The DiaSorin Molecular Simplexa™ COVID-19 Direct is 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 tor 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 Description

The 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 that has not undergone nucleic acid extraction. The system consists of the Simplexa COVID-19 Direct reaction mix, the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories. 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 ORF 1ab 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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study proving the device meets them, based on the provided text:

Device: Simplexa™ COVID-19 Direct

1. Table of Acceptance Criteria and Reported Device Performance

For the Simplexa™ COVID-19 Direct assay, the primary acceptance criteria revolve around its accuracy in detecting SARS-CoV-2 (COVID-19) RNA in patient samples, as well as its reproducibility, analytical sensitivity (Limit of Detection), analytical reactivity (ability to detect various strains), and specificity (cross-reactivity and interference).

Acceptance Criteria Category	Specific Acceptance Criteria (Implicit from Study Design)	Reported Device Performance (Simplexa™ COVID-19 Direct)
Clinical Agreement (Total Specimens)	High Percent Positive Agreement (PPA) and Negative Percent Agreement (NPA) compared to an EUA NAAT Composite Reference Method.	PPA: 98.2% (108/110) (95% CI: 93.6% to 99.5%)NPA: 99.6% (897/901) (95% CI: 98.9% to 99.8%)
Clinical Agreement (NPS)	High PPA and NPA for Nasopharyngeal Swabs.	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 Agreement (NS)	High PPA and NPA for Nasal Swabs.	PPA: 98.0% (48/49) (95% CI: 89.3% to 99.6%)NPA: 99.5% (660/663) (95% CI: 98.7% to 99.8%)
Reproducibility (Low Positive)	High agreement with expected results across sites and operators for low positive samples.	S gene: 94.4% (85/90) agreement; Avg. Ct (All Sites) 31.6 ± 0.95 (3.0%)ORF1ab: 95.6% (86/90) agreement; Avg. Ct (All Sites) 32.2 ± 0.97 (3.0%)Total (algorithm based): 98.9% (89/90) agreement
Reproducibility (Moderate Positive)	High agreement with expected results across sites and operators for moderate positive samples.	S gene: 95.6% (86/90) agreement; Avg. Ct (All Sites) 30.5 ± 0.80 (2.6%)ORF1ab: 100.0% (90/90) agreement; Avg. Ct (All Sites) 31.3 ± 0.87 (2.8%)Total (algorithm based): 100.0% (90/90) agreement
Reproducibility (Negative)	100% agreement with expected results for negative samples.	S gene: 100.0% (90/90) agreementORF1ab: 100.0% (90/90) agreementTotal (algorithm based): 100.0% (90/90) agreement
Reproducibility (Positive Control)	100% agreement with expected results for positive control.	S gene: 100.0% (90/90) agreementORF1ab: 100.0% (90/90) agreementTotal (algorithm based): 100.0% (90/90) agreement
Analytical Sensitivity / Limit of Detection (NPS)	LoD confirmed as the lowest concentration with ≥95% positivity.	500 copies/mL (100% detection for total algorithm based)
Analytical Sensitivity / Limit of Detection (NS)	LoD confirmed as the lowest concentration with ≥95% positivity.	242 copies/mL (100% detection for total algorithm based)
Analytical Sensitivity / LoD (WHO International Standard)	LoD confirmed as the lowest concentration with ≥95% positivity (IU/mL).	500 IU/mL (97.5% detection)
Analytical Reactivity / Inclusivity	Ability to detect various SARS-CoV-2 strains and variants.	All 5 wet-tested strains (Hong Kong, England, South Africa, Japan, hCoV19/USA) detected at 100% (3/3 replicates) at 1000 copies/mL. In silico analysis showed 98.6% - 99.99% sequence homology with broad variant coverage (Omicron BA.4/BA.5, BA.2.12.1, BA.2.75).
Cross-Reactivity	No false positives when challenged with common respiratory pathogens or human nucleic acid.	0.0% detection across 47 tested organisms (viruses, bacteria, fungi, human genomic DNA, pooled human nasal fluid) for S gene and ORF1ab targets. IC detected at 100%. MERS-CoV showed 0.0% detection.
Potential Interfering Substances	No false negatives for COVID-19 detection in the presence of common nasal/respiratory substances.	100% detection for most substances (antibiotics, antivirals, nasal corticosteroids, etc.). Saliva showed 83.3% detection at 10% (v/v) but 100% at 5% (v/v), indicating interference at higher concentrations. Zanamivir 83.3% IC detection for 5/6 replicates.
Interference by Other Microorganisms	No inhibition of SARS-CoV-2 detection by other microorganisms.	100% detection of SARS-CoV-2 at 2x LoD for 46/47 co-present organisms. Lactobacillus plantarum 17-5 showed interference above 5x10^5 CFU/mL.
Carry-Over Contamination	No evidence of carry-over contamination.	No carry-over contamination observed during testing with high positive and negative samples.

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

Clinical Agreement Test Set:
- Total Samples: 1,150 prospective (fresh and/or frozen) samples collected.
- Samples analyzed: 1,011 samples (114 excluded due to insufficient evidence for media types, 24 invalid results, 1 indeterminate CRM result).
- Breakdown: 299 Nasopharyngeal Swabs (NPS) and 712 Nasal Swabs (NS).
- Provenance: Collected from four (4) geographically diverse collection sites, one of which was outside the United States (OUS). Samples were prospective (fresh and/or frozen).
- Timeframe: October 2020 to April 2021.
Reproducibility Test Set:
- Total replicates: 90 replicates per panel member (4 panel members), totaling 360 individual tests.
- Panel members: 2 contrived low positive (LP), 2 contrived moderate positive (MP), 1 positive control, 1 negative (UTM).
- Provenance: Tested at two (2) external clinical sites and one (1) internal site.
- Study Design: Each panel member tested in triplicate per run, for 2 runs per day, for 5 non-consecutive testing days. Each site had two operators.
Analytical Sensitivity (LoD) Test Set:
- NPS: 40 replicates for confirmation.
- NS: 20 replicates for confirmation.
- WHO International Standard: 40 replicates for confirmation.
Analytical Reactivity (Wet testing) Test Set:
- 3 replicates per strain for 5 SARS-CoV-2 strains.
Cross-Reactivity Test Set:
- 3 replicates per organism for 47 different viruses, bacteria, and fungi (some 6 replicates for Leptospira interrogans).
Potential Interfering Substances Test Set:
- 3 replicates per substance (some 6 replicates for saliva and Zanamivir).
Interference by Other Microorganisms Test Set:
- 3 replicates per organism (some 6 replicates for Lactobacillus plantarum 17-5).

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

The ground truth for the clinical agreement test set was established using a "Composite Reference Method (CRM)" based on three (3) COVID-19 EUA approved NAAT assays. The rule for CRM agreement was: "Two out of three positive results determined 'Detected' CRM and two out of three negative results determined 'Not Detected' CRM."

The document does not specify the number or qualifications of experts (e.g., medical technologists, clinical lab scientists, or physicians) who performed these NAAT assays or interpreted their results for the CRM. It's implied that these were standard laboratory personnel qualified to run EUA-approved molecular diagnostic tests.

For analytical studies (LoD, reproducibility, reactivity, cross-reactivity, interference), the ground truth was based on the known composition and concentration of the samples (e.g., spiked RNA, cultured organisms, negative matrix). No external experts beyond the study design team would have been needed for this type of ground truth establishment.

4. Adjudication Method for the Test Set

For the clinical agreement test set, the adjudication method for the ground truth (CRM) was clearly defined: "Two out of three positive results determined 'Detected' CRM and two out of three negative results determined 'Not Detected' CRM." This is a form of consensus-based adjudication, specifically a majority rule.

For other analytical studies, adjudication was not described as it involved pre-defined positive/negative samples rather than interpretive human judgment.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study focuses on the in vitro diagnostic performance of a molecular assay (RT-PCR) in a laboratory setting, not on the interpretative performance of human readers (e.g., radiologists) with or without AI assistance. Therefore, there is no discussion of human readers or an effect size of AI assistance.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) Was Done

Yes, the primary clinical performance and analytical studies are standalone (algorithm only) performance. The Simplexa™ COVID-19 Direct is an RT-PCR assay. Its "performance" refers to its ability to detect SARS-CoV-2 RNA based on its set algorithms for signal detection (Ct values for S gene and ORF1ab targets) and interpretation. The results (detected/not detected) are determined directly by the instrument and its software, not by a human interpreting images or complex patterns. The human involvement is in sample preparation and loading, and reviewing the qualitative output from the instrument.

7. The Type of Ground Truth Used

Clinical Agreement Test Set: Composite Reference Method (CRM) using results from three (3) COVID-19 EUA approved NAAT assays, with a "two out of three" majority rule for determining "Detected" or "Not Detected." This is a form of expert consensus based on other validated diagnostic tests.
Analytical Studies (Reproducibility, LoD, Reactivity, Cross-Reactivity, Interference): Known analytical truth established by spiking known concentrations of inactivated viral particles or other organisms into negative matrices. This is a laboratory-controlled ground truth.

8. The Sample Size for the Training Set

The provided document describes a premarket notification (510(k)) for an in vitro diagnostic device. For such devices, particularly RT-PCR assays, the "training set" typically refers to internal development and optimization data, rather than a distinct, formally defined "training set" for machine learning algorithms that would be tested on a separate "test set."

The document does not specify a numerical sample size for a training set. The assay's design (primers, probes, conditions) would have been developed and optimized internally by DiaSorin Molecular using various samples and experiments, but these are not enumerated as a specific "training set" in this regulatory submission. The "in silico inclusivity analysis" section points to the use of GISAID databases (millions of sequences) which could be considered a form of "training data" for validating the generalizability of the primer/probe design, but not as a conventional, labeled "training set" in a machine learning context.

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

Since a formal "training set" with established ground truth is not explicitly detailed in the way a machine learning model's training data would be, we can infer the following:

Assay Development & Optimization: The ground truth for the development phase would have been based on known positive and negative samples, viral loads, and various SARS-CoV-2 strains or synthetic genetic material. This involves standard molecular biology techniques where the presence or absence of the target nucleic acid, and its concentration, are experimentally determined and controlled.
In Silico Inclusivity: For the evaluation of primer/probe design against genetic variants, the "ground truth" is the published, annotated SARS-CoV-2 genome sequences available in the GISAID database. This involves bioinformatic analysis to determine sequence homology and potential binding efficacy.

Summary

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September 13, 2022

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

DiaSorin Molecular LLC Tara Viviani Director, Regulatory Affairs 11331 Valley View Street Cypress, California 90630

Re: K212147

Trade/Device Name: Simplexa COVID-19 Direct Regulation Number: 21 CFR 866.3981 Regulation Name: Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test Regulatory Class: Class II Product Code: OOX Dated: August 12, 2022 Received: August 15, 2022

Dear Tara Viviani:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Himani Bisht. Ph.D. Assistant Director Viral Respiratory and HPV Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K212147

Device Name Simplexa™ COVID-19 Direct

Indications for Use (Describe)

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.

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)

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Image /page/3/Picture/0 description: The image shows the logo for DiaSorin Molecular. The logo features a stylized DNA double helix in shades of green and blue on the left. To the right of the DNA graphic are the words "DiaSorin" in dark blue on the top line and "Molecular" in green on the bottom line.

Applicant	DiaSorin Molecular LLC.11331 Valley View StreetCypress, California 90630USA
Establishment Registration No.	2023365
Contact Person	Tara VivianiSenior Director, Molecular Regulatory AffairsTel. 562.240.6271Tara. Viviani@DiaSorin.com
Summary Date	September 13, 2022
Proprietary Name	Simplexa™ COVID-19 Direct
US Product Codes/Names andRegulation Numbers	QQX- Device to detect and identify nucleic acid targets in respiratoryspecimens from microbial agents that cause the SARS-CoV-2respiratory infection and other microbial agents when in a multi-target test (21 CFR § 866.3981)OOI - Instrumentation for clinical multiplex test systems (21 CFR §862.2570)
Classification	Class II
Predicate Devices	BioFire COVID-19 Test 2 (K211079)

Intended Use

Simplexa™ COVID-19 Direct REF MOL4150

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.

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 Description

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Image /page/4/Picture/0 description: The image shows the logo for DiaSorin Molecular. The logo consists of a stylized DNA helix in shades of green and blue on the left, followed by the text "DiaSorin" in dark blue on the top line and "Molecular" in green on the bottom line. The logo is clean and modern, suggesting a focus on biotechnology and molecular diagnostics.

associated accessories. 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 ORF 1ab 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.

Simplexa™ COVID-19 Direct REF MOL4150

Component Name	REF	EC Symbolon Label	AbbreviatedName	CapColor	Numberof Vials	ReactionsperVial/Kit	Volumeper Vial
Simplexa™ COVID-19Direct Reaction Mix	MOL4151	REAG	Co19	Brown	24	1/24	50 $ μ $ L

Simplexa™ COVID-19 Direct Components and Descriptions

Kit Component	Contents
Simplexa™COVID-19 DirectReaction Mix (RM)	DNA polymerase, reverse transcriptase, RNase inhibitor, buffer, dNTPs, encapsulated RNA template (Internal Control), fluorescent probes and corresponding forward and reverse primers specific for detection of SARS-CoV-2 RNA and for the RNA Internal Control.
	Target	ProbeFluorophore(Dye)	Excitation (nm)	Emission(nm)	Targeted Gene
	S gene	FAM	445-505	507-533	S gene
	ORF1ab	JOE	503-543	547-573	ORF1ab
	InternalControl RNA(IC)	Q670	622-658	652-708	N/A
Simplexa™COVID-19 DirectBarcode	Assay specific parameters and lot information

Materials Supplied Separately

Direct Amplification Disc Kit (REF MOL1455) - Direct Amplification Discs for use on the LIAISON® MDX

Materials Required But Not Supplied

LIAISON® MDX with LIAISON® MDX Studio Software version 1.1 or higher.

Simplexa™ COVID-19 Positive Control Gen II Pack (REF MOL4165).

50 µL fixed volume pipette (VWR Signature™ Fixed Volume Ergonomic High-Performance Pipettor Model VWR FE50 or equivalent).

Sterile, nuclease-free disposable pipette tips with filters (Extra Long tips ≥ 91 mm are recommended for pipetting directly from primary collection tubes).

Freezer (manual defrost) at -10 to -30 °C (for kit component and/or specimen frozen storage).

Refrigerator at 2 to 8 °C (for specimens).

Disposable, powder-free gloves.

Vortex for mixing patient samples.

Centrifuge for collecting contents to bottom of tubes.

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510(k) Summary Simplexa™ COVID-19 Direct MOL4150 September 12, 2022 Page 3 of 13

Comparison to Predicate Device

Comparison toPredicate Device	Predicate Device:BioFire COVID-19 Test 2 (K211079)	Candidate Device:Simplexa COVID-19 DirectSimplex COVID-19 Positive Control Gen II Pack
Product Code	QQX	Same
RegulationNumber	21 CFR 866.3981	Same
OrganismDetected	SARS-CoV-2	Same
Measurand	RNA from SARS-CoV-2	Same
Intended Use Kit	The BioFire COVID-19 Test 2 is aqualitative nested multiplexed RT-PCR invitro diagnostic test intended for use withthe BioFire FilmArray 2.0 and BioFireFilmArray Torch Systems. The BioFireCOVID-19 Test 2 detects nucleic acids fromsevere acute respiratory syndromecoronavirus 2 (SARS-CoV-2) innasopharyngeal swabs (NPS) fromsymptomatic individuals suspected ofCOVID-19 by their healthcare provider.Results are for the identification of SARS-Co V-2 RNA. The SARS-Co V-2 RNA isgenerally detectable in NPS specimensduring the acute phase of infection. Positiveresults are indicative of the presence ofSARS-CoV-2 RNA; clinical correlation withpatient history and other diagnosticinformation is necessary to determinepatient infection status. Positive results donot rule out co-infection with otherpathogens. Results are meant to be used inconjunction with other clinical,epidemiologic, and laboratory data, inaccordance with the guidelines provided bythe relevant public health authorities. TheBioFire COVID-19 Test 2 is intended for useby trained medical and laboratoryprofessionals in a laboratory setting orunder the supervision of a trained laboratoryprofessional.	Simplexa™ COVID-19 Direct REF MOL4150The DiaSorin Molecular Simplexa™ COVID-19Direct is a real-time RT-PCR assay intended foruse on the LIAISON® MDX instrument for the invitro qualitative detection of nucleic acid fromsevere acute respiratory syndrome coronavirus2 (SARS-CoV-2) in nasopharyngeal swabs(NPS) and nasal swabs (NS) from symptomaticindividuals suspected of COVID-19 by theirhealthcare provider. The Simplexa™ COVID-19Direct assay is an aid in the diagnosis of SARS-CoV-2 infection.Positive results are indicative of the presence ofSARS-CoV-2 RNA. Clinical correlation withpatient history and other diagnostic informationis necessary to determine patient infectionstatus. Positive results do not rule out co-infection with other pathogens. Negative resultsdo not preclude SARS-CoV-2 infection andshould not be used as the sole basis for patientmanagement decisions.Results are meant to be used in conjunctionwith other clinical, epidemiologic, and laboratorydata, in accordance with the guidelinesprovided by the relevant public healthauthorities.
AutomatedSystem (Sampleto Answer)	Automated	Same
Instrumentation	BioFire® FilmArray® 2.0 or BioFire®FilmArray® Torch Systems	LIAISON MDX
Sample Types	Nasopharyngeal Swab	Nasopharyngeal Swab, Nasal Swab

CLINICAL AGREEMENT

The clinical performance of the Simplexa™ COVID-19 Direct was established in multi-site clinical evaluation. A total of 1,150 prospective fresh and/or prospective frozen Nasopharyngeal (NPS) and/or

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510(k) Summarv Simplexa™ COVID-19 Direct MOL4150 September 12, 2022 Page 4 of 13

Nasal Swabs (NS) samples were collected from four (4) geographically diverse collection sites, one of which was outside the United States (OUS). A total of 409 NPS samples and a total of 741 nasal swabs were collected. One hundred fourteen (114) samples were excluded from the analysis due to insufficient evidence to include certain media types, 24 were excluded due to invalid results, and one (1) specimen was excluded due to an indeterminate result from the composite reference method. The Simplexa™ COVID-19 Direct Clinical Agreement testing was performed at three (3) external clinical sites and one (1) internal site from October 2020 to April 2021. Three COVID-19 EUA approved NAAT assays were used to establish a comparator reference method (CRM) to evaluate the performance of the candidate device. Two out of three positive results determined "Detected" CRM and two out of three negative results determined "Not Detected" CRM. The clinical performance of Simplexa™ COVID-19 Direct is summarized in the Tables 1-3 below.

Table 1: Total Specimen Clinical Summary: Simplexa™ COVID-19 Direct vs. EUA NAAT CRM

Simplexa™ COVID-19 DirectProspective	CRM
Upper Respiratory	Detected	Not Detected	Total
Detected	108	4	112
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)95% CI: 98.9% to 99.8%

Table 2: NPS Clinical Summary: Simplexa™ COVID-19 Direct vs. EUA NAAT CRM

Simplexa™ COVID-19 DirectProspective NPS	CRM
	Detected	Not Detected	Total
Detected	60	1a	61
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%

ª One sample positive by Simplexa was detected one of the NAATs included in the CRM.

CRM = Composite Reference Method, PPA = Percent Positive Agreement, NPA = Negative Percent Agreement

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Image /page/7/Picture/0 description: The image shows the logo for DiaSorin Molecular. The logo consists of a stylized DNA double helix on the left, with the words "DiaSorin" in dark blue on the right. Below "DiaSorin" is the word "Molecular" in green. The DNA helix is colored with a gradient from green to blue.

Table 3: NS Collection: Simplexa™ COVID-19 Direct vs. EUA NAAT CRM

Simplexa™ COVID-19 DirectProspectiveNS	CRM
	Detected	Not Detected	Total
Detected	48	3a	51
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%

a One sample positive by Simplexa was positive by one of the NAATs in the CRM.

CRM = Composite Reference Method, PPA = Percent Positive Agreement, NPA = Negative Percent Agreement

REPRODUCIBILITY

The Simplexa™ COVID-19 Direct reproducibility study was performed at two (2) external clinical sites and one (1) internal site to evaluate repeatability, between operator, between site and total reproducibility of the assay. The panel consisted of a total of four (4) panel members including two (2) contrived panel samples, one (1) positive control and one (1) universal transport media (UTM) neqative sample. The contrived panel members were prepared at approximately one to two times (1x to 2x) the Limit of Detection (LoD) for a low positive (LP) and three to five times (3x to 5x) LoD for a moderate positive (MP). Both contrived panel members were built with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 spiked into native negative nasopharyngeal swab matrix. Each panel member was tested in triplicate per run for two (2) runs per day for a total of five (5) non-consecutive testing days at three (3) testing sites for a total of ninety (90) replicates. Each site had two (2) operators who each assayed the entire panel once per day, for a total of two (2) sets of data per day. The quantitative summary of variance components for each panel member based on the algorithm is in Table 4 below.

		Table 4. Simplexa™ COVID-19 Direct Reproducibility

		Site 1		Site 2		Site 4		All Sites
Sample		Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	95% CI
NPS_WA-1_LP	S gene	93.3%(28/30)	31.6 ±0.95(3.0%)	90.0%(27/30)	32.4 ±1.14(3.5%)	100.0%(30/30)	31.6 ±0.72(2.3%)	94.4%(85/90)	88% -98%
	ORF1ab	90.0%(27/30)	32.2 ±0.97(3.0%)	96.7%(29/30)	32.7 ±1.18(3.6%)	100.0%(30/30)	31.9 ±1.09(3.4%)	95.6%(86/90)	89% -98%
	Totala	100.0%(30/30)	N/A	96.7%(29/30)	N/A	100.0%(30/30)	N/A	98.9%(89/90)	94% -100%
NPS_WA-1_MP	S gene	100.0%(30/30)	30.5 ±0.80(2.6%)	90.0%(27/30)	30.8 ±1.11(3.6%)	96.7%(29/30)	31.1 ±1.14(3.7%)	95.6%(86/90)	89% -98%

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Simplexa™ COVID-

Sample		Site 1		Site 2		Site 4		All Sites
		Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	Avg.Ct ±SD(%CV)	Agree-ment withExpectedResults	95% CI
	ORF1ab	100.0%(30/30)	31.3 ±0.87(2.8%)	100.0%(30/30)	31.6 ±1.39(4.4%)	100.0%(30/30)	30.7 ±0.98(3.2%)	100.0%(90/90)	96% -100%
	Totala	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(90/90)	96% -100%
Negative(UTM)	S gene	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(90/90)	96% -100%
	ORF1ab	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(30/30)	0.0 ±0.00(N/A%)	100.0%(90/90)	96% -100%
	Totala	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(90/90)	96% -100%
PositiveControl(PC)	S gene	100.0%(30/30)	25.8 ±0.26(1.0%)	100.0%(30/30)	25.7 ±0.18(0.7%)	100.0%(30/30)	25.7 ±0.42(1.6%)	100.0%(90/90)	96% -100%
	ORF1ab	100.0%(30/30)	26.3 ±0.32(1.2%)	100.0%(30/30)	26.0 ±0.12(0.5%)	100.0%(30/30)	25.8 ±0.36(1.4%)	100.0%(90/90)	96% -100%
	Totala	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(30/30)	N/A	100.0%(90/90)	96% -100%
Total	S gene	98.3% (118/120)		95.0% (114/120)		99.2% (119/120)		97.5%(351/360)	95% -99%
	ORF1ab	97.5% (117/120)		99.2% (119/120)		100.0% (120/120)		98.9%(356/360)	97% -100%
	Totala	100.0% (120/120)		99.2% (119/120)		100.0% (120/120)		99.7%(359/360)	98% -100%

"Total based on algorithm that at least one target (S gene or ORF lab) is Detected for the sample to be Detected for SARS-CoV-2.

ANALYTICAL SENSITIVITY/LIMIT OF DETECTION

The Limit of Detection (LoD) for NPS and NS was determined to be the lowest detectable concentration of inactivated viral particles (copies/mL) for strain 2019-nCoV/USA-WA1/2020 at which ≥ 95% of all replicates tested positive. Initially, the tentative LoD was identified with serial dilutions of the inactivated viral particles in five (5) replicates for NPS matrix in UTM or four (4) replicates for NS matrix in UTM. The lowest concentration at which all replicates were positive was interpreted as the tentative LoD. For NPS, the LoD was then confirmed by testing forty (40) replicates with concentrations at the tentative LoD while for NS, the LoD was confirmed by testing twenty (20) replicates with concentrations at the tentative LoD. For both NPS and NS, the final LoD was confirmed to be the lowest concentration resulting in positive detection with a minimum 95% positivity. Table 5 shows the results of the studies.

The final LoD for NPS, according to the assay results interpretation was 500 copies/mL. The LoD for NS, according to the assay results interpretation was 242 copies/mL.

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Simplexa™ COVID

Table 5: Analytical Sensitivity/Limit of Detection for Inactivated Viral Particles in NPS or NS Matrix in UTM

SpecimenType	SARS-CoV-2LoD(copies/mL)	% Detection(# Detected / # Tested)	S gene (FAM)% Detection(# Detected/#Tested)	Mean Ct ± SD(%CV)	ORF1ab (JOE)% Detection(# Detected/#Tested)	Mean Ct ± SD(%CV)
NPS	500	100% (40/40)a	90% (36/40)	32.5 ± 1.60(4.9%)	100% (40/40)	31.6 ± 1.56(4.9%)
NS	242	100% (20/20)a	80% (16/20)	34.0 + 0.83(2.4%)	80% (16/20)	32.9 + 0.75(2.3%)

Ct = Cycle threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation

ª % Detection based on algorithm that at least one target (S gene or ORF1ab) is Detected for SARS-CoV-2.

The Limit of Detection (LoD) using inactivated SARS-CoV-2 WHO International Standard viral particles in NPS matrix was determined. The tentative LoD was identified with serial dilutions of the viral particles. The final LoD was confirmed to be the lowest concentration quantified in International Units per milliliter (U/mL) resulting in positive detection with a minimum 95% positivity. Table 6 shows the results of the study.

The final LoD for NPS, according to the assay results interpretation was 500 IU/mL..

		Table 6: Analytical Sensitivity/Limit of Detection for SARS-CoV-2 WHO International Standard

SARS-CoV-2WHOInternationalStandard(IU/mL)	% Detection (#Detected / # Tested)	S gene (FAM)		ORF1ab (JOE)
		% Detection (#Detected / #Tested)	Mean Ct $ \pm $ SD(%CV)	% Detection (#Detected / #Tested)	Mean Ct $ \pm $ SD (%CV)
500	97.5% (39/40)	95% (38/40)	33.2 $ \pm $ 1.28 (3.8%)	85% (34/40)	33.6 $ \pm $ 1.36 (4.1%)

Ct = Cycle threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, IU = International Units

FDA SARS-CoV-2 Reference Panel Testing: The evaluation of sensitivity and MERS-CoV cross-reactivity was performed using reference material (T1), testing blinded samples and according to a standard protocol provided by the FDA. The study included a range finding study and a confirmatory study for the Limit of Detection (LoD). Blinded sample testing was used to establish specificity and to confirm the LoD. The results are summarized in Table 7.

	Table 7: Summary of LoD Confirmation Result using the FDA SARS-CoV-2 Reference Panel

Reference Materials Provided by FDA	Specimen Type	Product LoD	Cross-Reactivity
SARS-CoV-2	NPS	6 x 103 NDU/mL	N/A
MERS-CoV		N/A	ND

NDU/mL = RNA NAAT detectable units/mL, N/A = Not applicable, ND = Not detected, NPS = Nasopharyngeal Swab

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Simplexa™ COVID ge 8 of 13

REACTIVITY/INCLUSIVITY

In Silico

An in silico inclusivity analysis of the COVID-19 target primers and probes in the Simplexa™ COVID-19 Direct assay was performed. All primer and probe sets designed for detection of the ORF1ab and S gene were tested against the complete SARS-CoV-2 genome sequences available in the GISAID database submitted from November 01, 2021 to January 31, 2022. The analysis included 2,170,584 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 2,170,382 sequences (~99.99%) and were predicted to be detected by the assay based on sequence homology. There were 202 sequences (~0.01%) with mismatches in at least one primer or probe binding region, region in either ORF1ab or S gene target region. A melting temperature (Tm) analysis was conducted for those sequences with mismatches in the binding sites of both gene assay target regions. A Tm calculation was performed with assay-specific conditions using a Tm Mismatch Bioinformatics Tool. Tm values observed above their respective annealing temperature had mismatches that were not located at the 3' end for the primers; as such, detection of these sequences is not affected by the mismatches.

An additional in silico inclusivity analysis was performed for complete SARS-CoV-2 genome sequences available in the GISAID database submitted from February 01, 2022 to April 30, 2022 including sequences of the Omicron BA.4 and BA.5 subvariants. The analysis included 377,668 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 372,411 sequences (~98.6%) and were predicted to be detected by the assay based on sequence homology. There were 5213 (~1.4%) sequences with no mismatches for one gene oligo set (either ORF1ab or S gene), and there were 44 sequences (~0.01%) 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 as described above and the results demonstrated that detection of these sequences is not affected by the mismatches. Table 8 below summarizes the Tm analysis results.

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 (~98.7%) and were predicted to be detected by the assay based on sequence homology. There were 2602 (~1.2%) sequences with no mismatches for one gene oligo set (either ORF1ab or S gene), and there were 40 sequences (~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 as described above and the results demonstrated that detection of these sequences is not affected by the mismatches. Table 8 below summarizes the Tm analysis results.

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510(k) S Simplexa™ COVID-19 Direct September Page 9 of 13

Table 8. Summary of Tm Analysis Results
Timeframe of SequencesAnalyzed	Number ofaccessions in GISAIDDatabase for thetimeframe	Number of sequenceswhere at least onetarget oligo set meetsTm 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

Analytical reactivity was evaluated using five (5) strains of SARS-CoV-2 viral particles that were available. All five (5) strains of SARS-CoV-2 (Hong Kong/VM200001061/2020, England/204820464/2020, South Africa/KRISP-EC-K005325/2020, Japan/TY7-503/2021, hCoV19/USA/PHC658/2021) were detected at a concentration of 1000 copies/mL All replicates of the test samples had a result of "Detected" according to the interpretation algorithm.

Table 9. Analytical Reactivity Results

COVID-19 Strain	Tested Concentration	COVID-19 Qualitative Results% Detection (# Detected/#Tested)
Hong Kong/VM200001061/2020	1000 copies/mL	100% (3/3)
England/204820464/2020	1000 copies/mL	100% (3/3)
South Africa/KRISP-ECK005325/2020	1000 copies/mL	100% (3/3)
Japan/TY7-503/2021	1000 copies/mL	100% (3/3)
hCoV19/USA/PHC658/2021	1000 copies/mL	100% (3/3)

CROSS-REACTIVITY

Cross-reactivity of the Simplexa™ COVID-19 Direct assay was evaluated using both in silico analysis and by testing whole organisms or purified nucleic acid from other organisms. Analytical specificity/crossreactivity was tested using 47 different viruses, bacteria, and fungi that could be found in nasopharyngeal swab specimens. In addition, pooled human nasal fluid was tested to represent diverse microbial flora. Test specimens for laboratory testing were prepared by spiking cultured isolates/inactivated organisms/purified nucleic acids (whole genome) (i.e., a minimum of 10° CFU/mL or higher for bacteria and typically 105 TCIDsolmL or PFU/mL or higher for viruses) into COVID-19 negative pooled NPS matrix collected in UTM All samples were tested in three replicates. The results from the cross-reactivity testing are summarized in Table 10.

Organism	TestedConcentration1	Qualitative Results: % Detection(# Detected/#Tested)
		S gene(FAM)	ORF1ab(JOE)	IC(Q670)
Adenovirus C (Type 1)	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Adenovirus 7A	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Bordetella pertussis	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Candida albicans	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Chlamydophila pneumoniae	1 x 106 IFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Chlamydophila psittaci (genomic DNA)	1 x 106 copies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
	Tested	Qualitative Results: % Detection
Organism		(# Detected/#Tested)
	Concentration1	S gene(FAM)	ORF1ab(JOE)	IC(Q670)
Corynebacterium diphtheriae	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Coxiella burnetii (genomic DNA)	1 x 106 copies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Cytomegalovirus	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Enterovirus 68	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Epstein-Barr virus	1 x 105 copies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Escherichia coli	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Haemophilus influenzae	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human coronavirus 229E*	3 x 104 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human coronavirus HKU1 (RNA)	1 x 105 genomecopies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human coronavirus NL63*	3 x 104 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human coronavirus OC43	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human genomic DNA (Leukocytes)	1 x 106 cells/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Human metapneumovirus (hMPV)*	3 x 104 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Influenza A/Perth/16/2009	1 x 105 EID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Influenza B/Florida/02/06	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Lactobacillus plantarum 17-5	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Legionella longbeachae	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Legionella pneumophila	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Leptospira interrogans	1:10 Dilution	0.0% (0/6)	0.0% (0/6)	100.0% (6/6)
Measles	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
MERS-coronavirus	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Moraxella catarrhalis	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Mumps	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Mycobacterium tuberculosis (genomicDNA)	1 x 106 copies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Mycoplasma pneumoniae	1 x 106 CCU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Neisseria elongata	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Neisseria meningitidis	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Parainfluenza virus 1	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Parainfluenza virus 2	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Parainfluenza virus 3	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Parainfluenza virus 4	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Parechovirus 3	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Pseudomonas aeruginosa	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Respiratory syncytial Virus A	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Respiratory syncytial Virus B	1 x 105 TCID50/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Rhinovirus	1 x 105 U/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
SARS-coronavirus (RNA)	1 x 105 copies/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Staphylococcus aureus	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Staphylococcus epidermidis	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Streptococcus pneumoniae	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Streptococcus pyogenes	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Streptococcus salivarius	1 x 106 CFU/mL	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)
Pooled Human Nasal Fluid	1:1 Dilution	0.0% (0/3)	0.0% (0/3)	100.0% (3/3)

Table 10: Laboratory Tested Cross-Reactivity Analysis

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510(k) Summary Simplexa™ COVID-19 Direct MOL4150 September 12, 2022

Page 10 of 13

*A lower concentration was tested due to inability to obtain stock material with high titer

1CCU/mL = Color Changing Units/milliter, CFU/mL = Colony Forming Units/milliliter, IFU/mL = Infectious units/milliliter, U/mL = Units/milliliter, TCID50/mL = Tissue Culture Infectious Dose/milliliter

Bacillus anthracis, Influenza C and Pneumocystis jirovecii were not available. In addition, human coronavirus 229E, human coronavirus NL63 and human metapneumovirus (hMPV) were not available at a high enough concentration to permit wet testing at the concentration levels stated above. As a result of in silico BLAST analysis, the assay was found not to cross-react with these organisms.

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POTENTIAL INTERFERING SUBSTANCES

Potential interfering substances from respiratory specimens were tested for ability to generate false negative results. The test samples contained inactivated SARS-CoV-2 at a concentration of 3x LoD in pooled negative nasopharyngeal swab matrix. Testing was performed with three (3) replicates per substance. The FluMist nasal vaccine was not tested as an interfering substance since it was unavailable at the time of the study.

Potentially InterferingSubstance	ActiveIngredient	TestedConcentration	COVID-19QualitativeResults: %Detection(# Detected/#Tested)	IC QualitativeResults: %Detection(# Detected/#Tested)
Antibiotic nasal ointment(Mupirocin)	Mupirocin	6.6 mg/mL	100.0% (3/3)	100.0% (3/3)
Anti-viral drug (Oseltamivir)	Oseltamivir	3.3 mg/mL	100.0% (3/3)	100.0% (3/3)
Cold Eeze (Throat lozenges,Oral anesthetic and analgesic)	Zincumgluconicum 2X	2.5% (w/v)	100.0% (3/3)	100.0% (3/3)
Homeopathic allergy reliefmedicine	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)	Luffaopperculata,Galphimiaglauca,histaminumhydrochloricum,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)
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)

Table 11: Potential Interfering Substances

*Interference from saliva was observed at a concentration above 5%

mg = milligram, mL = milliliter, v/y = volume, w/v = weight to volume, ug = microgram, NA = Not Applicable

INTERFERENCE BY OTHER MICROORGANISMS

The Simplexa™ COVID-19 Direct assay was evaluated by testing the ability to identify SARS-CoV-2 when other potentially inhibitory organisms were present. The panel of forty-seven (47) potentially inhibitory organisms was individually spiked into a pool with a low concentration at two times (2x) LoD of inactivated COVID-19 viral particles in NPS matrix. In addition, pooled human nasal fluid was tested to represent diverse microbial flora. For organisms not titered in CFU/mL, other industry acceptable units were used as indicated. Samples were assayed in triplicate to screen for potential inhibition. No inhibition by other organisms was observed at the concentrations indicated in Table 12. Lactobacillus plantarum 17-5 showed interference when tested above (5 x 105 CFU/mL).

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510(k) Summary Simplexa™ COVID-19 Direct MOL4150 September 12, 2022 Page 12 of 13

Organism	TestedConcentration¹	COVID-19 QualitativeResults: % Detection(# Detected /#Tested)
Adenovirus C (Type 1)	1 x 10⁵ U/mL	100.0% (3/3)
Adenovirus 7A	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
Bordetella pertussis	1 x 10⁶ CFU/mL	100.0% (3/3)
Candida albicans	1 x 10⁶ CFU/mL	100.0% (3/3)
Chlamydia pneumoniae	1 x 10⁶ IFU/mL	100.0% (3/3)
Chlamydophila psittaci (genomic DNA)	2.5 x 10⁵ copies/mL*	100.0% (3/3)
Corynebacterium diphtheriae	1 x 10⁶ CFU/mL	100.0% (3/3)
Coxiella burnetii (genomic DNA)	1 x 10⁶ copies/mL	100.0% (3/3)
Cytomegalovirus	1 x 10⁵ U/mL	100.0% (3/3)
Enterovirus 68	1 x 10⁵ U/mL	100.0% (3/3)
Epstein-Barr virus	1 x 10⁵ copies/mL	100.0% (3/3)
Escherichia coli	1 x 10⁶ CFU/mL	100.0% (3/3)
Haemophilus influenzae	1 x 10⁶ CFU/mL	100.0% (3/3)
Human coronavirus 229E	1.5 x 10⁴ TCID₅₀/mL*	100.0% (3/3)
Human coronavirus HKU1 (RNA)	1 x 10⁵ genome copies/mL	100.0% (3/3)
Human coronavirus NL63	1.5 x 10⁴ U/mL*	100.0% (3/3)
Human coronavirus OC43	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
Human genomic DNA (Leukocytes)	1 x 10⁶ cells/mL	100.0% (3/3)
Human metapneumovirus (hMPV)	1.5 x 10⁴ TCID₅₀/mL*	100.0% (3/3)
Influenza A/Perth/16/2009	1 x 10⁵ EID₅₀/mL	100.0% (3/3)
Influenza B/Phuket/3073/2013	1 x 10⁵ CEID₅₀/mL	100.0% (3/3)
Lactobacillus plantarum 17-5**	5 x 10⁵ CFU/mL	100.0% (6/6)
Legionella longbeachae	1 x 10⁶ CFU/mL	100.0% (3/3)
Legionella pneumophila	1 x 10⁶ CFU/mL	100.0% (3/3)
Leptospira interrogans	1:10 Dilution	100.0% (6/6)
Measles	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
MERS-coronavirus	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
Moraxella catarrhalis	1 x 10⁶ CFU/mL	100.0% (3/3)
Mumps	1 x 10⁵ U/mL	95.0% (19/20)
Mycobacterium tuberculosis (genomic DNA)	1 x 10⁶ copies/mL	100.0% (3/3)
Mycoplasma pneumoniae	1 x 10⁶ CCU/mL	100.0% (3/3)
Neisseria elongata	1 x 10⁶ CFU/mL	100.0% (3/3)
Neisseria meningitidis	1 x 10⁶ CFU/mL	100.0% (3/3)
Parainfluenza virus 1	1 x 10⁵ U/mL	100.0% (3/3)
Parainfluenza virus 2	1 x 10⁵ U/mL	100.0% (3/3)
Parainfluenza virus 3	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
Parainfluenza virus 4	1 x 10⁵ U/mL	100.0% (3/3)
Parechovirus 3	1 x 10⁵ U/mL	100.0% (3/3)
Pseudomonas aeruginosa	1 x 10⁶ CFU/mL	100.0% (3/3)
Respiratory syncytial virus A	5 x 10⁴ TCID₅₀/mL*	100.0% (3/3)
Respiratory syncytial virus B	1 x 10⁵ TCID₅₀/mL	100.0% (3/3)
Rhinovirus	1 x 10⁵ U/mL	100.0% (3/3)
SARS-Coronavirus (RNA)	1 x 10⁵ copies/mL	100.0% (3/3)
Staphylococcus aureus	1 x 10⁶ CFU/mL	100.0% (3/3)
Staphylococcus epidermidis	1 x 10⁶ CFU/mL	100.0% (3/3)
Streptococcus pneumoniae	1 x 10⁶ CFU/mL	100.0% (3/3)
Streptococcus pyogenes	1 x 10⁶ CFU/mL	100.0% (3/3)
Streptococcus salivarius	1 x 10⁶ CFU/mL	100.0% (3/3)

Table 12. Simplexa™ COVID-19 Direct – Microbial Interference

*A lower concentration was tested due to inability to obtain stock material with high titer

**Interfernce with Lactobacillus plantarum 17-5 was observed at a concentration above 5 x 105 CFU/mL.

1CCU = Color changing units/milliliter, CFU/mL = Colony forming units/milliiter, IFU/ml = Infectious units/milliliter, U/mL = Units/milliliter, TCID50/mL = Tissue Culture Infectious Dose per milliliter

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CARRY-OVER CONTAMINATION

Amplification carry-over for the Simplexa™ assays has been assessed against existing assays that use the same sample matrices, workflow and specimen type, and therefore no carry-over is anticipated. The study was designed by alternately placing high positive and negative samples on each disc. No evidence of carry-over contamination was observed.

Conclusion

The analytical and method comparison studies have demonstrated that the Simplexa™ COVID-19 Direct is Substantially Equivalent to the predicate device (K211079). The device labeling is compliant with 21 CFR ട്ട 809.10.

Regulation Number and Section

§ 866.3981 Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test.

(a)
Identification. A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
e.g., BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
e.g., when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
e.g., recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
e.g., regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
e.g., saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads if infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to State or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
i.e., H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.