Simplexa COVID-19 Direct

K212147 · Diasorin Molecular, LLC · QQX · Sep 13, 2022 · Microbiology

Device Facts

• Record ID: K212147
• Device Name: Simplexa COVID-19 Direct
• Applicant: Diasorin Molecular, LLC
• Product Code: QQX · Microbiology
• Decision Date: Sep 13, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3981
• Device Class: Class 2

Indications for Use

The DiaSorin Molecular Simplexa™ COVID-19 Direct is a real-time RT-PCR assay intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) and nasal swab specimens from symptomatic individuals suspected of COVID-19 by their healthcare provider. The Simplexa™ COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection. 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 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 other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities.

Device Story

System performs real-time RT-PCR for qualitative detection of SARS-CoV-2 RNA from nasopharyngeal or nasal swabs without nucleic acid extraction. Components include reaction mix, LIAISON MDX instrument, and Direct Amplification Disc. Primers/probes target ORF1ab and S genes; RNA internal control monitors for inhibition/failure. Used in laboratory settings by trained professionals. Instrument processes samples, amplifies cDNA, and detects fluorescence (FAM, JOE, Q670). Output provides qualitative detection status. Results assist clinicians in diagnosing SARS-CoV-2 infection when combined with patient history, epidemiology, and other laboratory data.

Clinical Evidence

Multi-site clinical evaluation of 1,150 prospective samples (NPS and NS). Compared against a composite reference method (CRM) using three EUA-approved NAAT assays. Total PPA was 98.2% (95% CI: 93.6-99.5%) and NPA was 99.6% (95% CI: 98.9-99.8%). NPS-specific PPA was 98.4% and NS-specific PPA was 98.0%. Reproducibility study across three sites showed high agreement (99.7% total).

Technological Characteristics

Real-time RT-PCR assay; no-extraction workflow. Components: Reaction mix (DNA polymerase, reverse transcriptase, RNase inhibitor, primers/probes), Direct Amplification Disc (DAD) with microfluidic channels/laser-activated valves, and Positive Control Pack. Detection: FAM (S gene), JOE (ORF1ab), Q670 (Internal Control). Instrument: LIAISON MDX thermocycler. Connectivity: External computer with LIAISON MDX Studio Software. Software: Automated result interpretation.

Indications for Use

Indicated for symptomatic individuals suspected of COVID-19 by a healthcare provider. For use with nasopharyngeal and nasal swab specimens to aid in the diagnosis of SARS-CoV-2 infection.

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)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K212147

B Applicant

DiaSorin Molecular LLC

C Proprietary and Established Names

Simplexa COVID-19 Direct

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 nucleic acids

C Type of Test:

Simplexa COVID-19 Direct is a real-time RT-PCR test for use with LIAISON MDX instrument for the qualitative in vitro detection and identification of nucleic acids from severe acute

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) and nasal (NS) swabs from individuals with symptoms of upper respiratory tract infection suspected of COVID-19.

## 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 Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) and nasal swabs (NS)] from symptomatic individuals suspected of COVID 19 by their healthcare provider. The Simplexa COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection.

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 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 other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities.

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

Rx - For Prescription Use Only

### D Special Instrument Requirements:

LIAISON MDX Instrument with LIAISON MDX Studio Software.

## Device/System Characteristics:

### A Device Description:

Simplexa COVID-19 Direct is a real-time RT-PCR (rRT-PCR) system that enables the direct amplification and detection of SARS-CoV-2 (COVID-19) RNA from nasopharyngeal swab or nasal swab specimens that have not undergone nucleic acid extraction.

The test system consists of the following:

- Simplexa COVID-19 Direct reaction mix, (DNA polymerase, reverse transcriptase, RNase inhibitor, buffer, dNTPs, encapsulated RNA template (Internal Control),

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fluorescent probes and corresponding forward and reverse primers specific for detection of SARS-CoV-2 RNA and for the RNA Internal Control.)

- Direct Amplification Disc (DAD), which has eight (8) separate wedges where up to eight specimens (or controls) may be processed on each disc. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow, and a reaction chamber.
- Control Pack - Simplexa COVID-19 Positive Control Gen II Pack
- LIAISON MDX instrument, a real-time Polymerase Chain Reaction (PCR) thermocycler used for the identification of nucleic acids from biological specimens. The LIAISON MDX instrument is controlled by an external computer running the LIAISON MDX Studio Software and uses real-time fluorescence detection to identify targets within the sample wells of a Direct Amplification Disc (DAD).

- The LIAISON MDX, previously referred to as 3M Integrated Cycler, was cleared under K102314.

## B Principle of Operation:

The assay uses forward and reverse primers and associated fluorescent probe(s) included in the reaction mix to amplify SARS-CoV-2 cDNA reverse transcribed from RNA. The primers and probe sets are designed to detect SARS-CoV-2 ORF1ab and S gene from the viral RNA in nasopharyngeal swab or nasal swab. An RNA internal control, with associated primers and a fluorescent probe, is included in the reaction mix to detect RT-PCR failure and/or inhibition.

To start processing a patient sample, the user adds 50 μL of Reaction Mix to the reagent input well (R) on the DAD using a pipette, followed by 50 μL of unextracted specimen to the sample input well (SAMPLE). A DAD with reagents and sample(s) is subsequently loaded on the LIAISON MDX. Centrifugal force aboard the LIAISON MDX moves the fluid into the metering chamber of the DAD. The reagent chamber is specifically designed to measure 40 μL of reagent and the sample chamber is specifically designed to measure 10 μL of sample. Excess reagent and sample are forced into the waste chambers by centrifugal force.

After the centrifugal force has mixed the sample and reagent within the reaction chamber, coat protein denaturation, reverse transcription and PCR amplification cycles begin. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5' nuclease activity of DNA polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. The S gene probe has a FAM fluorophore and the ORF1ab gene probe has JOE fluorophore. Fluorescence intensity is monitored at each PCR cycle by detection modules in the LIAISON MDX. A sample is considered positive for a particular target if intensity of the optical reading crosses a particular threshold before a predetermined cut-off cycle. Results are obtained by using the LIAISON MDX Studio software version 1.1 or above. According to the result interpretation if at least one target (S gene or ORF1ab) is Detected for the sample then the sample is positive for SARS-CoV-2.

An RNA internal control, with associated primers and a fluorescent probe, is included in the reaction mix to detect RT-PCR failure and/or inhibition.

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After the run is complete the results are displayed by the software as shown in the following table:

| Results | | Interpretation |
| --- | --- | --- |
| SARS-Cov-2 Target | | |
| ORF1ab gene | S gene | |
| Detected | Detected | Result indicates the presence of SARS-CoV-2 RNA in the patient sample. |
| Detected | --- | Result indicates the presence of SARS-CoV-2 RNA in the patient sample. |
| --- | Detected | Result indicates the presence of SARS-CoV-2 RNA in the patient sample. |
| Not Detected | Not Detected | Result indicates the absence of SARS-CoV-2 RNA in the patient sample. |
| Invalid | | Result indicates inability to conclusively determine presence or absence of SARS-CoV-2 RNA in the patient sample. This result may be due to 1) Internal Control (IC) failure, or 2) failure to detect sufficient specimen volume. The sample needs to be retested. If the problem persists, contact Technical Service. |
| Results | | Interpretation |
| EC500 | | Data processing error due to noise, weak or late amplification in the signal. Repeat the sample. If the problem persists, contact Technical Service. |
| EC505 | | Insufficient information to determine whether amplification was present. If the problem persists, contact Technical Service. |
| EC515 | | Internal Control Amplification is not within specification. Result is invalid, repeat the sample. If the problem persists, contact Technical Service. |

## C Instrument Description Information:

1. **Instrument Name:**
LIAISON MDX instrument with LIAISON MDX Studio Software (version 1.1 or above)

2. **Specimen Identification:**
Specimen identification can be entered via barcode insert containing the assay definition.

3. **Specimen Sampling and Handling:**
Simplexa COVID-19 Direct is a no extraction RT-PCR assay. In this assay the operator adds 50 ul of the reaction mix to the direct amplification disc (DAD) and adds another 50 ul of unextracted sample in designated wells. The disc is then loaded with the sample and necessary controls are run on the LIAISON MDX instrument.

4. **Calibration:**
No calibration is required.

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5. Quality Control:

External controls are 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): | K212147 | K211079 |
| --- | --- | --- |
| Device Trade Name | Simplexa COVID-19 Direct | BioFire COVID-19 Test 2 |
| General Device Characteristic Similarities | | |
| Intended Use/Indications for Use | The DiaSorin Molecular Simplexa COVID-19 Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) and nasal swabs (NS) from symptomatic individuals suspected of COVID 19 by their healthcare provider. The Simplexa COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection. Positive results are indicative of the | 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. |

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K212147 - Page 6 of 22
| | 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 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 other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. | 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 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 |
| --- | --- | --- |
| Assay principle | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens. | same |
| Analyte | RNA | same |
| Test interpretation | Automated test interpretation | same |
| Controls | Assay contains an internal control for PCR function. | Same |
| Organism Detected | SARS-CoV-2 | same |
| **General Device Characteristic Differences** | | |
| Instrumentation | LIAISON MDX | FilmArray2.0 or Film Array Torch |
| Specimen type | Nasopharyngeal and Nasal swabs collected | Nasopharyngeal swabs collected in transport media |

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VI Standards/Guidance Documents Referenced:

Class II Special Controls as per 21 CFR 866.3981.

VII Performance Characteristics:

A Analytical Performance:

1. Precision/Reproducibility:

Reproducibility of the Simplexa COVID-19 Direct

The reproducibility study was conducted at three intended use sites (two external and one internal /in-house), over a period of five testing days, using one lot of reagents, with testing performed by two operators at each site. The test panel consisted of five panel members: one low positive (1-2x LoD), one medium positive (3-5x LoD), contrived with COVID-19 strain USA-WA1/2020, a negative sample (UTM as NTC), and one positive Control Sample prepared in clinical (NPS) matrix. Each sample was tested in three replicates once per day by each operator, for a total of 90 measurements per sample (3 sites x 2 users x 5 days x 3 reps=90 measurements). A total of five LIAISON MDX instruments (at least one per site) were used in the study. The table below summarizes the results from the reproducibility study, showing the mean CT values and calculated SD and %CV for each panel member.

Reproducibility Study, Summary of Qualitative Results

| Sample Panel Member | Expected Qualitative Result | % Agreement with Expected Result (# detected/#tested) |
| --- | --- | --- |
| Low positive sample (1-2x) | COVID-19 Detected | 98.9% (89/90)
95% CI: 94-100.0% |
| Moderate positive sample (3-5x) | COVID-19 Detected | 100.0% (90/90)
95% CI: 95.9-100.0% |
| Negative Control (UTM) | COVID-19 Not Detected | 100.0% (90/90)*
95% CI: 95.9-100.0% |
| Positive Control | COVID-19 Detected | 100.0% (90/90)
95% CI: 95.9-100.0% |

*The expected result for the Negative Panel Member is "Not Detected"

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Reproducibility Study, Summary of Results

| Analyte | Sample Panel Member | N | Mean Ct | Repeatability | | Between-Day | | Between-Operator | | Between-Site | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| S gene (FAM) | NPS_Low positive | 89a | 32.2 | 0.53 | 1.7 | 0.00 | 0.0 | 0.40 | 1.2 | 1.87 | 5.8 | 1.99 | 6.2 |
| | NPS_Moderate positive | 90b | 31.2 | 1.63 | 5.2 | 1.41 | 4.5 | 0.00 | 0.0 | 0.31 | 1.0 | 2.18 | 7.0 |
| | UTM | 90 | 0.0 | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A |
| | Positive Control as is | 90 | 25.7 | 0.27 | 1.1 | 0.14 | 0.6 | 0.00 | 0.0 | 0.00 | 0.0 | 0.31 | 1.2 |
| ORF1ab (JOE) | NPS_Low positive | 89c | 32.5 | 0.50 | 1.5 | 0.00 | 0.0 | 0.34 | 1.0 | 1.71 | 5.3 | 1.81 | 5.6 |
| | NPS_Moderate positive | 90 | 31.2 | 1.01 | 3.3 | 0.45 | 1.4 | 0.00 | 0.0 | 0.37 | 1.2 | 1.17 | 3.7 |
| | UTM | 90 | 0.0 | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A |
| | Positive Control as is | 90 | 26.0 | 0.27 | 1.0 | 0.08 | 0.3 | 0.02 | 0.1 | 0.24 | 0.9 | 0.37 | 1.4 |
| Internal Control (Q670) | NPS_Low positive | 90d | 32.0 | 1.16 | 3.6 | 0.88 | 2.7 | 0.00 | 0.0 | 0.82 | 2.6 | 1.67 | 5.2 |
| | NPS_Moderate positive | 90 | 31.8 | 0.92 | 2.9 | 0.60 | 1.9 | 0.00 | 0.0 | 0.61 | 1.9 | 1.26 | 3.9 |
| | UTM | 90 | 32.0 | 0.79 | 2.5 | 0.41 | 1.3 | 0.00 | 0.0 | 0.75 | 2.4 | 1.17 | 3.6 |
| | Positive Control as is | 90 | 31.6 | 0.45 | 1.4 | 0.31 | 1.0 | 0.00 | 0.0 | 1.09 | 3.4 | 1.21 | 3.8 |

a – 4/89 were not detected for S gene but had an interpretation of positive, therefore assigned a Ct of 40 for calculation. One of the replicates was not detected and therefore not used to calculate variances.
b – 4/90 were not detected for S gene but had an interpretation of positive, therefore assigned a Ct of 40 for calculation.
c – 3/89 were not detected for ORF1ab gene but was positive, therefore assigned a Ct of 40 for calculation. One of the replicates was not detected and therefore not used to calculate variances.
d – 2/90 were not detected for RNA IC but were positive for COVID-19 detection. A Ct for RNA IC is not applicable for positive samples and therefore was acceptable and assigned a Ct of 40 and used in the calculation of the variance components.

## Lot-to-lot Reproducibility of the Simplexa COVID-19 Direct Kit

The lot-to-lot reagent reproducibility of the Simplexa COVID-19 Direct kit was evaluated in a study conducted at one internal site, over a period of 12 non-consecutive days (18-23 and 25-30 January 2021), using three lots of Simplexa COVID-19 Direct kit, two lots of Simplexa COVID-19 Gen II Positive Control, with testing performed by two operators at the site. The test panel consisted of four panel members: one low positive (1-2x LoD) and one medium positive (3-5x LoD), each contrived with 2019-nCoV/USA-WA1/2020 strain, a negative sample (UTM as No Template Control), and one Positive Control sample (from Control Pack) prepared in negative clinical (NPS) matrix. Each sample was tested in duplicate per kit lot per run, in two runs per day, for a total of 12 non-consecutive days (2 replicates/kit lot x 3 kit lots/run x 2 runs/day x 12 days=144 /sample panel member). A total of two LIAISON MDX instruments were used in the study. The tables below summarize the results from the inter-lot precision study for the Simplexa COVID-19 Direct kit, showing the mean Ct values and calculated SD and %CV for each panel member.

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Qualitative Summary of -Lot-to-lot Reproducibility for Simplexa COVID-19 Direct

| Sample Panel Member | Observed Qualitative Result |
| --- | --- |
| Low positive sample (1-2x) | 99.3% (143/144) COVID-19 Detected 95% CI: (96.2% to 99.9%) |
| Moderate positive sample (3-5x) | 100.0% (144/144) COVID-19 Detected 95% CI: (97.5% to 100%) |
| Positive control | 100.0% (144/144) COVID-19 Detected 95% CI: (97.5% to 100%) |

Lot-to-lot Reproducibility, Summary of Results

| Analyte | Panel Member | Kit Lot-X9470N | | Kit Lot - 9586N | | Kit Lot - 9589N | | All Lots Combined | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) |
| S gene (FAM) | LP | 95.8% (46/48) | 32.2 ± 1.21 (3.7%) | 95.8% (46/48) | 31.9 ± 0.99 (3.1%) | 97.9% (47/48) | 32.0 ± 0.85 (2.6%) | 96.5% (139/144) | 32.0 ± 1.03 (3.2%) |
| | MP | 97.9% (47/48) | 30.9 ± 0.78 (2.5%) | 100.0% (48/48) | 30.8 ± 0.47 (1.5%) | 100.0% (48/48) | 30.8 ± 0.78 (2.5%) | 99.3% (143/144) | 30.8 ± 0.69 (2.2%) |
| | Negative | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/144) | N/A ± N/A (N/A%) |
| | PC | 100.0% (48/48) | 25.8 ± 0.36 (1.4%) | 100.0% (48/48) | 25.7 ± 0.29 (1.1%) | 100.0% (48/48) | 25.6 ± 0.28 (1.1%) | 100.0% (144/144) | 25.7 ± 0.33 (1.3%) |
| ORF1a b gene (JOE) | LP | 97.9% (47/48) | 31.9 ± 1.35 (4.2%) | 97.9% (47/48) | 31.5 ± 0.80 (2.5%) | 100.0% (48/48) | 31.8 ± 0.76 (2.4%) | 98.6% (142/144) | 31.7 ± 1.01 (3.2%) |
| | MP | 97.9% (47/48) | 30.6 ± 0.90 (2.9%) | 100.0% (48/48) | 30.5 ± 0.50 (1.6%) | 100.0% (48/48) | 30.5 ± 0.65 (2.1%) | 99.3% (143/144) | 30.5 ± 0.70 (2.3%) |
| | Negative | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/48) | N/A ± N/A (N/A%) | 0.0% (0/144) | N/A ± N/A (N/A%) |
| | PC | 100.0% (48/48) | 26.0 ± 0.52 (2.0%) | 100.0% (48/48) | 25.9 ± 0.38 (1.5%) | 100.0% (48/48) | 25.9 ± 0.37 (1.4%) | 100.0% (144/144) | 26.0 ± 0.43 (1.7%) |
| Internal Control (Q670) | LP | 97.9% (47/48) | 31.5 ± 0.63 (2.0%) | 100.0% (48/48) | 30.9 ± 0.58 (1.9%) | 100.0% (48/48) | 31.0 ± 0.39 (1.3%) | 99.3% (143/144) | 31.1 ± 0.60 (1.9%) |
| | MP | 97.9% (47/48) | 31.7 ± 1.17 (3.7%) | 100.0% (48/48) | 30.9 ± 0.34 (1.1%) | 100.0% (48/48) | 31.2 ± 0.64 (2.1%) | 99.3% (143/144) | 31.2 ± 0.86 (2.8%) |
| | Negative | 100.0% (48/48) | 31.1 ± 0.37 (1.2%) | 100.0% (48/48) | 30.5 ± 0.38 (1.2%) | 100.0% (48/48) | 30.4 ± 0.48 (1.6%) | 100.0% (144/144) | 30.7 ± 0.52 (1.7%) |
| | PC | 100.0% (48/48) | 31.2 ± 0.51 (1.6%) | 100.0% (48/48) | 30.6 ± 0.34 (1.1%) | 100.0% (48/48) | 30.5 ± 0.37 (1.2%) | 100.0% (144/144) | 30.7 ± 0.50 (1.6%) |

LP= low positive; MP=moderate positive; PC=positive control

Lot-to-lot Reproducibility of the Simplexa COVID-19 Direct Gen II Positive Control Kit

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The precision of the Simplexa COVID-19 Direct Gen II Positive Control kit for the lot-to-lot reagent reproducibility were evaluated in a study conducted at one internal site, over a period of 12 non-consecutive days (18-23 and 25-30 January 2021) using one lot of Simplexa COVID-19 Direct kit, three lots of Simplexa COVID-19 Gen II Positive Control with testing performed by two operators at the site, using one LIAISON MDX instrument. Forty eight (48) replicates of each of three lots of the Simplexa COVID-19 Direct Gen II Positive Control were tested for both channels (S gene [FAM] and ORF1ab gene [JOE]) and were detected in each of the 144 total replicates.

The table below summarizes the results from the inter lot precision study (Simplexa COVID-19 Direct Gen II Positive Control kit), showing the mean Ct values and calculated SD and $\% \mathrm{CV}$ for each panel member.

Qualitative Summary of Inter-lot Reproducibility of Simplexa COVID-19 Gen II Positive Control

| Sample Panel Member | Expected Qualitative Result | Observed Qualitative Result |
| --- | --- | --- |
| Positive Control | 100% of the tested replicate results are COVID-19 Positive | 100.0% (144/144) COVID-19 Positive 95% CI: 97.5 to 100.0% |

Lot-to-lot Reproducibility Test Results for Simplexa COVID-19 Gen II Positive Control

| Analyte | Panel Member | PC Lot - V9878N | | PC Lot - V9881N | | PC Lot - V9882N | | All Lots Combined | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) | Qualitative Results | Mean Ct ± SD (%CV) |
| S gene (FAM) | Positive Control | 100% (48/48) | 30.3 ± 0.61 (2.0%) | 100% (48/48) | 29.2 ± 0.67 (2.3%) | 100% (48/48) | 27.8 ± 0.52 (1.9%) | 100% (144/144) | 29.1 ± 1.19 (4.1%) |
| ORF1ab gene (JOE) | | 100% (48/48) | 30.2 ± 0.69 (2.3%) | 100% (48/48) | 29.3 ± 0.69 (2.4%) | 100% (48/48) | 28.1 ± 0.62 (2.2%) | 100% (144/144) | 29.2 ± 1.09 (3.7%) |
| Internal Control (Q670) | | 97.9% (47/48) | 31.7 ± 0.6 (1.9%) | 100.0% (48/48) | 31.9 ± 0.42 (1.3%) | 100.0% (48/48) | 31.8 ± 0.68 (2.1%) | 99.3% (143/144) | 31.8 ± 0.57 (1.8%) |

# 2. Linearity:

Not applicable; this is a qualitative assay.

# 3. Analytical Specificity/Interference:

# Cross Reactivity:

The potential for assay cross-reactivity was evaluated by addition of a panel of micro-organisms selected based on their possible presence in respiratory specimens. Exclusivity of the Simplexa COVID-19 Direct was evaluated by spiking viruses at $1 \times 10^{5} \mathrm{TCID}_{50} / \mathrm{mL}$ , and

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bacteria or fungi at 1×10⁶ CFU/mL (where possible). Each organism was tested in triplicate. All three SARS-CoV-2 assay targets were expected to be negative in each replicate and any invalid runs due to failure of controls or instrument/software errors were to be repeated. A total of 47 different organisms were tested in addition to the human nasal fluid which closely represents diverse microbial flora and human genomic DNA.

In silico (BLAST) analysis was performed for Bacillus anthracis, Influenza C and Pneumocystis jirovecii as these organisms were not available for wet testing. Human coronavirus 229E, human coronavirus NL63, human metapneumovirus (hMPV) and Leptospira interrogans could not be sourced at the specified concentration so it was supplemented by in silico analysis.

A summary of the cross-reactivity evaluation is shown below. No cross-reactivity was detected for any of the organisms tested (i.e., 0/3 detection for each assay/replicate/analyte).

Simplexa COVID-19 Direct Cross-Reactivity (Analytical Specificity)

| Organisms/Virus | Test Concentration | Cross-Reactivity Detected |
| --- | --- | --- |
| Adenovirus C (Type 1) | 1 x 10⁵ U/mL | None |
| Adenovirus 7A | 1 x 10⁵ TCID50 /mL | None |
| Bordetella pertussis | 1 x 10⁶ CFU/mL | None |
| Candida albicans | 1 x 10⁶ CFU/mL | None |
| Chlamydophila pneumoniae | 1 x 10⁶ IFU/mL | None |
| Chlamydophila psittaci (genomic DNA) | 1 x 10⁶ copies/mL | None |
| Corynebacterium diphtheriae | 1 x 10⁶ CFU/mL | None |
| Coxiella burnetii (genomic DNA) | 1 x 10⁶ copies/mL | None |
| Cytomegalovirus | 1 x 10⁵ U/mL | None |
| Enterovirus 68 | 1 x 10⁵ U/mL | None |
| Epstein-Barr virus | 1 x 10⁵ copies/mL | None |
| Escherichia coli | 1 x 10⁶ CFU/mL | None |
| Haemophilus influenzae | 1 x 10⁶ CFU/mL | None |
| Human coronavirus 229E* | 3 x 10⁴ TCID50 /mL | None |
| Human coronavirus HKU1 (RNA) | 1 x 10⁵ genome copies/mL | None |
| Human coronavirus NL63* | 3 x 10⁴ U/mL | None |
| Human coronavirus OC43 | 1 x 10⁵ TCID50 /mL | None |
| Human genomic DNA (Leukocytes) | 1 x 10⁶ cells/mL | None |
| Human metapneumovirus (hMPV)* | 3 x 10⁴ TCID50 /mL | None |
| Influenza A/Perth/16/2009 | 1 x 10⁵ EID50 /mL | None |
| Influenza B/Florida/02/06 | 1 x 10⁵ U/mL | None |
| Lactobacillus plantarum 17-5 | 1 x 10⁶ CFU/mL | None |
| Legionella longbeachae | 1 x 10⁶ CFU/mL | None |
| Legionella pneumophila | 1 x 10⁶ CFU/mL | None |
| Leptospira interrogans | 1:10 Dilution | None |
| Measles | 1 x 10⁵ TCID50 /mL | None |
| MERS-coronavirus | 1 x 10⁵ TCID50 /mL | None |
| Moraxella catarrhalis | 1 x 10⁶ CFU/mL | None |
| Mumps | 1 x 10⁵ U/mL | None |
| Mycobacterium tuberculosis (genomic DNA) | 1 x 10⁶ copies/mL | None |
| Mycoplasma pneumoniae | 1 x 10⁶ CCU/mL | None |
| Neisseria elongata | 1 x 10⁶ CFU/mL | None |
| Neisseria meningitidis | 1 x 10⁶ CFU/mL | None |
| Parainfluenza virus 1 | 1 x 10⁵ U/mL | None |

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| Parainfluenza virus 2 | 1 x 10^5 U/mL | None |
| --- | --- | --- |
| Parainfluenza virus 3 | 1 x 10^5 TCID50 /mL | None |
| Parainfluenza virus 4 | 1 x 10^5 U/mL | None |
| Parechovirus 3 | 1 x 10^5 U/mL | None |
| Pseudomonas aeruginosa | 1 x 10^6 CFU/mL | None |
| Respiratory syncytial Virus A | 1 x 10^5 TCID50 /mL | None |
| Respiratory syncytial Virus B | 1 x 10^5 TCID50 /mL | None |
| Rhinovirus | 1 x 10^5 U/mL | None |
| SARS-coronavirus (RNA) | 1 x 10^5 copies/mL | None |
| Staphylococcus aureus | 1 x 10^6 CFU/mL | None |
| Staphylococcus epidermidis | 1 x 10^6 CFU/mL | None |
| Streptococcus pneumoniae | 1 x 10^6 CFU/mL | None |
| Streptococcus pyogenes | 1 x 10^6 CFU/mL | None |
| Streptococcus salivarius | 1 x 10^6 CFU/mL | None |
| Pooled Human Nasal Fluid | 1:1 Dilution | None |

*A lower concentration was tested due to inability to obtain stock material with high titer
1 CCU/mL = Color Changing Units/milliliter, CFU/mL = Colony Forming Units/milliliter, IFU/mL = Infectious units/milliliter, U/mL = Units/milliliter, TCID50/mL = Tissue Culture Infectious Dose/milliliter

## Interference

Simplexa COVID-19 Direct was tested for potential interference by substances that may be present in respiratory specimens. Test samples were contrived in clinical NPS matrix, spiked with SARS-CoV-2(2019-nCoV/USA-WA1/2020 strain at 3x LoD (1500cp/ml) and adding the potentially interfering substances at concentrations shown below. Each test sample was run in triplicate at one site using four LIAISON MDX instruments by three operators, over the course of six days. No interference was observed at the tested concentrations. The following table provides the substances and concentrations tested. The FluMist nasal vaccine was not tested as it was unavailable at the time of the study. A limitation regarding FluMist evaluation is added to the assay labeling.

Interfering Substances Tested on the Simplexa COVID-19 Direct

| Potentially Interfering Substance | Active Ingredient | Tested Concentration | SARS-CoV-2 Qualitative Results: % Detection (# Detected/#Tested) | IC Qualitative Results: % Detection (# Detected/#Tested) |
| --- | --- | --- | --- | --- |
| Antibiotic nasal ointment (Mupirocin) | Mupirocin | 6.6 mg/mL | 100% (3/3) | 100.0% (3/3) |
| Anti-viral drug (Oseltamivir) | Oseltamivir | 3.3 mg/mL | 100% (3/3) | 100.0% (3/3) |
| Cold Eeze (Throat lozenges, Oral anesthetic and analgesic) | Zincum gluconicum 2X | 2.5% (w/v) | 100% (3/3) | 100.0% (3/3) |
| Homeopathic allergy relief medicine | N/A | 10% (v/v) | 100.0% (3/3) | 100.0% (3/3) |
| Mucin (Bovine submaxillary gland, type I-S) | N/A | 5 mg/mL | 100.0% (3/3) | 100.0% (3/3) |
| Nasal corticosteroids (Fluticasone) | Fluticasone | 5% (v/v) | 100.0% (3/3) | 100.0% (3/3) |
| Allergy Relief Swabs (Nasal Gel, Zicam) | Luffa opperculata, Galphimia glauca, histaminum hydrochloricum, Sulphur | 5% (w/v) | 100.0% (3/3) | 100.0% (3/3) |
| Nasal spray or drops (Oxymetazoline) | Oxymetazoline | 15% (v/v) | 100.0% (3/3) | 100.0% (3/3) |

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| Saliva | N/A | 10% (v/v) | 83.3% (5/6) | 83.3% (5/6) |
| --- | --- | --- | --- | --- |
| | N/A | 5% (v/v)* | 100.0% (6/6) | 100.0% (6/6) |
| Systemic antibacterial (Tobramycin) | Tobramycin | 4 μg/mL | 100.0% (3/3) | 100.0% (3/3) |
| Whole Blood | N/A | 2% (v/v) | 100.0% (3/3) | 100.0% (3/3) |
| Zanamivir | N/A | 3 mg/mL | 100.0% (6/6) | 83.3% (5/6) |

*Interference from saliva was observed at a concentration a above 5%
mg = milligram, mL = milliliter, v/v = volume to volume, w/v = weight to volume, μg = microgram, NA = Not Applicable

## Microbial Interference:

Simplexa COVID-19 Direct was evaluated for ability to identify SARS-CoV-2 when other potentially cross-reacting organisms were present. Potentially cross-reacting organisms were individually spiked into a pool of negative nasopharyngeal swab matrix with a low concentration of inactivated SARS-CoV-2 at approximately two times the limit of detection (2x LoD). Except for Lactobacillus plantarum 17-5, no inhibition by other organisms was observed at the tested concentration. Testing Lactobacillus plantarum 17-5 at a lower concentration (5 x 10⁵ CFU/mL) resulted in no inhibition.

Microbial Inhibition Tested on the Simplexa COVID-19 Direct

| Organism | Tested Concentration | COVID-19 Qualitative Results: % Detection (# Detected /#Tested) | IC (Q670) | |
| --- | --- | --- | --- | --- |
| | | | % Detection (# Detected / # Tested) | Mean Ct ± SD (%CV) |
| Adenovirus C | 1 x 10⁵ TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.0 ± 0.6 (1.9%) |
| Adenovirus 7A | 1 x 10⁵ TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | |
| Bordetella pertussis | 1 x 10⁶ CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 30.8 ± 0.2 (0.6%) |
| Candida albicans | 1 x 10⁶ CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.5 (1.6%) |
| Chlamydia pneumoniae | 1 x 10⁶ IFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.2 ± 0.2 (0.6%) |
| Chlamydophila psittaci (genomic DNA) | 2.5 x 10⁵ copies/mL* | 100.0% (3/3) | 100.0% (3/3) | 31.1 ± 0.0 (0.0%) |
| Corynebacterium diphtheriae | 1 x 10⁶ CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.3 ± 0.5 (1.6%) |
| Coxiella burnetii (genomic DNA) | 1 x 10⁶ copies/mL | 100.0% (3/3) | 100.0% (3/3) | 31.3 ± 0.1 (0.3%) |
| Cytomegalovirus | 1 x 10⁵ U/mL | 100.0% (3/3) | 100.0% (3/3) | 31.3 ± 0.4 (1.3%) |
| Enterovirus 68 | 1 x 10⁵ TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.6 ± 0.3 (0.9%) |
| Epstein-Barr virus | 1 x 10⁵ copies/mL | 100.0% (3/3) | 100.0% (3/3) | 32.0 ± 0.2 (0.6%) |
| Escherichia coli | 1 x 10⁶ CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.6 ± 1.0 (3.2%) |
| Haemophilus influenzae | 1 x 10⁶ CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.2 ± 0.4 (1.3%) |

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| Human coronavirus 229E | 1.5 x 10^{4} TCID50/mL* | 100.0% (3/3) | 100.0% (3/3) | 31.5 ± 0.0 (0.0%) |
| --- | --- | --- | --- | --- |
| Human coronavirus HKU1 (RNA) | 1 x 10^{5} genome copies/mL | 100.0% (3/3) | 100.0% (3/3) | 32.6 ± 0.2 (0.6%) |
| Human coronavirus NL63 | 1.5 x10^{4} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.6 ± 0.2 (0.6%) |
| Human coronavirus OC43 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.0 ± 0.7 (2.3%) |
| Human genomic DNA (Leukocytes) | 1 x 10^{6} copies/mL | 100.0% (3/3) | 100.0% (3/3) | 31.1 ± 0.3 (1.0%) |
| Human Metapneumovirus (hMPV) | 1.5 x 10^{4} TCID50/mL* | 100.0% (3/3) | 100.0% (3/3) | 31.9 ± 0.4 (1.3%) |
| Influenza A/Perth/16/2009 | 1 x 10^{5} EID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.8 ± 0.3 (1.0%) |
| Influenza B/Phuket/3073/2013 | 1 x 10^{5} CEID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.3 (1.0%) |
| Lactobacillus plantarum 17-5 | 1 x 10^{6} CFU/mL | 90.0% (18/20) | 100.0% (20/20) | 31.5 ± 0.4 (1.3%) |
| | 5 x 10^{5} CFU/mL** | 100.0% (6/6) | 100.0% (6/6) | 32.1 ± 0.9 (2.8%) |
| Legionella longbeachae | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.7 ± 0.3 (0.9%) |
| Legionella pneumophila | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 30.7 ± 0.3 (1.0%) |
| Leptospira interrogans | 1:10 Dilution | 100.0% (6/6) | 100.0% (6/6) | 33.2 ± 1.1 (3.3%) |
| Measles | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.3 ± 0.2 (0.6%) |
| MERS-coronavirus | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.8 ± 0.3 (1.0%) |
| Moraxella catarrhalis | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.4 ± 0.3 (1.0%) |
| Mumps | 1 x 10^{5} U/mL | 95.0% (19/20) | 100.0% (20/20) | 31.9 ± 0.4 (1.3%) |
| Mycobacterium tuberculosis (genomic DNA) | 1 x 10^{6} copies/mL | 100.0% (3/3) | 100.0% (3/3) | 30.7 ± 0.1 (0.3%) |
| Mycoplasma pneumoniae | 1 x 10^{6} CCU/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.4 (1.3%) |
| Neisseria elongata | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 32.5 ± 0.4 (1.2%) |
| Neisseria meningitidis | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 32.4 ± 0.3 (0.9%) |
| Parainfluenza virus 1 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.7 ± 0.7 (2.3%) |
| Streptococcus pyogenes | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.1 ± 0.1 (0.3%) |

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| Streptococcus salivarius | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.3 ± 0.5 (1.6%) |
| --- | --- | --- | --- | --- |
| Pooled human nasal fluid | 1:1 Dilution | 100.0% (3/3) | 100.0% (3/3) | 31.6 ± 0.4 (1.3%) |
| Parainfluenza virus 2 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.8 ± 0.9 (2.8%) |
| Parainfluenza virus 3 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.1 ± 0.4 (1.3%) |
| Parainfluenza virus 4 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.2 (0.6%) |
| Parechovirus 3 | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.8 ± 0.7 (2.2%) |
| Pseudomonas aeruginosa | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.6 (1.9%) |
| Respiratory syncytial virus A | 5 x 10^{4} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.9 ± 0.2 (0.6%) |
| Respiratory syncytial virus B | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 30.8 ± 0.5 (1.6%) |
| Rhinovirus | 1 x 10^{5} TCID50/mL | 100.0% (3/3) | 100.0% (3/3) | 31.0 ± 0.3 (1.0%) |
| SARS-Coronavirus (RNA) | 1 x 10^{5} copies/mL | 100.0% (3/3) | 100.0% (3/3) | 32.3 ± 0.3 (0.9%) |
| Staphylococcus aureus | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.0 ± 0.2 (0.6%) |
| Staphylococcus epidermidis | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.5 ± 0.2 (0.6%) |
| Streptococcus pneumoniae | 1 x 10^{6} CFU/mL | 100.0% (3/3) | 100.0% (3/3) | 31.1 ± 0.1 (0.3%) |

* A lower concentration was tested due to inability to obtain stock material with high titer
** Interference with Lactobacillus plantarum 17-5 was observed at a concentration above 5 x 10⁵ CFU/mL.
¹ CCU = Color changing units/milliliter
CFU/mL = Colony forming units/milliliter
IFU/ml = Infectious units/milliliter
U/mL = Units/milliliter
TCID50/mL = Tissue Culture Infectious Dose per milliliter

4. Assay Reportable Range:

Not applicable; this is a qualitative assay.

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

Assay Controls:

The assay contains an internal control for PCR function. DiaSorin Molecular offers optional external QC materials that are intended for use with the assay. The positive control is marketed as Simplexa COVID-19 Positive Control Gen II Pack. Controls are packaged in single use aliquots and stored frozen; once thawed the controls are stable for thirty (30) minutes at ambient laboratory temperature.

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The following table shows daily controls, including count, mean, standard deviation (SD), $\% \mathrm{CV}$ , minimum and maximum for all valid PC and NTC results during the clinical study.

| Ctrl | S gene (FAM) | | | | | | ORF1ab (JOE) | | | | | | Internal Control (Q670) | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | N | Mean | SD | CV | Min | Max | N | Mean | SD | CV | Min | Max | N | Mean | SD | CV | Min | Max |
| NTC | 7 | 0.0 | 0.0 | N/A | 0.0 | 0.0 | 7 | 0.0 | 0.0 | N/A | 0.0 | 0.0 | 7 | 31.1 | 0.69 | 2.2% | 30. | 33.9 |
| PC | 7 | 28.1 | 2.0 | 7.3 | 25. | 31. | 7 | 28.3 | 2.0 | 7.2% | 25.1 | 32. | 7 | 31.2 | 0.82 | 2.6% | 29. | 34.6 |

## Fresh versus frozen Study:

A fresh vs. frozen study was designed for the candidate device to determine the assay performance testing NPS swabs in UTM and saline after freezing. SARS-CoV2 negative NPS swab samples in UTM/saline were used to prepare negative or positive samples spiked with heat-inactivated SARS-COV-2 viral particles from the 2019-nCoV/USA-WA1/2020 strain at two concentrations (10 samples at $5\mathrm{x}$ LoD and 30 samples at $2\mathrm{x}$ LoD). All samples were tested fresh prior to freezing for 24 hrs at $\leq 70^{\circ}$ ; up to three freeze/thaw cycles following the storage condition, as described below.


The Fresh vs. Frozen study was conducted for each time point and showed a $100\%$ detection (positivity) for samples prepared at 5x LoD (except for time point C, one replicate at 5X LoD at time point C gave negative result initially; the same replicate was repeated on the same day on two different runs with successful detection of all targets.), $\geq 95\%$ detection (positivity) for samples prepared at 2x LoD and $0\%$ detection of all negative (un-spiked) samples.

## Specimen Stability:

Nasopharyngeal swabs (NPS) collected in UTM and in saline were tested as negative or as contrived positive samples by spiking the heat-inactivated SARS-CoV-2 viral particles from the 2019-nCoV/USA-WA1/2020 strain (ATCC, Manassas, VA) at two different concentrations: $2\mathrm{x}$ and $5\mathrm{x}$ LoD. Samples were tested fresh, then stored refrigerated $(2 - 8^{\circ}\mathrm{C})$ and tested in 10 replicates at Day 3, Day 5, Day 7, and Day 10.

Each sample were frozen and tested for up to three days for three freeze-thaw cycles. The study data supported sample storage at $2 - 8^{\circ}\mathrm{C}$ for up to 7 days post collection and if there is a delay the specimens can be stored at $-70^{\circ}\mathrm{C}$ for up to three days. for three freeze thaw cycles

## 6. Detection Limit:

The Limit of Detection (LoD) in NPS and NS samples was determined using heat inactivated SARS-CoV-2 (USA_WA1/2020) spiked into clinical NPS and NS specimens in UTM. LoD is defined as the lowest concentration of SARS-CoV-2 RNA that can be detected at a rate of at least $95\%$ . Tentative LoD was determined using serial dilutions of inactivated virus in NPS

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matrix in UTM and NS matrix in UTM. The tentative LoD in NPS and NS is then confirmed by testing 40 and 20 replicates respectively.

Simplexa COVID-19 Direct Limit of Detection – Nasopharyngeal Swab

| Copies/ml | Interpretation* | S gene | ORF1ab gene |
| --- | --- | --- | --- |
| 500 | 100% (40/40) | 90% (36/40) | 100% (40/40) |

*Interpretation as per the Results interpretation algorithm.

Simplexa COVID-19 Direct Limit of Detection – Nasal Swab

| Copies/ml | Interpretation* | S gene | ORF1ab gene |
| --- | --- | --- | --- |
| 242 | 100% (20/20) | 80% (16/20) | 80% (16/20) |

*Interpretation as per the Results interpretation algorithm.

The LoD was confirmed at 500 cp/ml for the NPS matrix and 242 cp/ml for NS matrix with heat inactivated SARS-CoV-2.

**Study Results with WHO International Standard Material:**

An additional LoD study was done using the SARS-CoV-2 WHO International Standard virus particles to further evaluate the sensitivity of the Simplexa COVID-19 Direct. The tentative LoD was identified with serial dilutions of the viral particles in NPS matrix. The final LoD was confirmed to be the lowest concentration quantified in International Units per milliliter (IU/mL) resulting in positive detection with a minimum 95% positivity.

The following table shows the confirmatory LoD study results with additional replicates.

LoD Confirmation with SARS-CoV-2 WHO International Standard

| IU/mL | S gene (% Detection) | ORF1ab gene (% Detection) | RNA IC (% Detection) | Result Interpretation (%Detection) |
| --- | --- | --- | --- | --- |
| 1500 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) |
| 500 | 95% (38/40) | 85% (34/40) | 100% (40/40) | 97.5% (39/40) |
| 167 | 40% (8/20) | 30% (6/20) | 100% (20/20) | 55% (11/20) |

The results confirmed that the LoD with SARS-CoV-2 WHO International Standard virus particles in NPS matrix is at 500 IU/ml with 97.5% detection.

7. Analytical Reactivity:

**In silico**

An *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 available in the GISAID database submitted from November 01, 2021 to January 31, 2022 and February 01, 2022 to April 30, 2022. The analysis included 2,170,584 and 377,668 sequences in the amplicon regions of the ORF1ab and S gene oligo sets, respectively. Only target sequences with full coverage of all three oligo-binding regions (forward primer, reverse primer, and

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probe) are included in the analyses for both oligo sets. Partial target sequences and sequences with ambiguous or degenerate bases in an oligo binding region are excluded from this inclusivity analysis.

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 Tm values of each oligo sequence to its binding region on each SARS-CoV-2 sequence, it is predicted that the Simplexa COVID-19 Direct will detect all analyzed SARS-CoV-2 sequences from the GISAID database, including sequences of the Omicron BA.1, Omicron BA, Omicron BA.2.12.1, BA.4 and BA.5 subvariants 2 and IHU variants.

An additional in silico inclusivity analysis was performed for complete SARS-CoV-2 genome sequences available in the GISAID 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 primer/probe regions. Only target sequences with full coverage of all three ORF1ab and S gene forward and reverse primer as well as probe region were included in the analyses. The analysis showed that the Simplexa COVID-19 Direct target regions had no mismatch to 208,582 sequences ( $\sim 98.7\%$ ) and were predicted to be detected by the assay based on sequence homology. There were 2602 ( $\sim 1.2\%$ ) sequences with no mismatches for one gene oligo set (either ORF1ab or S gene), and there were 40 sequences ( $\sim 0.02\%$ ) with mismatches in at least one primer or probe binding region, region in either ORF1ab or S gene target region.

A Tm analysis was conducted, and results are summarized below:

Summary of Tm Analysis Results

| Timeframe of Sequences Analyzed | Number of accessions in GISAID Database for the timeframe | Number of sequences where at least one target oligo set meets Tm criteria | Identity to SARS-CoV-2 gene design |
| --- | --- | --- | --- |
| Nov. 1, 2021, to Jan. 31, 2022 | 2,170,584 | 2,170,584 | 100% |
| Feb. 1, 2022, to Apr. 30, 2022 | 377,668 | 377,668 | 100% |
| May 1, 2022, to July 31, 2022 | 211,224 | 211,224 | 100% |

Wet Testing

The analytical reactivity was evaluated using the following five strains (of SARS-CoV-2) for wet testing-

- Hong Kong/VM200001061/2020
- England/204820464/2020
- South Africa/KRISP-EC-K005325/2020
- Japan/TY7-503/2021
- hCoV19/USA/PHC658/2021

The SARS-CoV-2 viral particles listed above were diluted at 1,000 copies/ml (2X LoD) in NPS clinical matrix, in triplicate. All replicates tested "Positive" for COVID-19.

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Summary of Analytical Reactivity

| COVID-19 Strain | Tested Concentration | SARS-CoV-2 Qualitative Results: % Detection (# Detected /#Tested) | IC Qualitative Results: % Detection (# Detected /#Tested) |
| --- | --- | --- | --- |
| Hong Kong/VM200001061/2020 | 1000 copies/mL | 100% (3/3) Positive | 100.0% (3/3) |
| England/204820464/2020 | 1000 copies/mL | 100% (3/3) Positive | 100.0% (3/3) |
| South Africa/KRISP-EC-K005325/2020 | 1000 copies/mL | 100% (3/3) Positive | 100.0% (3/3) |
| Japan/TY7-503/2021 | 1000 copies/mL | 100% (3/3) Positive | 100.0% (3/3) |
| hCoV19/USA/PHC658/2021 | 1000 copies/mL | 100% (3/3) Positive | 100.0% (3/3) |

8. Assay Cut-Off:

Simplexa COVID-19 Direct test kit can be only used with LIAISON MDX System. It is a thermocycler which is capable of heating, cooling, mixing of sample and reagents. The system can detect up to four fluorophores at distinct wavelengths and the sensors on the instrument can monitor the primary functions.

The assay cut-off for this device was established by adjusting the target fluorescent channel threshold and the RT-PCR cycle threshold (Ct) cut-offs to values that allow for sensitivity while maintaining analyte specificity. The fluorescent channel threshold was optimized with the RT-PCR Ct cut-offs for the S gene (FAM), ORF1ab gene (JOE), and Internal Control (Q670), during the Development, Verification and Transfer studies during the assay development, according to the sponsor's internal protocols. The MDX system is designed to interpret the test data and automatically report the test results to the operator.

9. Carry-Over:

Since carry-over studies with high positive NPS samples followed by negative samples have been previously performed for other FDA-cleared Simplexa assays, e.g., Simplexa Flu A/B & RSV Direct Gen II (K201505), a carry-over study was not performed for this submission. In support of the Simplexa COVID-19 Direct assay. The FDA-cleared Simplexa Flu A/B & RSV Direct Gen II assay utilizes identical DAD discs to those used for the Simplexa COVID-19 Direct assay. For details on the carry-over study performed for the cleared Simplexa Flu A/B & RSV Direct Gen II, please refer to file K201505

B Comparison Studies:

1. Method Comparison with Predicate Device:

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

2. Matrix Comparison:

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Not applicable.

## C Clinical Studies:

The clinical study for Simplexa COVID-19 Direct was conducted with prospective fresh and/or prospective frozen NPS and/or NS specimens, collected from four geographically diverse collection sites and four testing sites (three external clinical sites and one internal site). The study utilized leftover, de-identified specimens prospectively collected in transport media (Copan UTM, Cepheid UTM, BD VTM and saline 0.9%), between October 14, 2020, and April 30, 2021, from individuals with signs and symptoms of respiratory tract infection.

The performance of the assay was compared to the test results obtained with three commercially available high-performing FDA EUA authorized SARS-CoV-2 molecular RT-PCR assays.

Specimens were collected from 1150 patients of which, 409 were NPS specimens and 741 were NS specimens. All the patient samples were enrolled as all comers that met the study protocol inclusion and exclusion criteria

Each specimen, after collection in the media, was tested with Simplexa COVID-19 Direct. For comparison, the leftover samples were also tested with three comparator methods according to the IFU for each of the assays, where the third assay was only used for samples which gave discrepant results between the other two methods, for a two-out-of-three result interpretation. There were 114 samples that were collected in media other than UTM or saline that were excluded from analysis. Additionally, 139 specimens were excluded from the performance analysis due to instrument failure (LIASON MDX) or user error (24), or an indeterminate comparator result (1).

Among the 1011 specimens available for the final calculations of performance estimates, 443 (43.8%) were males ranging in age from 76 days old to 96 years old, and 568 (56.2%) were females ranging in age from 51 days old to 91 years old.

The clinical performance of the Simplexa COVID-19 Direct assay when compared with the comparator reference method (CRM) across the four sites combined is shown below.

Clinical Performance of Simplexa COVID-19 Direct vs. CRM (NPS and NS combined)

| Prospective Sample | CRM Detected | CRM Not detected | Total |
| --- | --- | --- | --- |
| Simplexa COVID-19 Direct Detected | 108 | 4 | 112 |
| Simplexa COVID-19 Direct Not Detected | 2 | 897 | 899 |
| Total | 110 | 901 | 1011 |
| PPA | 98.2% (108/110)
95% CI: (93.6% to 99.5%) | | |
| NPA | 99.6% (897/901) | | |

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95% CI: (98.9% to 99.8%)

Clinical Performance of Simplexa COVID-19 Direct vs. CRM (NPS only)

| Prospective NPS Sample | CRM Detected | CRM Not detected | Total |
| --- | --- | --- | --- |
| Simplexa COVID-19 Direct Detected | 60 | 1 | 61 |
| Simplexa COVID-19 Direct Not Detected | 1 | 237 | 238 |
| Total | 61 | 238 | 299 |
| PPA | 98.4% (60/61)
95% CI: (91.3% to 99.7%) | | |
| NPA | 99.6% (237/238)
95% CI: (97.7% to 99.9%) | | |

Clinical Performance of Simplexa COVID-19 Direct vs. CRM (NS specimens)

| Prospective NS Sample | CRM Detected | CRM Not Detected | Total |
| --- | --- | --- | --- |
| Simplexa COVID-19 Direct Detected | 48 | 3 | 51 |
| Simplexa COVID-19 Direct Not Detected | 1 | 660 | 661 |
| Total | 49 | 663 | 712 |
| PPA | 98.0% (48/49)
95% CI: (89.3% to 99.6%) | | |
| NPA | 99.5% (660/663)
95% CI: 98.7% to 99.8% | | |

D Clinical Cut-Off:

Not applicable

E Expected Values/Reference Range:

The expected values for the Simplexa COVID-19 Direct, based on the positivity rate observed during the clinical study, are presented stratified by site in the table below. Overall, SARS-CoV-2 was detected in 10.7% (108/1011) of specimens. The positivity rates across sites ranged between 6.28% (Site 4) to 30.5% (Site 2).

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Positivity by Simplexa COVID-19 Direct Observed During the Study, Stratified by Site

| SARS-CoV-2 | Overall (n=1011) | | 1 - LabCorp South Bend, IN (n=261) | | 2 - TriCore Albuquerque, NM (n=59) | | 3 - UCLA Los Angeles, CA (n=182) | | 4 Cerba Xpert France (n=509) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | N | % | N | % | N | % | N | % | N | % |
| Positive | 110 | 10.7% | 38 | 14.55% | 18 | 30.5% | 22 | 12.1% | 32 | 6.28% |
| Negative | 901 | 88.7% | 223 | 84.44% | 41 | 69.5% | 160 | 87.91% | 477 | 93.71% |

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.

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