← Product Code [QOF](/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF) · K220963

# Simplexa COVID-19 & Flu A/B Direct (K220963)

_Diasorin Molecular, LLC · QOF · Mar 17, 2023 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K220963

## Device Facts

- **Applicant:** Diasorin Molecular, LLC
- **Product Code:** [QOF](/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF.md)
- **Decision Date:** Mar 17, 2023
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3981
- **Device Class:** Class 2
- **Review Panel:** Microbiology

## Indications for Use

The DiaSorin Molecular Simplexa™ COVID-19 & Flu A/B Direct is a real-time RT-PCR assay intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus and influenza B virus in nasopharyngeal swabs (NPS) from individuals with signs and symptoms of respiratory tract infection. The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A and influenza B infection. Negative results do not preclude SARS-CoV-2, influenza B infection and should not be used as the sole basis for patient management decisions. Positive results do not rule out coinfection with other organisms. Results should be combined with clinical observations, patient history, and epidemiological information. The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.

## Device Story

The Simplexa™ COVID-19 & Flu A/B Direct is a real-time RT-PCR assay for use on the LIAISON® MDX instrument. It processes unprocessed nasopharyngeal swabs (NPS) without prior nucleic acid extraction. The system uses fluorescent probes and primers to amplify and detect SARS-CoV-2 (S and ORF1ab genes), influenza A (matrix gene), and influenza B (matrix gene) RNA, alongside an internal RNA control. The device is operated by trained clinical laboratory personnel in a laboratory setting. The LIAISON® MDX instrument performs the amplification and detection, providing qualitative results for the three targets. These results aid clinicians in the differential diagnosis of respiratory infections, helping to guide patient management and infection control decisions. The device benefits patients by providing rapid, simultaneous detection of multiple respiratory pathogens from a single specimen.

## Clinical Evidence

Clinical performance was evaluated using over 1400 prospective and archived NPS specimens. Prospective study (Aug 2021–Mar 2022) showed PPA of 91.9% (Flu A), 98.5% (SARS-CoV-2) and NPA of 99.8% (Flu A), 100% (Flu B), 97.4% (SARS-CoV-2). Retrospective study showed PPA of 97.6% (Flu A), 98.2% (Flu B) and NPA of 100% for all targets. Comparators included FDA-cleared NAATs and a composite reference method for SARS-CoV-2.

## Technological Characteristics

Real-time RT-PCR assay; utilizes LIAISON® MDX instrument and Direct Amplification Disc. Targets: SARS-CoV-2 (S and ORF1ab genes), Flu A (matrix gene), Flu B (matrix gene). Employs fluorescent probes (TaqMan technology). Connectivity: LIAISON® MDX Studio Software. No nucleic acid extraction required.

## Regulatory 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](/device/DEN200031.md))

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

{0}

FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K220963

B Applicant

DiaSorin Molecular LLC

C Proprietary and Established Names

Simplexa COVID-19 & Flu A/B Direct

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  |
|  OOI | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry  |

## II Submission/Device Overview:

A Purpose for Submission:

This submission is a Traditional 510(k) to obtain clearance for a new device, the Simplexa COVID-19 & Flu A/B Direct.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

{1}

B Measurand:

Viral RNA from Influenza A (Flu A), Influenza B (Flu B), and SARS-CoV-2

C Type of Test:

Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) system, qualitative

III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

The DiaSorin Molecular Simplexa COVID-19 & Flu A/B Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus and influenza B virus in nasopharyngeal swabs (NPS) from individuals with signs and symptoms of respiratory tract infection.

The Simplexa COVID-19 & Flu A/B Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A and influenza B infection. Negative results do not preclude SARS-CoV-2, influenza A or influenza B infection and should not be used as the sole basis for patient management decisions. Positive results do not rule out coinfections with other organisms. Results should be combined with clinical observations, patient history, and epidemiological information.

The Simplexa COVID-19 & Flu A/B Direct assay is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.

C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

D Special Instrument Requirements:

LIAISON MDX instrument with LIAISON MDX Studio Software

IV Device/System Characteristics:

A Device Description:

The system consists of the Simplexa COVID-19 & Flu A/B assay reagents (supplied in quantity for 24 reactions), the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories. The LIAISON MDX instrument is a real-time Polymerase Chain Reaction (PCR) thermocycler used for the identification of nucleic acid from biological specimens. The Direct Amplification Disc (DAD) is compartmentalized into eight separate wedges and up to eight separate specimens or controls may be processed on each disc.

K220963 - Page 2 of 24

{2}

Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow, and a reaction chamber. The user adds 50 μL of Reaction Mix to the reagent input well and 50 μL of unextracted specimen to the sample input well. The reverse transcription, amplification and detection are performed automatically by the instrument.

## B Principle of Operation:

The Simplexa COVID-19 & Flu A/B Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of SARS-CoV-2 RNA, human influenza A (Flu A) virus RNA and human influenza B (Flu B) virus RNA from unprocessed nasopharyngeal swabs (NPS) that have not undergone nucleic acid extraction. The system consists of the Simplexa COVID-19 & Flu A/B Direct assay, the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa COVID-19 & Flu A/B Direct assay, fluorescent probes are used together with corresponding forward and reverse primers to amplify SARS-CoV-2, Flu A, Flu B and internal control RNA targets. For COVID-19 detection, the assay targets two different regions specific to the SARS-CoV-2 genome; the S gene which encodes the spike glycoprotein and the ORF1ab region which encodes wellconserved non-structural proteins and therefore is less susceptible to recombination. For Flu detection the assay targets conserved regions of influenza A viruses (matrix gene) and influenza B viruses (matrix gene). The assay provides three results; COVID-19 (ORF1ab and/or S gene detection), influenza A viruses (matrix gene detection) and influenza B viruses (matrix gene detection). An RNA internal control is used to detect RT-PCR failure and/or inhibition.

## C Instrument Description Information:

1. Instrument Name:
Liaison MDX System

2. Specimen Identification:
Barcode scanner or manual entry

3. Specimen Sampling and Handling:
Simplexa COVID-19 & Flu A/B Direct is a no extraction RT-PCR assay. The operator adds 50 μL of unextracted sample to the sample input well and adds 50 μL of the reaction mix to the reagent input well in the direct amplification disc (DAD).

4. Calibration:
Each reagent kit comes with a barcode card, which contains assay specific parameters and lot information. The barcode card is scanned prior to each run.

K220963 - Page 3 of 24

{3}

K220963 - Page 4 of 24

5. Quality Control:

The Simplexa COVID-19 & Flu A/B Positive Control Pack may be used as an external positive control. Universal transport media (UTM) may be used as a negative external control (No Template Control).

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):

Table 1. Comparison of Simplexa COVID-19 & Flu A/B Direct with Predicate

|  Device & Predicate Device(s): | K220963 | DEN200031  |
| --- | --- | --- |
|  Device Trade Name | Simplexa COVID-19 & Flu A/B Direct | BioFire Respiratory Panel 2.1  |
|  General Device Characteristic Similarities |  |   |
|  Product Code | QOF | QOF  |
|  Regulation Number | 21 CFR 866.3981 | 21 CFR 866.3981  |
|  Intended Use/Indications For Use | The DiaSorin Molecular Simplexa COVID-19 & Flu A/B Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus and influenza B virus in nasopharyngeal swabs (NPS) from individuals with signs and symptoms of respiratory tract infection.
The Simplexa COVID-19 & Flu A/B Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A and influenza B infection. Negative results do not preclude SARS-CoV-2, influenza A or influenza B infection and should not be used as the sole basis for | The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire FilmArray Torch systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19. The following organism types and subtypes are identified using the BioFire RP2.1: Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus (SARS CoV-2), Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, including  |

{4}

K220963 - Page 5 of 24
|   | patient management decisions. Positive results do not rule out coinfection with other organisms. Results should be combined with clinical observations, patient history, and epidemiological information.

The Simplexa COVID-19 & Flu A/B Positive control Pack intended to be used as a control with the Simplexa COVID-19 & Flu A/B Direct kit for use on the LIAISON MDX instrument. This control is not intended for use with other assays or systems. | 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 Pneumoniae

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  |
| --- | --- | --- |

{5}

K220963 - Page 6 of 24
|   |  | necessary when evaluating a patient with possible respiratory tract infection.  |
| --- | --- | --- |
|  Sample to answer system | Automated | Automated  |
|  Specimen Types | Nasopharyngeal Swab | Nasopharyngeal Swab  |
|  **General Device Characteristic Differences** |  |   |
|  Device Technology | RT-PCR using Direct Amplification Disc (DAD) moves reagents through reaction chambers using laser-operated valve opening and centrifugal forces | RT-PCR using disposable pouch module with blister packs to separate chemical steps and move reagents via mechanical force  |
|  Organisms Detected | SARS-CoV-2
Influenza A
Influenza B | SARS-CoV-2
Influenza A
Influenza B
Adenovirus
Coronavirus 229E
Coronavirus HKU1
Coronavirus NL63
Coronavirus OC43
Human Metapneumovirus
Human Rhinovirus/Enterovirus
Parainfluenza Virus 1
Parainfluenza Virus 2
Parainfluenza Virus 3
Parainfluenza Virus 4
Respiratory Syncytial Virus
Bordetella parapertussis
Bordetella pertussis
Chlamydia pneumoniae
Mycoplasma pneumoniae  |
|  Measurand | RNA from SARS-CoV-2, Influenza A, Influenza B | Nucleic acid from SARS-CoV-2, Influenza A, Influenza B, Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae  |
|  Instrument | LIAISON MDX | BioFire FilmArray 2.0 or BioFire FilmArray Torch  |

{6}

VI Standards/Guidance Documents Referenced:

Class II Special Controls as per 21 CFR 866.3981

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Analytical Reactivity:

The analytical reactivity of the Simplexa COVID-19 & A/B Direct with various strains of influenza A, influenza B and SARS-CoV-2, was evaluated by testing dilutions of quantified virus stocks prepared by spiking into pooled negative nasopharyngeal swab matrix (swabs collected in UTM) at concentrations near the LoD. A total of 63 Flu A strains, 21 Flu B strains and five SARS-CoV-2 strains were tested in three replicates. Samples that were not detected in all three replicates at the initially contrived concentrations were retested at a higher concentration. Tables 2, 3, and 4 below show the strains that were positive in all three replicates at the concentrations shown.

Table 2. Inclusivity for Simplexa COVID-19 & Flu A/B Direct – Influenza A

|  Subtype | Influenza A Strain | Tested Concentration | # Detected / # Tested  |
| --- | --- | --- | --- |
|  H7N9 | A/Anhui/1/2013 | 1:100,000 dilution | 3/3  |
|  H1N9 | A/American green-winged teal/Mississippi/300/2010 | 100 CEID_{50}/mL | 3/3  |
|  H13N6 | A/black-legged kittiwake/Quebec/02838-1/2009 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 pdm09 | A/Brisbane/02/2018 | 100 EID_{50}/mL | 3/3  |
|  H3N2 | A/Brisbane/10/07 | 100 TCID_{50}/mL | 3/3  |
|  H1N1 | A/Brisbane/59/07 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/California/02/2014 | 100 TCID_{50}/mL | 3/3  |
|  H1N1 pdm09 | A/California/4/2009 | 100 TCID_{50}/mL | 3/3  |
|  H10N7 | A/chicken/Germany/N/49 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 pdm09 | A/Christ Church/16/2010 | 100 EID_{50}/mL | 2/3  |
|   |   |  1000 EID_{50}/mL | 3/3  |
|  H3N8 | A/duck/Chabarovsk/1610/1972 | 100 CEID_{50}/mL | 3/3  |
|  H4N6 | A/duck/Czechoslovakia/1956 | 100 CEID_{50}/mL | 0/3  |
|   |   |  5000 CEID_{50}/mL | 3/3  |
|  H12N6 | A/duck/Wisconsin/480/1979 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 pdm09 | A/Guangdong-Maonan/1536/2019 | 100 EID_{50}/mL | 3/3  |
|  H1N1 | A/Hawaii/15/2001 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Hong Kong/267/2019 | 100 EID_{50}/mL | 3/3  |
|  H9N2 | A/Hong Kong/33982/2009(H9N2)-PR8-IDCDC_RG26 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Kansas/14/2017 | 100 EID_{50}/mL | 3/3  |
|  H10N7 | A/mallard/Illinois/10OS4334/2010 | 100 CEID_{50}/mL | 3/3  |
|  H12N5 | A/mallard/Wisconsin/4218/2009 | 100 CEID_{50}/mL | 3/3  |
|  H10N1 | A/mallard/Wisconsin/4230/2009 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 pdm09 | A/Massachusetts/15/2013 | 100 CEID_{50}/mL | 3/3  |

K220963 - Page 7 of 24

{7}

K220963 - Page 8 of 24
|  H1N1
pdm09 | A/Mexico/4108/2009 | 100 CEID_{50}/mL | 3/3  |
| --- | --- | --- | --- |
|  H1N2 | A/Minnesota/19/2011 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 | A/New Caledonia/20/99 | 100 TCID_{50}/mL | 3/3  |
|  H1N1
pdm09 | A/New York/18/2009 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/New York/55/2004 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Perth/16/2009 | 100 EID_{50}/mL | 3/3  |
|  H10N8 | A/quail/Italy/1117/1965 | 100 CEID_{50}/mL | 3/3  |
|  H1N8 | A/red knot/Delaware Bay/240/1994 | 100 CEID_{50}/mL | 3/3  |
|  H4N6 | A/red knot/Delaware/541/1988 | 100 CEID_{50}/mL | 1/3  |
|   |  | 1000 CEID_{50}/mL | 3/3  |
|  H3N6 | A/redhead/Alberta/192/2002 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Rhode Island/01/2010 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Santiago/7981/2006 | 100 CEID_{50}/mL | 3/3  |
|  H1N3 | A/shorebird/Delaware Bay/211/1994 | 100 CEID_{50}/mL | 2/3  |
|   |  | 1000 CEID_{50}/mL | 3/3  |
|  H16N3 | A/shorebird/Delaware/172/2006 | 100 CEID_{50}/mL | 2/3  |
|   |  | 1000 CEID_{50}/mL | 3/3  |
|  H1N1 | A/Solomon Island/3/2006 | 100 TCID_{50}/mL | 3/3  |
|  H1N1 | A/Swine/1976/31 | 100 TCID_{50}/mL | 3/3  |
|  H1N1 | A/Swine/Iowa/15/30 | 100 TCID_{50}/mL | 3/3  |
|  H1N2 | A/swine/Ohio/09SW1477/2009 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/swine/Ohio/09SW83E/2009 | 100 CEID_{50}/mL | 3/3  |
|  H1N1 | A/Taiwan/42/06 | 100 TCID_{50}/mL | 3/3  |
|  H6N2 | A/turkey/Massachusetts/3740/1965 | 100 CEID_{50}/mL | 1/3  |
|   |  | 2000 CEID_{50}/mL | 3/3  |
|  H1N1 | A/WS/33 | 100 TCID_{50}/mL | 3/3  |
|  H1N1
pdm09 | A/California/7/2009 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/Hong Kong/4801/2014 | 100 TCID_{50}/mL | 3/3  |
|  H3N2v | A/Indiana/08/2011 | 100 TCID_{50}/mL | 3/3  |
|  H3N2v | A/Minnesota/11/2010 | 100 CEID_{50}/mL | 3/3  |
|  H1N1
pdm09 | A/NY/02/09 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/Ohio/02/2012 | 100 CEID_{50}/mL | 3/3  |
|  H3N2 | A/Port Chalmers/1/1973 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/Singapore/INFIMH-16-0019/2016 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/Texas/50/2012 | 100 TCID_{50}/mL | 3/3  |
|  H3N2 | A/Wisconsin/67/05 | 100 TCID_{50}/mL | 3/3  |
|  H1N1 | A/PR/8/34 | 100 TCID_{50}/mL | 3/3  |
|  H7N9 | A/Anhui/1/2013 | 1:100,000 dilution | 3/3  |
|  H5N1 | A/chicken/Vietnam/NCVD-016/2008(H5N1)-PR8-IDCDC-RG12 | 1:100,000 dilution | 3/3  |
|  H5N1 | A/Egypt/N03072/2010(H5N1)-PR8-IDCDC-RG29 | 1:100,000 dilution | 3/3  |
|  H5N1 | A/Hubei/1/2010(H5N1)-PR8-IDCDC-RG30 | 1:100,000 dilution | 3/3  |
|  H5N1 | A/India/NIV/2006(H5N1)-PR8-IBCDC-RG7 | 1:100,000 dilution | 3/3  |
|  H7N7 | A/mallard/Netherlands/12/2000(H7N7)/PR8-IBCDC-1 | 1:100,000 dilution | 3/3  |
|  H5N2 | A/pheasant/New Jersey/1355/1998(H5N2)-PR8-IBCDC-4 | 1:100,000 dilution | 3/3  |

{8}

Table 3. Inclusivity for Simplexa COVID-19 & Flu A/B Direct – Influenza B

|  Lineage | Influenza B Strain | Tested Concentration | # Detected / # Tested  |
| --- | --- | --- | --- |
|  Victoria | B/Brisbane/33/2008 | 100 CEID_{50}/mL | 3/3  |
|  Victoria | B/Brisbane/60/2008 | 100 TCID_{50}/mL | 3/3  |
|  Victoria | B/Colorado/06/2017 | 100 TCID_{50}/mL | 3/3  |
|  Victoria | B/Florida/02/2006 | 100 TCID_{50}/mL | 3/3  |
|  Victoria | B/Michigan/09/2011 | 100 EID_{50}/mL | 3/3  |
|  Victoria | B/Nevada/03/2011 | 100 CEID_{50}/mL | 3/3  |
|  Victoria | B/Texas/02/2013 | 100 TCID_{50}/mL | 3/3  |
|  Victoria | B/Victoria/304/2006 | 100 CEID_{50}/mL | 3/3  |
|  Victoria | B/Washington/02/2019 | 100 EID_{50}/mL | 3/3  |
|  Yamagata | B/ChristChurch/33/2004 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/Florida/04/2006 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/Florida/07/04 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/Guangdong-Liwan/1133/2014 | 100 CEID_{50}/mL | 1/3  |
|   |   |  1000 CEID_{50}/mL | 3/3  |
|  Yamagata | B/Maryland/1/59 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/Massachusetts/02/2012 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/New Hampshire/01/2016 | 100 EID_{50}/mL | 3/3  |
|  Yamagata | B/Panama/45/90 | 100 TCID_{50}/mL | 3/3  |
|  Yamagata | B/Texas/81/2016 | 100 EID_{50}/mL | 3/3  |
|  Yamagata | B/Utah/09/2014 | 100 CEID_{50}/mL | 3/3  |
|  Yamagata | B/Wisconsin/01/2010 | 100 CEID_{50}/mL | 3/3  |
|  Unknown | B/Great Lakes/1739/54 | 100 TCID_{50}/mL | 3/3  |

Table 4. Inclusivity for Simplexa COVID-19 & Flu A/B Direct – SARS-CoV-2

|  SARS-CoV-2 Strain | Tested Concentration | Flu A Detection # Detected / # Tested | Flu B Detection # Detected / # Tested | SARS-CoV-2 Detection # Detected / # Tested  |
| --- | --- | --- | --- | --- |
|  Hong Kong/VM200001061/2020 | 1000 copies/mL | 0/3 | 0/3 | 3/3  |
|  England/20480464/2020 | 1000 copies/mL | 0/3 | 0/3 | 3/3  |
|  South Africa/KRISP-EC-K005325/2020 | 1000 copies/mL | 0/3 | 0/3 | 3/3  |
|  Japan/TY7-503/2021 | 1000 copies/mL | 0/3 | 0/3 | 3/3  |
|  hCoV19/USA/PHC658/2021 | 1500 copies/mL | 0/3 | 0/3 | 3/3  |

Additional testing was performed using the CDC panels and results are shown below in Tables 5-8.

K220963 - Page 9 of 24

{9}

Table 5. 2018-2019 CDC Influenza Panel Strains Tested with Simplexa COVID-19 & Flu A/B Direct

|  Virus | Subtype | Organism  |
| --- | --- | --- |
|  Flu A | A
(H3N2) | A/Perth/16/2009  |
|   |   |  A/Singapore/INFIMH-16-0019/2016*  |
|   |  A
(H1N1) pdm09 | A/California/07/2009  |
|   |   |  A/Michigan/45/2015*  |
|  Flu B | B
(Victoria lineage) | B/Brisbane/60/2008  |
|   |   |  B/Colorado/06/2017*  |
|   |  B
(Yamagata lineage) | B/Wisconsin/01/2010  |
|   |   |  B/Phuket/3073/2013*  |

*WHO recommended vaccine strains

Table 6. 2019-2020 CDC Influenza Panel Strains Tested with Simplexa COVID-19 & Flu A/B Direct

|  Virus | Subtype | Organism  |
| --- | --- | --- |
|  Flu A | A
(H3N2) | A/Perth/16/2009  |
|   |   |  A/Kansas/14/2017*  |
|   |  A
(H1N1) pdm09 | A/Christ Church/16/2010  |
|   |   |  A/Brisbane/02/2018*  |
|  Flu B | B
(Victoria lineage) | B/Michigan/09/2011  |
|   |   |  B/Colorado/06/2017*  |
|   |  B
(Yamagata lineage) | B/New Hampshire/01/2016  |
|   |   |  B/Phuket/3073/2013*  |

*WHO recommended vaccine strains

Table 7. 2020-2021 CDC Influenza Panel Strains Tested with Simplexa COVID-19 & Flu A/B Direct

|  Virus | Subtype | Organism  |
| --- | --- | --- |
|  Flu A | A
(H3N2) | A/Perth/16/2009  |
|   |   |  A/Hong Kong/2671/2019*  |
|   |  A
(H1N1) pdm09 | A/Christ Church/16/2010  |
|   |   |  A/Guangdong-Maonan/1536/2019*  |
|  Flu B | B
(Victoria lineage) | B/Michigan/09/2011  |
|   |   |  B/Washington/02/2019*  |
|   |  B
(Yamagata lineage) | B/Texas/81/2016  |
|   |   |  B/Phuket/3073/2013*  |

*WHO recommended vaccine strains

K220963 - Page 10 of 24

{10}

Inclusivity (in silico)

An in silico inclusivity analysis of the assay oligo sequences for SARS-CoV-2 was performed against SARS-CoV-2 sequences available in the GISAID EpiCoV database submitted from May 01, 2022 to July 31, 2022, including sequences of the Omicron BA.2.12.1, BA.2.75, BA.4 and BA.5 subvariants. The analysis included 211, 224 sequences in the amplicon regions of the ORF1ab and S gene oligo sets. Only target sequences with full coverage of all three oligo-binding regions (forward primer, reverse primer, and probe) are included in the analyses for both oligo sets. In this sequence set, there are 208, 582 (~98.7%) sequences with no mismatches in the oligo binding regions for both genes, 2602 (~1.2%) sequences with no mismatches for one gene oligo set (either ORF1ab or S gene), and 40 (~0.02%) sequences with mismatches in at least one oligo binding region for both gene oligo sets. Based on in silico analysis of the percent homology between assay oligos and target sequences, potential impact of location of the mismatches on extension and/or binding, and the mismatch melt temperature (Tm) values of the oligo sequence to its binding region on each analyzed SARS-CoV-2 sequence, it is predicted that the assay will detect 100% of the 211,224 SARS-CoV-2 sequences, including sequences of all defined variants of concern or variants of interest, available in the GISAID EpiCoV database from May 01, 2022 to July 31, 2022.

An additional in silico inclusivity analysis of the oligonucleotide (oligo) sequences for the SARS-CoV-2 ORF1ab and S gene sets were performed against all SARS-CoV-2 sequences (Human host, Complete, High Coverage) submitted to the GISAID EpiCoV database from November 1, 2022 to February 8, 2023. Based on in silico inclusivity analysis of the SARS-CoV-2 oligo sequences of both gene oligo sets, it is predicted that 100% of analyzed sequences (n = 12,378) can be detected by the assay.

For Influenza A, in silico inclusivity analysis was performed using human host sequences available from the GISAID EpiFlu database and collected between November 1, 2022 and February 8, 2023. Based on in silico inclusivity analysis of the Influenza A oligos, it is predicted that the assay can detect ~99% (n = 9150) of all human host Influenza A sequences collected between November 1, 2022 and February 8, 2023.

2. Precision/Reproducibility:

The device was evaluated in a reproducibility study testing a panel of eight samples. The panel members were prepared by diluting viral stocks of influenza A, influenza B and SARS-CoV-2 strains into pooled negative clinical matrix (NP swabs collected in UTM). The panel included a Low Positive (2x LoD) and a Moderate Positive (4x LoD) level for each virus; a Positive Control and a negative sample consisting of UTM (no-template control) were also included.

K220963 - Page 11 of 24

{11}

Table 8. Reproducibility Sample Panel for Simplexa COVID-19 & Flu A/B Direct

|  Panel Member or Strain | Panel Relative Testing Level | Target LoD Level | Concentration (copies/mL)  |
| --- | --- | --- | --- |
|  2019-nCoV/USA-WA1/2020 | Low Positive | 2x | 1000  |
|  2019-nCoV/USA-WA1/2020 | Moderate Positive | 4x | 2000  |
|  Influenza A/Hong Kong/8/68 | Low Positive | 2x | 1000  |
|  Influenza A/Hong Kong/8/68 | Moderate Positive | 4x | 2000  |
|  Influenza B/Malaysia/2506/04 | Low Positive | 2x | 500  |
|  Influenza B/Malaysia/2506/04 | Moderate Positive | 4x | 1000  |
|  UTM as NTC | Negative | N/A | N/A  |
|  PC | Positive | N/A | N/A  |

UTM = Universal Transport Media, NTC = No Template Control, PC = Positive Control/ N/A = Not applicable

The study was conducted at two external and one internal testing sites with each site having two designated LIAISON MDX instruments and two dedicated operators. Each of the two operators used a unique lot of the reaction mix (for a total of two lots). Each panel member was tested in three replicates, in two runs per day, over five days, at three locations. A total of 90 measurements (3 replicates x 2 runs/day x 5 days x 3 sites) were generated for each panel member. The study design allowed for evaluation of multiple components of variance. The data was assessed for (a) qualitative results, i.e., percent agreement with expected results, and (b) quantitative analysis of variance components, i.e., average Ct values and calculated SD and %CV.

Table 9. Reproducibility Qualitative Results

|  Panel Member | Site 1 | Site 2 | Site 3 | All Sites | 95% CI  |
| --- | --- | --- | --- | --- | --- |
|   |  Agreement with Expected Results | Agreement with Expected Results | Agreement with Expected Results | Agreement with Expected Results  |   |
|  Flu A Hong Kong - LP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  Flu A Hong Kong - MP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  Flu B Malaysia - LP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  Flu B Malaysia - MP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  2019-nCoV LP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  2019-nCoV MP | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  PC Flu A | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  PC Flu B | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  PC nCoV | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  NTC Flu A | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  NTC Flu B | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |
|  NTC nCoV | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 95.9%-100.0%  |

LP = Low Positive, MP = Moderate Positive, nCoV = SARS-CoV-2, UTM = Universal Transport Media, NTC = No Template Control, PC = Positive Control/ N/A = Not applicable

K220963 - Page 12 of 24

{12}

Table 10. Reproducibility Variance Results by Site

|  Panel Member | Site 1 |   | Site 2 |   | Site 3 |   | All Sites  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Avg. Ct | % CV | Avg. Ct | % CV | Avg. Ct | % CV | Avg. Ct | % CV | 95% CI  |
|  Flu A Hong Kong - LP | 32.3 | 1.7% | 32.9 | 3.7% | 32.8 | 1.7% | 32.7 | 2.6% | 95.9-100.0%  |
|  Flu A Hong Kong - MP | 31.7 | 1.5% | 31.5 | 1.4% | 31.9 | 1.4% | 31.7 | 1.5% | 95.9-100.0%  |
|  Flu B Malaysia - LP | 30.8 | 2.2% | 30.9 | 1.9% | 31.5 | 2.0% | 31.1 | 2.2% | 95.9-100.0%  |
|  Flu B Malaysia - MP | 30.0 | 1.2% | 29.9 | 1.5% | 30.4 | 1.6% | 30.1 | 1.6% | 95.9-100.0%  |
|  2019-nCoV LP | 30.2 | 1.4% | 29.5 | 1.4% | 30.2 | 2.2% | 30.0 | 2.0% | 95.9-100.0%  |
|  2019-nCoV MP | 29.0 | 1.6% | 28.8 | 1.6% | 29.4 | 2.1% | 29.1 | 1.9% | 95.9-100.0%  |
|  PC Flu A | 26.2 | 1.2% | 26.1 | 0.8% | 26.0 | 0.8% | 26.1 | 1.0% | 95.9-100.0%  |
|  PC Flu B | 27.6 | 3.2% | 28.2 | 1.4% | 27.1 | 0.8% | 27.6 | 2.6% | 95.9-100.0%  |
|  PC nCoV | 27.8 | 4.2% | 27.2 | 0.9% | 26.8 | 0.8% | 27.3 | 2.9% | 95.9-100.0%  |
|  NTC Flu A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 95.9-100.0%  |
|  NTC Flu B | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 95.9-100.0%  |
|  NTC nCoV | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 95.9-100.0%  |

LP = Low Positve, MP = Moderate Positive, nCoV = SARS-CoV-2, NTC = No Template Control, PC = Positive Control/ N/A = Not applicable

Table 11. Reproducibility Variance Components

|  Panel Member | N | Mean Ct | Between Day |   | Between Operator* |   | Between Site |   | Within Day |   | Total Reproducibility  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV  |
|  Flu A Hong Kong - LP | 90 | 32.7 | 0.24 | 0.7 | 0.00 | 0.0 | 0.00 | 0.0 | 0.83 | 2.5 | 0.87 | 2.7  |
|  Flu A Hong Kong - MP | 90 | 31.7 | 0.13 | 0.4 | 0.13 | 0.4 | 0.00 | 0.0 | 0.44 | 1.4 | 0.48 | 1.5  |
|  Flu B Malaysia - LP | 90 | 31.1 | 0.31 | 1.0 | 0.21 | 0.7 | 0.29 | 0.9 | 0.55 | 1.8 | 0.73 | 2.3  |
|  Flu B Malaysia - MP | 90 | 30.1 | 0.26 | 0.9 | 0.00 | 0.0 | 0.17 | 0.6 | 0.40 | 1.3 | 0.50 | 1.7  |
|  2019-nCoV LP | 90 | 30.0 | 0.37 | 1.2 | 0.00 | 0.0 | 0.13 | 0.4 | 0.49 | 1.6 | 0.63 | 2.1  |
|  2019-nCoV MP | 90 | 29.1 | 0.28 | 1.0 | 0.00 | 0.0 | 0.05 | 0.2 | 0.51 | 1.7 | 0.58 | 2.0  |
|  PC Flu A | 90 | 26.1 | 0.06 | 0.2 | 0.00 | 0.0 | 0.15 | 0.6 | 0.21 | 0.8 | 0.26 | 1.0  |
|  PC Flu B | 90 | 27.6 | 0.50 | 1.8 | 0.00 | 0.0 | 0.48 | 1.7 | 0.32 | 1.2 | 0.76 | 2.8  |
|  PC nCoV | 90 | 27.3 | 0.43 | 1.6 | 0.00 | 0.0 | 0.68 | 2.5 | 0.26 | 1.0 | 0.85 | 3.1  |

K220963 - Page 13 of 24

{13}

|  NTC Flu A | 90 | 0.0 | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  NTC Flu B | 90 | 0.0 | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A  |
|  NTC nCoV | 90 | 0.0 | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A  |

$\mathrm{LP} =$  Low Positive,  $\mathrm{MP} =$  Moderate Positive,  $\mathrm{nCoV} =$  SARS-CoV-2,  $\mathrm{NTC} =$  No Template Control,  $\mathrm{PC} =$  Positive Control/  $\mathrm{N / A} =$  Not applicable
* Since each operator used a different lot of reagents, Between Operator and Between Lot statistics are confounded variables.

# 3. Linearity:

Not applicable. This is a qualitative test.

# 4. Analytical Specificity/Interference:

Cross-reactivity

The Simplexa COVID-19 & Flu A/B Direct assay's analytical specificity was evaluated in a study testing samples containing organisms that are present as normal flora in nasopharyngeal passages and those that cause similar clinical symptoms as influenza A, influenza B and/or SARS-CoV-2. Forty-four different pathogens including bacteria, viruses and one fungus were included in the study, with each organism tested in three replicates. No cross reactivity was observed with the organisms at the concentrations tested, as shown below.

Table 12. Cross-reactivity for Simplexa COVID-19 & Flu A/B Direct

|  Organism | Test Concentration | Flu A % Detection (# positive/# tested) | Flu B % Detection (# positive/# tested) | SARS-CoV-2 % Detection (# positive/# tested) | IC % Detection (# positive/# tested)  |
| --- | --- | --- | --- | --- | --- |
|  Viruses  |   |   |   |   |   |
|  Adenovirus Type 1 | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Adenovirus Type 7a | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Cytomegalovirus | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Enterovirus Type 68 | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Enterovirus Type 71 | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Epstein-Barr Virus | 1 x 105copies/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Human Coronavirus 229E | 1 x 104TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Human Coronavirus NL63 | 1 x 104TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Human Coronavirus OC43 | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Human Coronavirus RNA HKU1 | 1 x 105genome copies/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Human Metapneumovirus 9 | 1 x 104TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Measles virus | 1 x 105TCID50/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |

K220963 - Page 14 of 24

{14}

K220963 - Page 15 of 24
|  MERS-Coronavirus | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
| --- | --- | --- | --- | --- | --- |
|  Mumps virus | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Parainfluenza Type 1 | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Parainfluenza Type 2 | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Parainfluenza Type 3 | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Parainfluenza Type 4 | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Parechovirus Type 3 | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Rhinovirus 1A | 1 x 10^{4} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  RSV-A | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  RSV-B | 1 x 10^{5} TCID_{50}/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Bacteria  |   |   |   |   |   |
|  Bordetella pertussis | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Chlamydia pneumoniae | 1 x 10^{6} IFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Corynebacterium diphtheriae | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Coxiella burnetii | 1 x 10^{6} copies/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Escherichia coli O157:H7 | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Haemophilus influenzae | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Lactobacillus plantarum | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Legionella longbeachae | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Legionella pneumophila | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Leptospira interrogans | 1 x 10^{6} copies/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Moraxella catarrhalis | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Mycobacterium tuberculosis DNA | 1 x 10^{6} copies/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Mycoplasma pneumoniae | 1 x 10^{6} CCU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Neisseria elongata | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Neisseria meningitidis | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Pseudomonas aeruginosa | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Staphylococcus aureus | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Staphylococcus epidermidis | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Streptococcus pneumoniae | 1 x 10^{6} CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |

{15}

|  Streptococcus pyogenes | 1 x 10^6 CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
| --- | --- | --- | --- | --- | --- |
|  Streptococcus salivarius | 1 x 10^6 CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |
|  Fungus  |   |   |   |   |   |
|  Candida albicans | 1 x 10^6 CFU/mL | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 100% (3/3)  |

$\mathrm{TCID}_{50} =$  Tissue Culture  $50\%$  Infectious Dose,  $\mathrm{CFU} =$  Colony-forming units,  $\mathrm{IFU} =$  Infectious units,  $\mathrm{CCU} =$  Color changing units

# Competitive Interference

The performance of the Simplexa COVID-19 & Flu A/B Direct assay was evaluated for its ability to accurately detect influenza A, influenza B, and SARS-CoV-2 in cases of co-infection with the assay's target organisms. Baseline samples were prepared by spiking COVID-19/USA-WA1/2020, Influenza A/Hong Kong/8/1968 or Influenza B/Phuket/3073/2013 at 4x LoD into nasopharyngeal swab (NPS) matrix in UTM. A high level (1,000x LoD) of each of the viruses was added to each of the baseline samples and tested. No interference was observed for the target analytes in the presence of potentially competing co-infection analytes at the concentrations and combinations tested.

Table 13. Competitive Interference of the Simplexa COVID-19 & Flu A/B Direct

|  Target Analyte | Interferent | Interferent Concentration | Flu A Detectiona | Flu A Mean Ct | Flu B Detection | Flu B Mean Ct | nCoV Detection | nCoV Mean Ct  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Flu A | None | N/A | 5/5 | 32.7 | 0/5 | N/A | 0/5 | N/A  |
|  Flu B | None | N/A | 0/5 | N/A | 5/5 | 31.0 | 0/5 | N/A  |
|  nCoV | None | N/A | 0/5 | N/A | 0/5 | N/A | 5/5 | 29.2  |
|  Flu A | Flu B | 7.5 x 10^5 | 3/3 | 32.5 | 3/3 | 22.5 | 0/3 | N/A  |
|  Flu A | nCoV | 5.0 x 10^5 | 3/3 | 32.7 | 0/3 | N/A | 3/3 | 21.6  |
|  Flu B | Flu A | 5.0 x 10^5 | 3/3 | 24.4 | 3/3 | 32.4 | 0/3 | N/A  |
|  Flu B | nCoV | 5.0 x 10^5 | 0/3 | N/A | 3/3 | 30.7 | 3/3 | 21.2  |
|  nCoV | Flu A | 5.0 x 10^5 | 3/3 | 24.2 | 0/3 | N/A | 3/3 | 29.4  |
|  nCoV | Flu B | 7.5 x 10^5 | 0/3 | N/A | 3/3 | 22.8 | 3/3 | 28.6  |

a "Detection" defined as # of positive tests / # tested nCoV = SARS-CoV-2, N/A = not applicable

# Microbial Interference

The performance of the Simplexa COVID-19 & Flu A/B Direct assay was evaluated for its ability to accurately detect influenza A, influenza B, and SARS-CoV-2 in the presence of other clinically relevant pathogens and related biological material. The same panel as above in the cross-reactivity study, consisting of forty-seven potentially inhibitory organisms, was individually spiked into a pool containing low concentrations (approximately 2x LoD) each of the influenza A, influenza B and SARS-CoV-2 viruses. Samples were assayed in three replicates. No inhibition was observed for influenza A, influenza B, or SARS-CoV-2 by the organisms at the concentrations shown below.

K220963 - Page 16 of 24

{16}

Table 14. Microbial Interference of the Simplexa COVID-19 & Flu A/B Direct

|  Organism | Test Concentration | Flu A % Detection (# positive/# tested) | Flu B % Detection (# positive/# tested) | SARS-CoV-2 % Detection (# positive/# tested) | IC % Detection (# positive/# tested)  |
| --- | --- | --- | --- | --- | --- |
|  Viruses  |   |   |   |   |   |
|  Adenovirus Type 1 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Adenovirus Type 7a | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Cytomegalovirus | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Enterovirus Type 68 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Enterovirus Type 71 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Epstein-Barr Virus | 1 x 105copies/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Human Coronavirus 229E | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Human Coronavirus NL63 | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Human Coronavirus OC43 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Human Coronavirus RNA HKU1 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Human Metapneumovirus 9 | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Measles virus | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  MERS-Coronavirus | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Mumps virus | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Parainfluenza Type 1 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Parainfluenza Type 2 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Parainfluenza Type 3 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Parainfluenza Type 4 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Parechovirus Type 3 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Rhinovirus 1A | 1 x 104TCID50/mL | 95% (19/20) | 100% (20/20) | 100% (20/20) | 100% (20/20)  |
|  RSV-A | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  RSV-B | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Bacteria  |   |   |   |   |   |
|  Bordetella pertussis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Chlamydia pneumoniae | 1 x 106IFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Corynebacterium diphtheriae | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |

K220963 - Page 17 of 24

{17}

|  Coxiella burnetii | 1 x 10^6 copies/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
| --- | --- | --- | --- | --- | --- |
|  Escherichia coli O157:H7 | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Haemophilus influenzae | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Lactobacillus plantarum | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Legionella longbeachae | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Legionella pneumophila | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Leptospira interrogans | 1 x 10^6 copies/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Moraxella catarrhalis | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Mycobacterium tuberculosis DNA | 1 x 10^6 copies/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Mycoplasma pneumoniae | 1 x 10^6 CCU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Neisseria elongata | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Neisseria meningitidis | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Pseudomonas aeruginosa | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Staphylococcus aureus | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Staphylococcus epidermidis | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Streptococcus pneumoniae | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Streptococcus pyogenes | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Streptococcus salivarius | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Fungus  |   |   |   |   |   |
|  Candida albicans | 1 x 10^6 CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Other Biological Material  |   |   |   |   |   |
|  Human genomic DNA | 1 x 10^6 cells/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  Pooled Human Nasal Wash | Neat (un-diluted) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |
|  SARS-CoV-1 Synthetic RNA | 1 x 10^5 genome copies/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3)  |

# Interfering Substances

The performance of the Simplexa COVID-19 & Flu A/B Direct assay was evaluated with potentially interfering substances that may be present in nasopharyngeal passages. A total of 12 potentially interfering substances were individually spiked into a pooled nasopharyngeal swab matrix containing SARS-CoV-2 (2019-nCoV/USA-WA1/2020), Flu A (Hong Kong/8/68) and Flu B (Malaysia/2506/04) inactivated viral particles, each at a targeted concentration approximately  $3\mathrm{x}$  LoD; each sample was tested in three replicates. There was one potential occurrence of interference with Cold Eeze oral analgesic at the higher concentration. All other substances showed no interference at the concentrations listed, including Cold Eeze oral

K220963 - Page 18 of 24

{18}

analgesic at the lower concentration. The FluMist nasal vaccine was not tested as an interfering substance due to its unavailability at the time of this study.

Table 15. Interference of Endogenous and Exogenous Substances on the Simplexa COVID-19 & Flu A/B Direct

|  Potential Interferent | Active Inredient | Test Concentration | Flu A detection | Flu B detection | nCoV detection | IC detection  |
| --- | --- | --- | --- | --- | --- | --- |
|  Afrin Nasal Spray | Oxymetazoline | 15% (v/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Antibacterial | Tobramycin | 4 μg/mL | 3/3 | 3/3 | 3/3 | 3/3  |
|  Nasal antibiotic | Mupirocin | 6.6 mg/mL | 3/3 | 3/3 | 3/3 | 3/3  |
|  Blood | N/A | 2% (v/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Bovine submaxillary gland, I-S | Purified Mucin Protein | 60 μg/mL | 3/3 | 3/3 | 3/3 | 3/3  |
|   |   |  5 mg/mL | 3/3 | 3/3 | 3/3 | 3/3  |
|  Cold Eeze oral analgesic | N/A | 2.5% (w/v) | 4/4 | 4/4 | 2/2* | 4/4  |
|   |   |  1.25% (w/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Nasal corticosteroid | Beclomethasone | 5% (w/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Nasal corticosteroid | Fluticasone | 5% (w/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Relenza | Zanamivir | 3.3 mg/mL | 3/3 | 3/3 | 3/3 | 3/3  |
|  Tamiflu | Oseltamivir | 1 μM | 3/3 | 3/3 | 3/3 | 3/3  |
|  Zicam Nasal Gel | Luffa opperculata, Galphimia glauca, histaminum hydrochloricum | 5% (w/v) | 3/3 | 3/3 | 3/3 | 3/3  |
|  Zicam Nasal Spray | N/A | 10% (v/v) | 3/3 | 3/3 | 3/3 | 3/3  |

nCoV = SARS-CoV-2, N/A = not applicable
* There was one error result for SARS-CoV-2 detection using Cold Eeze oral analgesic at the 2.5% concentration. The test was repeated and an error occurred again. Cold Eeze oral analgesic was retested at the 1.25% concentration and all analytes were detected as expected.

5. Assay Reportable Range:

Not applicable. This is a qualitative assay.

6. Traceability and Stability (Controls, Calibrators, or Methods):

Specimen Stability

For the specimen stability study, samples were contrived in individual negative samples as described for the Fresh vs. Frozen study (See below). The samples were tested, then stored at 2-8°C and tested after three days, after five days, after seven days, and after ten days. For this analysis, the results for the low positive samples (1.5x LoD and 2x LoD) were combined. Results for positive specimens for each target analyte are summarized in the table below:

K220963 - Page 19 of 24

{19}

Table 16. Specimen Stability for the Simplexa COVID-19 & Flu A/B Direct

|   |  | Timepoint  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Target | Sample Type | Fresh | 3 Days 8°C | 5 Days 8°C | 7 Days 8°C | 10 Days 8°C  |
|  N/A | Media | 0/10 | 0/10 | 0/10 | 0/10 | 0/10  |
|  Flu A | 5x LoD | 10/10 | 10/10 | 10/10 | 10/10 | 10/10  |
|   |  1.5x-2x LoD | 20/20 | 20/20 | 20/20 | 20/20 | 20/20  |
|  Flu B | 5x LoD | 10/10 | 10/10 | 10/10 | 10/10 | 10/10  |
|   |  1.5x-2x LoD | 18/20 | 18/20 | 19/20 | 19/20 | 20/20  |
|  SARS-CoV-2 | 5x LoD | 10/10 | 10/10 | 10/10 | 10/10 | 10/10  |
|   |  1.5x-2x LoD | 20/20 | 20/20 | 20/20 | 20/20 | 20/20  |

Although 90% detection was achieved for Influenza B after 3 days of storage, it was consistent with the results obtained on fresh samples and, therefore, was likely due to the concentration being close to the analytical LoD. Subsequent timepoints showed 95% to 100% detection for Influenza B. The package insert indicates that specimens can be stored for up to 7 days at 2-8 °C.

Fresh versus Frozen

The Simplexa COVID-19 & Flu A/B Direct assay was evaluated for the product's ability to detect low levels of SARS-CoV-2, influenza A and influenza B in nasopharyngeal swabs in UTM, which were stored below -70°C. Fresh vs frozen stability study showed that samples can be frozen for three days at or below -70°C for three freeze-thaw cycles.

Samples were stored at ≤ -70°C for at least twenty-four 24 hours before thawing and testing. Additional freeze/thaw timepoints were tested (i.e., thaws #2 and #3) with each test point being performed following at least a full 24-hour time frozen at -70°C between.

Table 17. Simplexa COVID-19 & Flu A/B Direct Fresh versus Frozen Equivalency

|  Target | Sample Type | Fresh | 1st Thaw | 2nd Thaw | 3rd Thaw  |
| --- | --- | --- | --- | --- | --- |
|  N/A | Media | 0/10 | 0/10 | 0/10 | 0/10  |
|  Flu A | 5x LoD | 10/10 | 10/10 | 10/10 | 10/10  |
|   |  1.5x-2x LoD | 20/20 | 20/20 | 20/20 | 19/20  |
|  Flu B | 5x LoD | 10/10 | 10/10 | 9/10a | 10/10  |
|   |  1.5x-2x LoD | 18/20b | 20/20 | 19/20 | 20/20  |
|  SARS-CoV-2 | 5x LoD | 10/10 | 10/10 | 10/10 | 10/10  |
|   |  1.5x-2x LoD | 20/20 | 20/20 | 20/20 | 18/20c  |

a Negative result was later retested and confirmed positive. 10/10 detections at the subsequent timepoint confirmed the frozen stability of that specimen type and concentration.
b Two samples tested negative, possibly due to virus loads being at or below LoD. Subsequent tests showed at least 95% detection for the three freeze/thaw timepoints.
c Two samples tested negative, possibly due to virus lots being at or below LoD. The experiment was repeated the next day after the 4th freeze thaw and obtained the expected results.

7. Detection Limit:

The Limit of Detection (LoD) study was performed using five LIAISON MDX Instruments, two lots of Reaction Mix and one lot of Positive Control. A total of 77 runs were performed by three operators over four days. The test samples were prepared by diluting viral stocks of influenza A, influenza B, and heat-inactivated SARS-CoV-2 into pooled nasopharyngeal swab (NPS) specimens collected in UTM. Two strains of each influenza virus and one strain of SARS-CoV-2

K220963 - Page 20 of 24

{20}

were tested. Each of the virus strains was tested at five or more concentrations near the expected LoD during the initial screening, followed by confirmation testing at one or more concentrations with two lots of the Reaction Mix, in 40 replicates. The LoD was defined as the concentration of the virus that resulted in at least 95% detection during confirmation testing for each of the virus strains. The confirmed LoD values are shown below.

Table 18. Simplexa COVID-19 & Flu A/B Direct Limit of Detection Summary

|  Simplexa COVID-19 & Flu A/B Direct Target | Strain | Limit of Detection | # Detected/# Tested | Mean Ct ±SD  |
| --- | --- | --- | --- | --- |
|  Flu A | Influenza A/Hong Kong/8/68 (H3N2) | 500 copies/mL | 40/40 | 34.0 ± 1.09  |
|   |  Influenza A/Michigan/45/2015 (H1N1) | 500 copies/mL | 39/40 | 33.1 ± 1.01  |
|  Flu B | Influenza B/Malaysia/2506/04 (Victoria) | 250 copies/mL | 39/40 | 32.0 ± 0.76  |
|   |  Influenza B/Phuket/3073/2013 (Yamagata) | 750 copies/mL | 39/40 | 31.9 ± 0.60  |
|  SARS-CoV-2 | COVID-19 (USA-WA1/2020) | 500 copies/mL | 40/40 | 30.8 ± 0.70  |
|  SARS-CoV-2 RNA | WHO International Standard | 651 IU/mL | 39/40 | 31.7 ± 1.40  |

8. Assay Cut-Off:

The fluorescent thresholds and assay Ct cutoffs remain unchanged from the settings determined in K201505 and K212147 for the Simplexa Flu A/B & RSV Direct Gen II and the Simplexa COVID-19 Direct, respectively.

9. Carry-Over:

Carry-over studies were performed as part of the analytical studies for K120413 and K201505 for the Simplexa Flu A/B & RSV Direct and Simplexa Flu A/B & RSV Direct Gen II.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Not applicable.

2. Matrix Comparison:

Not applicable.

C Clinical Studies:

1. Clinical Study Performance

The performance of Simplexa COVID-19 & Flu A/B Direct was evaluated in a clinical comparison study using prospective nasopharyngeal swab specimens, and retrospective

K220963 - Page 21 of 24

{21}

archived nasopharyngeal swab specimens from patients with signs and symptoms of respiratory tract infection.

Prospective specimens were collected from six (6) geographically diverse clinical sites within the United States between August 2021 and March 2022. There were a total of 1427 specimens enrolled for testing. Amplicon contamination at one testing site resulted in removal of 226 enrolled specimens. Total excluded samples were 257 which left 1170 prospective specimens suitable for testing. There were 1152/1170 valid results with the Simplexa COVID-19 & Flu A/B Direct after the first test resulting in an initial invalid rate of 1.5%. The final invalid rate after re-test (according to test protocols) was 0.2% (2/1170 invalid results). Evaluable specimens for comparator testing with Influenza A and Influenza B were 1168 and 1165, respectively.

Retrospective archived specimens consisted of 82 positive influenza A, 114 positive influenza B, and 62 negative specimens blinded and randomized for the study. There were no excluded samples in the retrospective study.

The Simplexa COVID-19 & Flu A/B Direct clinical agreement testing was performed at two (2) external clinical sites and one (1) internal site. The comparator for influenza A and B targets was an FDA cleared molecular test. For SARS-CoV-2 comparator testing, a composite test method (CRM) consisting of three COVID-19 Emergency Use Authorized RT-PCR assays was performed. Two out of three positive results determined "Detected" CRM and two out of three negative results determined "Not Detected" CRM. One of the three EUA-authorized tests became unavailable partway through the prospective clinical study and therefore only 496 of the prospective specimens were available for SARS-CoV-2 comparison testing.

Performance was evaluated using a multi-assay comparator algorithm (for SARS-CoV-2 performance) and an FDA-cleared comparator assay (for Influenza A and Influenza B). Tables 19-21 shows the results of the Simplexa COVID-19 & Flu A/B Direct assay and comparator assay results in the prospective study analysis and Tables 22-24 shows the results of the retrospective study analysis. The positive percent agreement (PPA) and negative percent agreement (NPA) were calculated for each target.

Table 19. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct - Prospective Specimens (Influenza A)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 57 | 2 | 59  |
|  Not Detected | 5 | 1104 | 1109  |
|  Total | 62 | 1106 | 1168  |
|  PPA = 91.9% (57/62)a | NPA = 99.8% (1104/1106)b  |   |   |
|  95% C.I.: 82.5%-96.5% | 95% C.I.: 99.3%-100%  |   |   |

a Five (5) specimens were negative by an additional FDA cleared NAAT.
b Two (2) specimens were positive by an additional FDA cleared NAAT. One of the two specimens (1/2) was tested with PCR followed by BDS and was positive.

K220963 - Page 22 of 24

{22}

Table 20. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct – Prospective Specimens (Influenza B)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 0 | 0 | 0  |
|  Not Detected | 0 | 1165 | 1165  |
|  Total | 0 | 1165 | 1165  |
|  PPA = N/A | NPA = 100% (1165/1165)  |   |   |
|  95% C.I.: N/A | 95% C.I.: 99.7%-100%  |   |   |

Table 21. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct – Prospective Specimens (SARS-CoV-2)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 67 | 11 | 78  |
|  Not Detected | 1 | 417 | 418  |
|  Total | 68 | 428 | 496  |
|  PPA = 98.5% (67/68)a | NPA = 97.4% (417/428)b  |   |   |
|  95% C.I.: 92.1%-99.7% | 95% C.I.: 95.5%-98.6%  |   |   |

a One specimen was positive by an additional FDA cleared NAAT.
b Nine of the eleven specimens (9/11) were positive by an additional FDA cleared NAAT and four (4) were positive by PCR followed by BDS.

Table 22. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct – Retrospective Specimens (Influenza A)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 80 | 0 | 80  |
|  Not Detected | 2 | 176 | 178  |
|  Total | 82 | 176 | 258  |
|  PPA = 97.6% (80/82) | NPA = 100% (176/176)  |   |   |
|  95% C.I.: 91.5%-99.3% | 95% C.I.: 97.9%-100%  |   |   |

Table 23. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct – Retrospective Specimens (Influenza B)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 112 | 0 | 112  |
|  Not Detected | 2 | 144 | 146  |
|  Total | 114 | 144 | 258  |
|  PPA = 98.2% (112/114) | NPA = 100% (144/144)  |   |   |
|  95% C.I.: 93.8%-99.5% | 95% C.I.: 97.4%-100%  |   |   |

Table 24. Clinical Performance for the Simplexa COVID-19 & Flu A/B Direct – Retrospective Specimens (SARS-CoV-2)

|  COVID-19 & Flu A/B Direct | Comparator |   | Total  |
| --- | --- | --- | --- |
|   |  Detected | Not Detected  |   |
|  Detected | 0 | 0 | 0  |
|  Not Detected | 0 | 252 | 252  |
|  Total | 0 | 252 | 252  |
|  PPA = N/A | NPA = 100% (252/252)  |   |   |
|  95% C.I.: N/A | 95% C.I.: 98.5%-100%  |   |   |

N/A = Not applicable

K220963 - Page 23 of 24

{23}

D Clinical Cut-Off:

Not applicable.

E Expected Values/Reference Range:

Table 25. Simplexa COVID-19 & Flu A/B Direct Observed Positivity Rate

|  Analyte | # Positive <5 Years | # Positive 6-21 Years | # Positive 22-65 Years | # Positive >65 Years | Total Positivity rate  |
| --- | --- | --- | --- | --- | --- |
|  Influenza A | 16 | 30 | 9 | 4 | 5.0% (59/1168)  |
|  Influenza B | 0 | 0 | 0 | 0 | 0.0% (0/1165)  |
|  SARS-CoV-2 | 0 | 10 | 63 | 5 | 15.7% (78/496)  |

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.

K220963 - Page 24 of 24

---

**Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K220963](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K220963)

**Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices. If you're preparing [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/), [get in touch](https://innolitics.com/contact).

**Cite:** Innolitics at https://innolitics.com