 100% | 100% |
| Chlamydia pneumoniae | 1.0E+06 TCID_{50}/mL | 100% | 100% |
| Corynebacterium diphtheriae | 1.0E+06 CFU/mL | 100% | 100% |
| Escherichia coli | 1.0E+06 CFU/mL | 100% | 100% |
| Haemophilus influenzae | 1.0E+06 CFU/mL | 100% | 100% |
| Lactobacillus gasseri | 1.0E+06 CFU/mL | 100% | 100% |
| Legionella pneumophila | 1.0E+06 CFU/mL | 100% | 100% |
| Legionella jordanis (non-pneumophila) | 1.0E+06 CFU/mL | 100% | 100% |
| Moraxella catarrhalis | 1.0E+06 CFU/mL | 100% | 100% |
| Mycobacterium tuberculosis | 1.0E+06 cells/mL | 100% | 100% |
| Neisseria elongata | 1.0E+06 CFU/mL | 100% | 100% |
| Neisseria meningitidis | 1.0E+06 CFU/mL | 100% | 100% |
| Pseudomonas aeruginosa | 1.0E+06 CFU/mL | 100% | 100% |
| Pneumocystis jirovecii | 1:20 of Patient Sample | 100% | 100% |
| Staphylococcus aureus | 1.0E+06 CFU/mL | 100% | 100% |
| Staphylococcus epidermidis | 1.0E+06 CFU/mL | 100% | 100% |
| Streptococcus pneumoniae | 1.0E+06 CFU/mL | 100% | 100% |
| Streptococcus pyrogenes | 1.0E+06 CFU/mL | 100% | 100% |
| Streptococcus salivarius | 1.0E+06 CFU/mL | 100% | 100% |
| Bordetella pertussis | 1.0E+06 CFU/mL | 100% | 100% |
| Mycoplasma pneumoniae | 1.0E+06 CFU/mL | 100% | 100% |

* Testing of SARS-CoV-1 generated an expected Pan-Sarbecovirus positive result.

b) Interfering substances study:

The potential for interference for the cobas SARS-CoV-2 Qualitative assay was evaluated with substances that may be present in respiratory specimens. A total of 20 potentially interfering substances were tested at or above clinically relevant levels in negative simulated clinical matrix stabilized in UTM in absence and presence of SARS-CoV-2 target (spiked at

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 12 of 20

{12}

~3x LoD). Each sample was tested in 10 replicates. The following table provides the substances and concentrations tested. The FluMist nasal vaccine was not tested.

Summary of Interfering Substances Study for Cobas SARS-CoV-2 Qualitative

| Name | Active Ingredients | Test concentration | Total N | Positivity % | |
| --- | --- | --- | --- | --- | --- |
| | | | | Target 1 (SARS-CoV-2) | Target 2 (Pan-Sarbeco) |
| Control | No Substance | N/A | 10 | 100 | 100 |
| Human Genomic DNA | - | 20 ng/μl | 10 | 100 | 100 |
| Mucus | - | One sputum swab/mL | 10 | 100 | 100 |
| Human PBMCs | - | 1.0E+03 cells/μL | 10 | 100 | 100 |
| Human Whole Blood | - | 1%, 2% and 5% (v/v) | 10 | 100 | 100 |
| Afrin Nasal Spray | Oxymetazoline | 0.011 mg/mL | 10 | 100 | 100 |
| Budesonide Nasal spray | Budesonide (glucocorticoid) | 0.039 mg/mL | 10 | 100 | 100 |
| Flonase Nasal Spray | Fluticasone Propionate | 0.167 mg/mL | 10 | 100 | 100 |
| Zicam Nasal Spray | Galphimia glauca, Luffa operculata, Sabadilla | 0.023 mg/mL | 10 | 100 | 100 |
| Saline Nasal Spray with Preservatives | 0.65% NaCl, phenylcarbino, Benzalkonium chloride | 1.0% (v/v) | 10 | 100 | 100 |
| Cepacol (Sore Throat Lozenges) | Benzocaine and menthol | 5.0 mg/mL | 10 | 100 | 100 |
| Chloraseptic | Phenol | 0.47 mg/mL | 10 | 100 | 100 |
| Liposomal NUMB520 Spray | Lidocaine and Phenylephrine | 2.68 mg/mL | 10 | 100 | 100 |
| Mupirocin ointment UPS | Mupirocin | 0.20 mg/mL | 10 | 100 | 100 |
| Relenza (Inhalation powder) | Zanamivir | 0.0015 mg/mL | 10 | 100 | 100 |
| Antiviral drug – Tamiflu | Oseltamivir | 0.0073 mg/mL | 10 | 100 | 100 |
| DMSO | N/A | N/A | 10 | 100 | 100 |
| Tobramycin ophthalmic Solution | Tobramycin | 0.018 mg/mL | 10 | 100 | 100 |
| Vaseline | Petroleum Jelly | 1% (w/v) | 10 | 100 | 100 |
| Snuff Tobacco | Nicotine | 1% (w/v) | 10 | 100 | 100 |
| Analgesic ointment (Vicks@VapoRub) | Camphor-synthetic eucalyptus oil and menthol ointment | 1% (w/v) | 10 | 100 | 100 |

* FluMist was not evaluated to assess potential interference.

## 4. Assay Reportable Range:

Not applicable; this is a qualitative assay.

## 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

### a) Quality Controls

The RNA Internal Control (RNA-IC), used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. In

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213804 - Page 13 of 20

{13}

addition, the test utilizes external controls- cobas SARS-CoV-2 Qualitative Control Kit (low titer positive control) and cobas Buffer Negative Control Kit (negative control), provided separately. Three lots of Positive Control were included in the clinical evaluation of the cobas SARS-CoV-2 Qualitative conducted at three testing sites. The summary of the Ct values for valid positive and negative control results obtained for each target, combined for sites and lots, is presented below.

Summary of the Ct values for Controls used in Clinical Studies

| Target Analyte | Total N | Mean Target Ct | Ct SD | Ct %CV |
| --- | --- | --- | --- | --- |
| SARS-CoV-2 | 131 | 33.66 | 0.21 | 0.66 |
| pan-Sarbeco | 131 | 35.56 | 0.34 | 1.00 |
| IC (positive samples) | 131 | 33.23 | 0.22 | 0.66 |
| IC (negative samples) | 131 | 33.1 | 0.24 | 0.7 |

b) Clinical Specimen stability:

The stability of SARS-CoV-2 viral targets during handling, transportation, and storage, was evaluated using NS and NPS specimens collected in different collection media. Five pools of NS specimens (collected in cobas PCR media, 0.9% saline and copan UTM) and five pools of NPS specimens (collected in UTM) were spiked with the inactivated quantified SARS-CoV-2 virus at 3xLoD. Three replicates per specimen type/storage condition were used. The study data supported storage for specimens collected in UTM and 0.9% physiological saline at 2-25°C for up to 48 hours followed by up to 3 days at 2-8°C and at ≤ -70°C for up to 30 days. Specimens collected in cobas PCR Media can be stored at 2-25°C for up to 24 hours followed by up to 3 days at 2-8°C and at ≤ -70°C for up to 30 days.

6. Detection Limit:

The Limit of Detection (LoD) for cobas SARS-CoV-2 Qualitative was determined using an inactivated quantified SARS-CoV-2 virus (WHO International Standard for SARS-CoV-2, NIBSC code: 20/146). LoD is defined as the lowest concentration of SARS-CoV-2 RNA that can be detected at a rate of at least 95%. A total of 5 concentration levels (500, 250, 125, 62.5 and 31.25 IU/ml) were prepared by diluting the SARS-CoV-2 target in negative simulated clinical matrix stabilized in UTM. Three independent dilution series with three lots of reagents were tested with a total of 24 replicates per concentration.

The concentration level with observed hit rates greater than or equal to 95% was determined to be the LoD for each of the two targets (SARS-CoV-2 and pan-Sarbecovirus) as described below.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 14 of 20

{14}

Summary of LoD for SARS-CoV-2 (Target-1)

| Viral Strain | Kit lot | Hit rate ≥ 95% [IU/mL] (no. of replicates detected) | Mean Ct at ≥ 95% Hit rate |
| --- | --- | --- | --- |
| WHO International Standard for SARS-CoV-2 | Lot 1 | 250 (24/24) | 33.2 |
| | Lot 2 | 125 (23/24) | 34.1 |
| | Lot 3 | 250 (23/24) | 33.2 |

The LoD was confirmed at 250 IU/mL for SARS-CoV-2 (Target 1). For all three reagent lots, at least 23/24 replicates detected the target at 250 IU/ml.

Summary of LoD for pan-Sarbecovirus (Target-2)

| Viral Strain | Kit lot | Hit rate ≥ 95% [IU/mL] (no. of replicates detected) | Mean Ct at ≥ 95% Hit rate |
| --- | --- | --- | --- |
| WHO International Standard for SARS-CoV-2 | Lot 1 | 125 (24/24) | 35.2 |
| | Lot 2 | 125 (24/24) | 36.0 |
| | Lot 3 | 125 (23/24) | 34.8 |

The LoD was confirmed at 125 IU/mL for pan-Sarbecovirus (Target 2). For all three reagent lots, at least 23/24 replicates detected the target at 125 IU/ml.

7. Matrix equivalency study:

Because some analytical studies were conducted using simulated matrix, equivalency between simulated and real clinical matrix was evaluated. SARS-CoV-2 target was diluted to make the test panel of four concentration levels (0x, 0.3x, 1x and 3x LoD) in each of three matrices (simulated clinical matrix, nasopharyngeal swab specimen and nasal swab specimen). 25 replicates were tested per each combination.

Summary of matrix equivalency study for cobas SARS-CoV-2 Qualitative

| Sample Type Matrix (Stabilized in UTM) | Panel Member | Level (x LoD) | Conc. IU/mL | Total Results (N) | Positive Results | Positivity % |
| --- | --- | --- | --- | --- | --- | --- |
| Nasal Swab | Above LoD | ~3x | 750 | 25 | 25 | 100% |
| | At LoD | ~1.0x | 250 | 25 | 25 | 100% |
| | Below LoD | ~0.3x | 83 | 25 | 25 | 100% |
| | Negative | 0x | 0 | 25 | 0 | 0% |
| | Above LoD | ~3x | 750 | 25 | 25 | 100% |
| | At LoD | ~1.0x | 250 | 25 | 25 | 100% |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 15 of 20

{15}

The results showed the performance of the assay with simulated clinical matrix, nasopharyngeal swab, and nasal swab matrices is equivalent.

## 8. Collection media equivalency (UTM, cobas PCR Media and 0.9% physiological saline)

Equivalency between copan UTM, cobas PCR Media (CPM) and 0.9% physiological saline was evaluated during the development of the subject IVD device. Paired individual negative nasal swab samples were collected and tested before and after spiking heat inactivated SARS-CoV-2 virus (WHO International Standard). At least 20 low positive (1.5-2x LoD) and 10 moderate positive (4x LoD) samples was prepared for each collection media. All negative and positive samples generated expected results for SARS-CoV-2 in the three collection media tested. The mean Ct differences between the three collection media was less than 2.

## 9. Analytical Reactivity (Inclusivity):

The inclusivity of cobas SARS-CoV-2 Qualitative assay for the detection of SARS-CoV-2 was confirmed by testing nine SARS-CoV-2 strains, including six variant strains. Qualified stocks of different SARS-CoV-2 were used to prepare concentration levels at and around the LoD. Each level was tested with four replicates. The lowest concentration at which all four replicates tested positive are reported in below.

Analytical Reactivity with SARS-CoV-2 Strains

| SARS-CoV-2 Strain | Target Material | Concentration tested with 100% Positivity |
| --- | --- | --- |
| Hong Kong/VM20001061/2020 | Inactivated Virus | 1.06E+02 cp/mL |
| Italy-INMI1 | Inactivated Virus | 1.00E+02 cp/mL |
| USA-WA1/2020 | Inactivated Virus | 5.03E+01 cp/mL |
| UK (B.1.1.7) | Inactivated Virus | 2.4E+01 cp/mL |
| Japan / Brazil (P.1) | Extracted RNA | 1.9E+02 cp/mL |
| South Africa (B.1.351) | Inactivated Virus | 2.4E+01 cp/mL |
| US NY (B.1.526) | Inactivated Virus | 1.9E+02 cp/mL |
| India (B.1.617.1) | Inactivated Virus | 2.5E+02 cp/mL |
| India (B.1.617.2) | Inactivated Virus | 7.0E+01 cp/mL |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 16 of 20

{16}

In silico analysis of additional SARS-CoV-2 sequences indicates that &gt;99.9% of known sequences for SARS-CoV-2 have no changes in primer/probe binding sites at both target regions simultaneously. All sequences are predicted to be detected by at least one of the two target regions.

In silico Analysis of cobas SARS-CoV-2 Test Oligo Design as of October 15, 2022

| Assay | Orf1ab (Target 1) | | | | E-gene (Target 2) | | | | Orf1ab & E-gene (Dual Targets) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Database | NCBI | | GISAID | | NCBI | | GISAID | | NCBI | | GISAID | |
| total | 6113594 | 100% | 12695489 | 100% | 6113594 | 100% | 12695489 | 100% | 6113594 | 100% | 12695489 | 100% |
| With mismatch | 251910 | 4.12% | 464658 | 3.66% | 32124 | 0.53% | 83852 | 0.66% | 1125 | 0.02% | 2971 | 0.02% |
| dCp>5 or Tm<65 | 211 | 0.00% | 493 | 0.00% | 34 | 0.00% | 1025 | 0.01% | 0 | 0.00% | 3 | 0.00% |

In Silico Analysis of SARS-CoV-2 Variants of Concern (VOC) as of October 10, 2022

| WHO label | Status | Pango Lineage | Nextstrain Clade | cobas SARS CoV-2 for use on the cobas 6800/8800 Systems |
| --- | --- | --- | --- | --- |
| Alpha | de-escalated | B.1.1.7 | 20I/501Y.V1 | detected |
| Beta | de-escalated | B.1.351 | 20H/501Y.V2 | detected |
| Gamma | de-escalated | P.1 | 20J/501Y.V3 | detected |
| Delta | Active VOC | B.1.617.2 | 21A,I,J | detected |
| Epsilon | de-escalated | B.1.427/B.1.429 | 20C/S:452R | detected |
| Zeta | de-escalated | P.2 | 20J | detected |
| Eta | de-escalated | B.1.525 | 20A/S:484K | detected |
| Theta | de-escalated | P.3 | 21E | detected |
| Iota | de-escalated | B.1.526 | 20C/S:484K | detected |
| Kappa | de-escalated | B.1.617.1 | 20A/S:154K | detected |
| Lambda | de-escalated | C.37 | 20D | detected |
| Mu | de-escalated | B.1.621 | 21H | detected |
| Omicron (BA.1 - BA.5, BQ.1, BQ.1.1, BF.7) | Active VOC | B.1.1.529 | 21K,L,M 22A,B,C,D,E | detected |

10. Assay Cut-Off:

The assay cut-off for this device was established based on variety of parameter thresholds taken together by the algorithm to make the qualitative decision whether a curve is to be considered positive or negative. These parameters (e.g., Ct, max. RFI, max. slope, sigmoidality of curve shape) are initially set by the algorithm. As the product development progresses, the data is annotated, and the algorithm is adjusted ("trained") accordingly. This iterative process in the end leads to a final set of parameter cut-offs. Selected data from testing of clinical specimens or cultures spiked into clinical background were used to confirm adequate separation between the latest Ct value observed in positive specimens in each target detection channel and the assay cut-off.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 17 of 20

{17}

11. Carry-Over:

Carry-over/cross contamination rate for cobas SARS-CoV-2 Qualitative assay was evaluated by testing SARS-CoV-2 high-positive samples and negative samples in a checkerboard configuration. Five runs, with each run containing 48 replicates of SARS-CoV-2 negative samples and 46 replicates of high positive samples were performed. A total of 230 SARS-CoV-2 high positive samples and 239 negative samples were tested. No cross-contamination was observed, as described in the table below.

Cross Contamination Run Results

| Run Number | Total Positive Results from Negative Samples | Total Samples | | | Cross Contamination Rate |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Negative | Controls | |
| Run 1 | 0 | 46 | 47 | 2 | 0.0 % |
| Run 2 | 0 | 46 | 48 | 2 | 0.0 % |
| Run 3 | 0 | 46 | 48 | 2 | 0.0 % |
| Run 4 | 0 | 46 | 48 | 2 | 0.0 % |
| Run 5 | 0 | 46 | 48 | 2 | 0.0 % |
| Total | 0 | 230 | 239 | 10 | 0.0 %
(upper one-sided 95% CI of 1.25%) |

B Comparison Studies:

1. Method Comparison with Predicate Device:

Not applicable. Refer to the Clinical Studies Section of this document.

C Clinical Studies:

Clinical Study Design

The performance of cobas SARS-CoV-2 Qualitative was evaluated in a multi-center study with three external testing sites evaluating prospectively collected clinical specimens in UTM-RT or UVT from individuals with signs and symptoms of respiratory infection. Participants from 12 geographically distributed enrollment centers each provided nasopharyngeal swab (NPS) and nasal swab (NS, anterior nares) specimens as part of a dual collection where (a) the collection order (first specimen collected) was alternated between the NPS and NS specimen, and (b) the collection method for NS specimens was also alternated with 50% of the NS specimens were self-collected on-site with healthcare provider (HCP) instructions while the other 50% were collected by the healthcare provider. The study used a composite comparator method wherein laboratory sites used up to three highly sensitive EUA SARS-CoV-2 molecular assays, testing NPS specimen from each subject. The composite comparator result was defined as the concordant results from two comparator assays (test A and test B). In case of discordance between the initial two comparator assays, the sample was tested by a third assay (test C) and the result of the third test determined the composite comparator result. The composite comparator

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 18 of 20

{18}

result was indetermined when valid results could not be obtained from two assays (i.e., insufficient volume for repeat testing of invalid/failed results).

From March to June 2021, a total of 1,154 participants were enrolled, of which samples from 968 participants were included in the evaluation. Samples from 186 participants were not included: 184 specimens were excluded due to issues associated with specimen shipments and/or being unable to complete testing within the times identified by manufacturer's instructions, and two subjects were excluded for being previously enrolled in the study (exclusion criteria). When self-reporting COVID-19 vaccination status, 207 (21.4%) of the 968 participants were fully vaccinated.

Of the 968 participants, 961 contributed a NPS specimen which resulted in 942 participants with a confirmed infected status. For NPS, 4 specimens had failed/invalid cobas SARS-CoV-2 Qualitative results, resulting in 938 evaluable NPS results. For NS, 8 specimens were invalid/missing cobas SARS-CoV-2 Qualitative NS results, resulting in 934 evaluable results.

When compared with the NPS composite comparator result, cobas SARS-CoV-2 Qualitative yielded a positive percent agreement (PPA) of 98.7% for NPS and 97.4% for NS specimens. The negative percent agreement (NPA) was 99.7% and 99.9% for NPS and NS specimens, respectively.

Summary of clinical performance of cobas SARS-CoV-2 Qualitative for nasopharyngeal (NPS) and nasal swabs (NS) versus the NPS composite comparator

| Specimen Type | Total (N) | PPA | PPA 2-sided 95% Score CI | NPA | NPA 2-sided 95% Score CI |
| --- | --- | --- | --- | --- | --- |
| Nasopharyngeal (NPS) | 938 | 98.7% (77/78) | (93.1 %, 99.8 %) | 99.7% (857/860) | (99.0 %, 99.9 %) |
| Nasal Swab (NS)* | 934 | 97.4% (76/78) | (91.1 %, 99.3 %) | 99.9% (855/856) | (99.3 %, 100 %) |

*Healthcare provider-collected nasal swab specimens and nasal swab specimens self-collected on-site with healthcare provider instructions

D Clinical Cut-Off:

Not applicable

E Expected Values/Reference Range:

The SARS-CoV-2 positivity determined by the cobas SARS-CoV-2 Qualitative is presented by enrollment site in table below for both NPS and NS specimens. Across the 12 enrollment sites from 8 states, the SARS-CoV-2 positivity ranged from 0% to 16.2% for NPS and from 0% to 15.8% for NS.

SARS-CoV-2 Prevalence by Collection Site based on cobas SARS-CoV-2 Qualitative

| Site location | NPS results | NPS Prevalence | NS results | NS Prevalence |
| --- | --- | --- | --- | --- |
| Oakland, CA | 114 | 0.0% (0/114) | 117 | 0.0% (0/117) |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 19 of 20

{19}

| Site location | NPS results | NPS Prevalence | NS results | NS Prevalence |
| --- | --- | --- | --- | --- |
| St.Louis, MO | 117 | 11.1% (13/117) | 117 | 10.3% (12/117) |
| Syracuse, NY | 11 | 9.1% (1/11) | 11 | 9.1% (1/11) |
| Bronx, NY | 64 | 7.8% (5/64) | 64 | 9.4% (6/64) |
| Nashville, TN | 92 | 12.0% (11/92) | 93 | 11.8% (11/93) |
| Easley, SC | 127 | 13.4% (17/127) | 128 | 12.5% (16/128) |
| Powdersville, SC | 117 | 16.2% (19/117) | 114 | 15.8% (18/114) |
| Dallas, TX | 147 | 6.8% (10/147) | 147 | 6.8% (10/147) |
| Dallas, TX | 31 | 0.0% (0/31) | 31 | 0.0% (0/31) |
| New Orleans, LA | 46 | 4.3% (2/46) | 46 | 2.2% (1/46) |
| Statesville, NC | 33 | 6.1% (2/33) | 34 | 5.9% (2/34) |
| Moorseville, NC | 42 | 0.0% (0/42) | 42 | 0.0% (0/42) |
| ALL | 941 | 8.5% (80/941) | 944 | 8.2% (77/944) |

Note: The table included all specimens with valid SARS-CoV-2 results on cobas SARS-CoV-2.
A positive SARS-CoV-2 is defined based on the cobas SARS-CoV-2.
NPS = nasopharyngeal swab specimen; NS = nasal swab specimen.

F Other Supportive Instrument Performance Characteristics Data:

Not applicable

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.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213804 - Page 20 of 20