Positive results are indicative of the presence of SARS-CoV-2 RNA. The Aptima SARS-CoV-2 Assay is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiological, and laboratory findings. Clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

Device Description

The Aptima SARS-CoV-2 Assay is a nucleic acid amplification in vitro diagnostic test developed for use on the fully automated Panther/Panther Fusion system to detect RNA from SARS-CoV-2 isolated and purified from nasopharyngeal and anterior nasal swab specimens collected into UTM/VTM or with the RespDirect Collection Kit.

The Aptima SARS-CoV-2 Assay combines the technologies of target capture, Transcription Mediated Amplification (TMA), and Dual Kinetic Assay (DKA).

Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the RNA target and protect them from degradation during storage. When the Aptima SARS-CoV-2 Assay is performed in the laboratory, the target RNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. The capture oligomer:target complex is then captured out of solution by decreasing the temperature of the reaction to room temperature. This temperature reduction allows hybridization to occur between the deoxyadenosine region on the capture oligomer and the poly-deoxythymidine molecules that are covalently attached to the magnetic particles. The microparticles, including the captured target molecules bound to them, are pulled to the side of the reaction vessel using magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima SARS-CoV-2 Assay replicates specific regions of the RNA from SARS-CoV-2 virus. Detection of the RNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent nucleic acid probes, which are unique and complementary to a region of each target amplicon and Internal Control (IC) amplicon, are labeled with different acridinium ester (AE) molecules. The AE-labeled probes combine with the amplicon to form stable hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from the unhybridized probe. During the detection step, light emitted from the labeled hybrids is measured as photon signals in a luminometer and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time.

The Aptima SARS-CoV-2 Assay amplifies and detects 2 conserved regions of the ORF1ab gene in the same reaction, using the "glower" kinetic type. The 2 regions are not differentiated and amplification of either or both regions lead to RLU signal. The assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

AI/ML Overview

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

Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the performance metrics presented as evidence of substantial equivalence to the predicate device. The key metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). While explicit numerical acceptance thresholds are not provided in the document, the reported performance demonstrates "comparable" results to the predicate device.

Performance Metric	Acceptance Criteria (Implicit)	Reported Device Performance (NP Swab, Overall)	Reported Device Performance (AN Swab, UTM/VTM)	Reported Device Performance (AN Swab, RespDirect eSTM)
Clinical Performance:
Positive Percent Agreement (PPA)	High Agreement	95.4% (90.3-97.9% CI)	96.5% (92.1-98.5% CI)	100% (96.6-100% CI)
Negative Percent Agreement (NPA)	High Agreement	99.5% (98.9-99.8% CI)	97.6% (96.5-98.4% CI)	98.0% (96.9-98.7% CI)

Analytical Performance (Key):
Limit of Detection (LoD) - Cultured Virus	Extremely Low Concentration	0.01 TCID50/mL	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Limit of Detection (LoD) - WHO Standard	Extremely Low Concentration	87.5 IU/mL	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Reactivity (detection of strains)	100% Positivity at low concentrations	Demonstrated at 0.03-0.151 TCID50/mL for tested strains; 99.98% by in silico analysis	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Specificity/Microbial Interference	No cross-reactivity/interference	No observed cross-reactivity/interference for 48 tested organisms	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Interfering Substances	No impact on performance	No impact on performance for tested substances	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Carryover Contamination Rate	Low/None	0% (0/294)	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)
Assay Precision (Total %CV of signal)	Low variability	2.75% to 3.84% (Negative, Low, Moderate Positive panels)	Not applicable (analytical, not specimen-type specific)	Not applicable (analytical, not specimen-type specific)

Study Details:

Sample Sizes Used for the Test Set and Data Provenance:
- Clinical Study 1 (NP Swab Specimens):
  - Total Enrolled: 1646 specimens
  - Evaluable (Final Data Set): 1495 NP swab specimens (1195 fresh, 300 frozen)
  - Provenance: Prospective multicenter study from four participating US pediatric/adolescent, private and/or university hospitals. Specimens collected between June-July 2020 and January-April 2023. Remnant specimens.
- Clinical Study 2 (Anterior Nasal Swab Specimens):
  - Total Enrolled Subjects: 2301
  - Evaluable Subjects: 2177 individuals (1159 with evaluable anterior nasal swab specimens in UTM/VTM, and 1018 with evaluable nasal swab specimens in eSTM).
  - Provenance: Prospective, multicenter clinical study at nine geographically and ethnically diverse US sites during the 2022-2023 respiratory season.
Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications:
- The document does not specify the number or qualifications of experts directly establishing the clinical ground truth. Instead, it relies on a Composite Comparator Algorithm (CCA). The CCA consists of "two highly sensitive US FDA EUA SARS-CoV-2 molecular tests" and a "validated PCR followed by bi-directional sequencing (PCR/BDS) assay." This implies that the 'expertise' comes from the validation and regulatory clearance of these comparator assays, rather than individual human experts adjudicating each case.
Adjudication Method for the Test Set:
- Composite Comparator Algorithm (CCA): "A final CCA result was assigned when two of the three comparator assay results were in concordance." This serves as the adjudication method for determining the true positive/negative status of the clinical samples.
If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done:
- No. This type of study (MRMC) is typically performed for diagnostic imaging devices where human readers interpret images with and without AI assistance. The Aptima SARS-CoV-2 Assay is an in vitro diagnostic (IVD), a laboratory test that detects nucleic acids, not an imaging device requiring human interpretation of visual data. Therefore, an MRMC study is not applicable here.
If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done:
- Yes, this is effectively a standalone performance study. The Aptima SARS-CoV-2 Assay is an automated nucleic acid amplification test run on the Panther/Panther Fusion system. Its performance is directly compared to the CCA, without human intervention in the interpretation of the device's results.
The Type of Ground Truth Used:
- Clinical Studies: Composite Comparator Algorithm (CCA) based on the concordance of results from two highly sensitive FDA EUA SARS-CoV-2 molecular tests and a validated PCR/BDS assay. This is a form of reference standard derived from established and highly sensitive laboratory methods.
- Analytical Studies (e.g., LoD, Reactivity, Specificity): The ground truth was established by known concentrations of SARS-CoV-2 virus strains or other microorganisms/interfering substances, prepared in controlled laboratory settings (e.g., "spiked with inactivated cultured SARS-CoV-2 virus").
The Sample Size for the Training Set:
- The document describes performance evaluation studies (analytical and clinical) for market clearance. It does not provide information on the specific training set size used for the development or training of the assay's internal algorithms (e.g., for the kinetic curve analysis or cut-off determination). This information would typically be part of the assay development and validation, not necessarily detailed in a 510(k) summary unless it significantly changed or impacted performance during the clearance process for the specific assay rather than the underlying platform. The focus here is on the performance of the final, already "trained" device.
How the Ground Truth for the Training Set Was Established:
- As noted above, details regarding the training set's ground truth establishment are not provided in this 510(k) summary. For IVD devices, ground truth for training internal algorithms typically involves using characterized positive and negative clinical samples, spiked samples with known viral loads, and potentially synthetic data, all carefully confirmed by highly sensitive reference methods or gold standard assays during the R&D phase of the product.

Summary

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Image /page/0/Picture/0 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.

February 13, 2025

Hologic, Inc. Katerina Capkova Regulatory Affairs Manager 10210 Genetic Center Dr. San Diego, California 92121

Re: K243396

Trade/Device Name: Aptima SARS-CoV-2 Assay 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: October 31, 2024 Received: October 31, 2024

Dear Katerina Capkova:

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 (the 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 available 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.

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (OS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 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 Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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 medical devices and radiation-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-regulatory

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assistance/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,

Anna M. Mielech -S

Anna Mielech, PhD. Deputy Branch Chief (Acting) Viral Respiratory and HPV Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics 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) K243396

Device Name Aptima SARS-CoV-2 Assay

Indications for Use (Describe)

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

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

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510(k) SUMMARY

Aptima SARS-CoV-2 Assay

Hologic, Inc. I. SUBMITTER: 10210 Genetic Center Drive San Diego, CA 92121

Contact Person: Katerina Capkova, PhD Regulatory Affairs Manager katerina.capkova@hologic.com Phone: 858.410.8167 Fax: N/A
Date Prepared: February 11, 2025

II. DEVICE

Proprietary Name:	Aptima SARS-CoV-2 Assay
Classification Name:	Respiratory Specimen Nucleic Acid SARS-CoV-2 Test
Regulation Number:	21 CFR 866.3981
Regulatory Class:	Class II
Product Code:	QQX

III. PREDICATE DEVICE

The predicate device for the Aptima SARS-CoV-2 Assay is the BioFire COVID-19 Test 2 (K211079; cleared November 1, 2021, BioFire Defense, LLC).

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IV. DEVICE DESCRIPTION

The Aptima SARS-CoV-2 Assay combines the technologies of target capture, Transcription Mediated Amplification (TMA), and Dual Kinetic Assay (DKA).

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hybridization. Single-stranded chemiluminescent nucleic acid probes, which are unique and complementary to a region of each target amplicon and Internal Control (IC) amplicon, are labeled with different acridinium ester (AE) molecules. The AE-labeled probes combine with the amplicon to form stable hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from the unhybridized probe. During the detection step, light emitted from the labeled hybrids is measured as photon signals in a luminometer and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time.

Assay Components

The Aptima SARS-CoV-2 Assay is available in 100-test and 250-test kit configuration. In both cases, the assay kit consists of two boxes containing nine reagents which are required for sample processing. In addition, the controls kit contains the positive and negative controls. RespDirect Collection Kit is an ancillary component of the assay. The Panther Fusion Specimen Lysis Tubes are also an ancillary kit to this assay required for processing of Viral Transport Media/Universal Transport Media (VTM/UTM) specimens prior to testing on the Panther/Panther Fusion system. In addition, there is one ancillary kit (Aptima Assay Fluids Kit) which is universally used with other commercialized Aptima assays.

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Table 1: Reagents Required to Perform the Aptima SARS-CoV-2 Assay

Box	Components Description
Assay Kit-Refrigerated	Amplification Reagent
	Enzyme Reagent
	Promoter Reagent
	Internal Control
Assay Kit-RoomTemperature	Amplification Reconstitution Solution
	Enzyme Reconstitution Solution
	Promoter Reconstitution Solution
	Selection Reagent
	Target Capture Reagent
Controls Kit-Refrigerated	Negative Control
	Positive Control

Table 2: Ancillary and Collection Kits Required to Perform the Aptima SARS-CoV-2 Assay

RespDirect Collection Kit
Panther Fusion Specimen Lysis Tubes
Aptima Assay Fluids Kit

Instrumentation

The Aptima SARS-CoV-2 Assay has been designed for and validated on the Panther/Panther Fusion system. The Panther Panther Fusion system is an integrated hardware and software system that together with the Aptima SARS-CoV-2 Assay fully automates all the steps necessary to perform the assay from sample preparation through amplification of nucleic acid, detection, data reduction and amplicon inactivation.

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V. INDICATIONS FOR USE

The Aptima® SARS-CoV-2 Assay is a nucleic acid amplification in vitro diagnostic test intended for the qualitative detection of RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated and purified from nasopharyngeal (NP) swab and anterior nasal (AN) swab specimens obtained from patients with signs and symptoms of COVID-19. Positive results are indicative of the presence of SARS-CoV-2 RNA. The Aptima SARS-CoV-2 Assay is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiological, and laboratory findings. Clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis

for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

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Image /page/9/Picture/0 description: The image is the logo for Hologic. The logo is in dark blue and consists of the word "HOLOGIC" in large, bold letters. To the right of the word is a registered trademark symbol. Below the word is a series of vertical lines, followed by the phrase "The Science of Sure" in a smaller font.

VI. COMPARISON OF TECHNOLOGICAL CHARACTERISTICS WITH THE PREDICATE DEVICE

A comparison of the Aptima SARS-CoV-2 Assay to the predicate BioFire COVID-19 Test 2 (K211079) is summarized in Table 3.

Table 3: Comparison of Similarities and Differences Between the Subject Device (Aptima SARS-CoV-2 Assay) and the Predicate Device (BioFire COVID-19 Test 2)

Item	AptimaSARS-CoV-2 Assay(Subject Device)	BioFire COVID-19 Test 2(Predicate Device)K211079
Prescription/over-the-counter use	Prescription Only	Same
PatientPopulation	Individuals with signs and symptoms of COVID-19	Same
Specimen Types	Nasopharyngeal (NP) and nasal swab specimens	Nasopharyngeal (NP) swab specimens
Intended User	Professional use	Same
TechnologyPrinciple ofOperation	Transcription-mediated amplification NAAT	Reverse transcriptase multiplexed polymerase chainreaction test
OrganismsDetected	SARS-CoV-2	Same
Assay Controls	Internal and run controls	Internal and external controls
Platform	Automated nucleic acid amplification platform.Uses Panther/Panther Fusion system for all stepsincluding nucleic acid extraction, amplification,detection, and result processing.	Automated nucleic acid amplification platform.Uses BioFire FilmArray 2.0 or BioFire FilmArrayTorch systems including integrated sample preparation,amplification, detection, and analysis.
Intended Use	The Aptima® SARS-CoV-2 Assay is a nucleic acidamplification in vitro diagnostic test intended for thequalitative detection of RNA from severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2)isolated and purified from nasopharyngeal (NP) swaband anterior nasal (AN) swab specimens obtained frompatients with signs and symptoms of COVID-19.Positive results are indicative of the presence ofSARS-CoV-2 RNA. The Aptima SARS-CoV-2 Assayis intended for use as an aid in the diagnosis ofCOVID-19 if used in conjunction with other clinical,epidemiological, and laboratory findings. Clinicalcorrelation with patient history and other diagnosticinformation is necessary to determine patient infectionstatus. Positive results do not rule out bacterialinfection or co-infection with other viruses.Negative results do not preclude SARS-CoV-2infection and should not be used as the sole basis forpatient management decisions. Negative results mustbe combined with clinical observations, patient history,and epidemiological information.	The BioFire COVID-19 Test 2 is a qualitative nestedmultiplexed RT-PCR in vitro diagnostic test intendedfor use with the BioFire FilmArray 2.0 and BioFireFilmArray Torch Systems. The BioFire COVID-19 Test2 detects nucleic acids from severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2) innasopharyngeal swabs (NPS) from symptomaticindividuals suspected of COVID-19 by their healthcareprovider.Results are for the identification of SARS-CoV-2 RNA.The SARS-CoV-2 RNA is generally detectable in NPSspecimens during the acute phase of infection. Positiveresults are indicative of the presence of SARS-CoV-2RNA; clinical correlation with patient history and otherdiagnostic information is necessary to determine patientinfection status. Positive results do not rule out co-infection with other pathogens.Results are meant to be used in conjunction with otherclinical, epidemiologic, and laboratory data, inaccordance with the guidelines provided by the relevantpublic health authorities. The BioFire COVID-19 Test 2is intended for use by trained medical and laboratoryprofessionals in a laboratory setting or under thesupervision of a trained laboratory professional.
Time to ObtainTest Results	Approximately 2.5 hours	Approximately 45 minutes

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VII. PERFORMANCE DATA

The following performance data (analytical and clinical) were provided in support of the substantial equivalence determination.

Brief Description of Analytical (Non-Clinical) Studies

The following analytical studies (non-clinical) were conducted to support the clearance of the Aptima SARS-CoV-2 Assay on the Panther/Panther Fusion System.

Analytical Sensitivity - Limit of Detection (LoD)

The analytical sensitivity (limit of detection or LoD) of the Aptima SARS-CoV-2 Assay was determined by testing dilutions of processed negative clinical NP swab VTM/UTM matrix spiked with inactivated cultured SARS-CoV-2 virus (USA-WA1/2020; BEI Resources; NR-52281) and WHO International Standard for SARS-CoV-2, NIBSC (20/146). For the cultured virus, ten replicates of each serial dilution were evaluated for each of two assay reagent lots across two Panther systems. The LoD was determined to be 0.01 TCID55/mL in the test sample (0.026 TCID50/mL in the neat, unprocessed sample) and verified by testing an additional minimum 20 replicates with one assay reagent lot. For the WHO International Standard, a minimum of 24 replicates were tested with each of the three reagent lots using Probit analysis for each lot and was confirmed with an additional 24 replicates using a single lot. The lowest concentration at which ≥95% detection was observed was 87.5 IU/mL . LoD confirmation was also performed with the RespDirect Collection Kit at 24 replicates with a single reagent lot and ≥95% detection was determined to be 87.5 IU/mL.

Note: The stated LoDs pertain to the concentrations in the tubes loaded onto the instrument. For samples collected in VTM/UTM, this is the concentration in the processed sample in an SLT. For samples collected using the RespDirect Collection kit, this is the concentration in the Enhanced Direct Load tube (RespDirect Collection Kit).

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Reactivity - Wet Testing

The reactivity of the Aptima SARS-CoV-2 Assay was determined by testing virus strains in processed negative clinical NP swab VTM/UTM matrix. Each strain was tested in triplicate at 3X LoD with one reagent lot. For strains not detected at 3X LoD, additional testing at higher concentrations was performed until 100% positivity was observed. Table 4 shows the lowest concentration of each strain in which 100% positivity was observed.

Description	Concentration
USA-WA1/2020*	0.03 TCID50/mL
USA-CA1/2020	0.03 TCID50/mL
USA-AZ1/2020	0.101 TCID50/mL
USA-WI1/2020	0.03 TCID50/mL
USA/OR-OHSU-PHL00037/2021 B.1.1.7	0.03 TCID50/mL
Uganda/MUWRP-20200195568/2020 A.23.1	0.03 TCID50/mL
USA/PHC658/2021 B.1.617.2	0.03 TCID50/mL
USA/MD-HP05285/2021 B.1.617.2	0.03 TCID50/mL
USA/CA/VRLC009/2021 B.1.427	0.03 TCID50/mL
USA/CA/VRLC012/2021 P.2	0.03 TCID50/mL
USA/MD-HP03056/2021 B.1.525	0.03 TCID50/mL
USA/CA-Stanford-15_S02/2021 B.1.617.1	0.03 TCID50/mL
Peru/un-CDC-2-4069945/2021 C.37	0.03 TCID50/mL
USA/MD-HP20874/2021 B.1.1.529	0.03 TCID50/mL
USA/GA-EHC-2811C/2021 B.1.1.529	0.03 TCID50/mL
USA/MD-HP30386/2022 BA.4	0.03 TCID50/mL
USA/COR-22-063113/2022 BA.5	0.03 TCID50/mL
South Africa/CERI-KRISP-K040013/2022 BA.5	0.03 TCID50/mL
USA/MD-HP38861/2022 BQ.1.1	0.03 TCID50/mL
USA/MD-HP40900/2022 XBB.1.5	0.101 TCID50/mL
USA/MD-HP47865/2023 XXB.2.3	0.03 TCID50/mL
USA/MD-HP46933/2023 EG.1.2	0.03 TCID50/mL
USA/MD-HP47946/2023 EG.5.1	0.03 TCID50/mL

Table 4: Analytical Reactivity Summary for SARS-CoV-2

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USA/CA-Stanford-139_S35/2023 XBB.1.9	0.101 TCID50/mL
USA/CA-Stanford-139_S23/2023 XBB.1.16	0.101 TCID50/mL
USA/MI-UM-10052670540/2023 BA.2.86	0.102 TCID50/mL
USA/New York-PV96109/2023 JN.1	0.151 TCID50/mL
USA/MD-HP49152/2023 HV.1	0.015 TCID50/mL

*Strain used to establish LoD.

1In silico analysis showed 100% homology to amplification regions.

21n silico analysis identified a single mismatch in the probe oligo for one region. Due to the mismatch and 100% homology to the second region, detection is not expected to be impacted. Virus stock degradation or error in TCID50mL quantification may have impacted the concentration at 100% detection.

Reactivity-In silico Analysis

The inclusivity of the Aptima SARS-CoV-2 Assay was evaluated using in silico analysis of the assay target capture oligos, amplification primers, and detection probes for the SARS-CoV-2 target systems in relation to sequences available in the NCBI and GISAID gene databases. Any sequence with missing or ambiguous sequence information was removed from the analysis for that region. Based on the in silico analysis of GISAID and NCBI sequences available for SARS-CoV-2 (10% random sampling of 16.553.661 million sequences up to July, 31, 2023 and all 508,436 sequences August 1, 2023 - January 31, 2024), the Aptima SARS-CoV-2 Assay is predicted to detect 99.98% (2,136,815/2,137,175 sequences) of all sequences evaluated.

The sequences evaluated included lineages and variants of concern (VOC) or variants under investigation (VUI) that may have important epidemiological, immunological, or pathogenic properties from the public health perspective. All lineages and variants of public health interest identified as of January 31, 2024 are predicted to be detected; new sequences and variants will continue to be monitored for impacts on detection by the Aptima SARS-CoV-2 Assay.

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Analytical Specificity and Microbial Interference

Analytical specificity (cross-reactivity) and microbial interference with the Aptima SARS-CoV-2 Assay were evaluated in the presence of closely related and non-targeted organisms. Panels consisting of 48 organisms (6) were tested in processed negative clinical NP swab VTM/ UTM matrix in the absence or presence of 3X LoD SARS-CoV-2. Bacteria were tested at 106 CFU/mL and viruses were tested at 105 TCID50/mL, except where noted. No crossreactivity or microbial interference was observed for any of the 48 organisms tested on the Aptima SARS-CoV-2 Assay at the indicated concentrations.

Microorganism	Concentration¹	Microorganism	Concentration¹
Adenovirus 1	1x105 TCID50/mL	Aspergillus fumigatus	1x106 CFU/mL
Adenovirus 7a	1x105 TCID50/mL	Bordetella parapertussis	1x106 CFU/mL
CMV Strain AD 169	5x103 TCID50/mL	Bordetella pertussis	1x106 CFU/mL
EBV	1x105 TCID50/mL	Candida albicans	1x106 CFU/mL
Enterovirus Type 71	1x105 TCID50/mL	Chlamydia pneumoniae	1x106 CFU/mL
Human coronavirus 229E	1x105 TCID50/mL	Corynebacterium diphtheriae	1x106 CFU/mL
Human coronavirus OC43	1x105 TCID50/mL	Escherichia coli	1x106 CFU/mL
Human coronavirus HKU12	1x106 copies/mL	Fusobacterium necrophorum	1x106 CFU/mL
Human coronavirus NL63	1x104 TCID50/mL	Haemophilus influenzae	1x106 CFU/mL
Human Metapneumovirus(hMPV)	1x106 TCID50/mL	Lactobacillus plantarum	1x106 CFU/mL
Influenza A	1x105 TCID50/mL	Legionella pneumophila	1x106 CFU/mL
Influenza B	2x103 TCID50/mL	Moraxella catarrhalis	1x106 CFU/mL
Measles	1x105 TCID50/mL	Mycobacterium tuberculosis	1x106 CFU/mL
MERS-coronavirus	1x104 TCID50/mL	Mycoplasma genitalium	1x106 CFU/mL
Mumps	1x105 TCID50/mL	Mycoplasma pneumoniae	1x106 CFU/mL
Parainfluenza virus 1	1x105 TCID50/mL	Neisseria gonorrhoeae	1x106 CFU/mL
Parainfluenza virus 2	1x105 TCID50/mL	Neisseria meningitides	1x106 CFU/mL
Parainfluenza virus 3	1x105 TCID50/mL	Neisseria mucosa	1x106 CFU/mL
Parainfluenza virus 4	1x103 TCID50/mL	Pneumocystis jirovecii (PJP)	1x106 nuclei/mL

Table 5: Aptima SARS-CoV-2 Analytical Specificity and Microbial Interference Microorganisms

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Respiratory syncytial virus	1x105 TCID50/mL	Pseudomonas aeruginosa	1x106 CFU/mL
Rhinovirus	1x104 TCID50/mL	Staphylococcus aureus	1x106 CFU/mL
SARS-coronavirus2	1x106 copies/mL	Staphylococcus epidermis	1x106 CFU/mL
Varicella Zoster Virus	1x104 TCID50/mL	Streptococcus pneumoniae	1x106 CFU/mL
Pooled human nasal wash3 – to represent diverse microbial flora in human respiratory tract	N/A	Streptococcus pyogenes	1x106 CFU/mL
		Streptococcus salivaris	1x106 CFU/mL

1CFU = Colony Forming Units; TCID50 = Median Tissue Culture Infectious Dose

²Cultured virus and whole genome purified nucleic acid for Human coronavirus were not readily available at the time testing was performed. Human coronavins IVTs corresponding to the ORFIab gene regions targeted by the assay were used to evaluate cross-reactivity and microbial interference.

31n place of evaluating pooled human nasal wash, testing of 30 individual negative clinical NP swab speciforned to represent diverse microbial flora in the human respiratory tract.

Interfering Substances

Interfering endogenous and exogenous substances (mucin, whole blood, potential medications and over-the-counter products) that may be present in the samples were evaluated in the Aptima SARS-CoV-2 assay. Clinically relevant concentrations of potentially interfering substances were added to pooled clinical negative NP swab VTM/UTM matrix and tested in the absence and presence of SARS-CoV-2 inactivated virus at 3X LoD. The substances and concentrations are shown in Table 6. No impact on the performance of the Aptima SARS-CoV-2 Assay was seen for any of the substances at the concentration tested.

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		Table 6: Potentially Interfering Substances

Substance Type	Substance Name	Active Ingredient(s)	Highest TestConcentration
Endogenous	Mucin	Purified mucin protein	60 µg/mL
Endogenous	Blood (human)	N/A	2% v/v
Nasal sprays ordrops	Neo-Synephrine®	Phenylephrine	15% v/v
Nasal sprays ordrops	Anefrin	Oxymetazoline	15% v/v
Nasal sprays ordrops	Saline	Sodium chloride	15% v/v
Nasal corticosteroids	Ventolin HFA2	Albuterol	45 ng/mL
Nasal corticosteroids	QVAR® Beconase AQ2	Beclomethasone	15 ng/mL
Nasal corticosteroids	Dexacort2	Dexamethasone	12 µg/mL
Nasal corticosteroids	Flonase	Fluticasone	5% v/v
Nasal corticosteroids	Nasacort	Triamcinolone	5% v/v
Nasal corticosteroids	Rhinocort	Budesonide	5% v/v
Nasal corticosteroids	Nasonex2	Mometasone	0.5 ng/mL
Nasal corticosteroids	AEROSPAN®2	Flunisolide	9.9 µg/mL
Nasal gel	Zicam® (Allergy Relief)	Luffa opperculata, Galphimia,Glauca, Histaminumhydrochloricum, Sulfur	5% v/v
Throat lozenges	Cepacol Extra Strength	Benzocaine, Menthol	0.7 mg/mL
Throat lozenges	Cold-Eeze throat lozenge	Zinc gluconate	0.7 mg/mL
Anti-viral drugs	Relenza2	Zanamivir	3.3 mg/mL
Anti-viral drugs	TamiFlu2	Oseltamivir	399 ng/mL
Anti-viral drugs	Virazole2	Ribavirin	10.5 µg/mL
Antibiotic, nasal	Bactroban cream2	Mupirocin	1.6 µg/mL
Antibacterial,	Tobramycin2	Tobramycin	33.1 µg/mL
Solvent Control	Water	N/A	5% v/v
Solvent Control	Dimethyl Sulfoxide (DMSO)	N/A	5% v/v

¹v/v: volume by volume

2Active ingredient tested, not substance

3T wo out of three positive results were observed for the positive pool containing Nasacort and retested and all replicates (3/3) were positive.

Carryover Contamination

The carryover contamination rate of the Aptima SARS-CoV-2 Assay for samples tested was assessed by testing high titer panels consisting of SARS-CoV-2 virus in negative clinical NP swab VTM/UTM matrix spiked 100 TCID56/mL (10,000 times the assay LoD). Positive panels were tested in a checkerboard pattern, alternating with negative panels. Testing consisted of 588

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negative and positive valid tests across three Panther systems. The Aptima SARS-CoV-2 Assay observed a carryover rate of 0% (0/294).

Assay Precision

The Aptima SARS-CoV-2 Assay within-lab precision was evaluated with a 4-member panel consisting of virus in negative clinical NP swab VTM/UTM matrix. The 4-member panel included a Negative, a High Negative (0.1X LoD), a Low Positive (1X LoD) and a Moderate Positive (5X LoD) panels were tested by two operators, using three reagent lots on three Panther systems over six days, at one site. Two runs were performed per operator per day for a total minimum of 36 runs. Each of the four panels was tested in three replicates per run for a total of 108 replicates per panel. The agreement with expected results was 100% in the Negative, Low Positive and Moderate Positive panel members. The High Negative panel member was 10X below the assay LoD, therefore a mix of positive and negative results were expected. This panel had 68/108 (63%) positive results. Agreement with expected results for all four panels is shown in Table 7.

PanelDescription	PanelComposition	Panel Conc.TCID50/mL	ExpectedResult	NPositive	NTested	MeankRLU	Agreementw/Expected(95% CI)
Negative	N/A	N/A	Negative	0	108	289	100%(96.6-100)
HighNegative	0.1xLoD	0.001	N/A	68	108	627	N/A
LowPositive	1.0xLoD	0.01	Positive	108	108	1131	100%(96.6-100)
ModeratePositive	5.0xLoD	0.05	Positive	108	108	1147	100%(96.6-100)

Table 7: Agreement of Aptima SARS-CoV-2 Assay Results with Expected Results

The total SARS-CoV-2 signal variability measured as %CV ranged from 2.75% to 3.84% in Negative, Low Positive, and Moderate Positive panel members. For the sources of variation all six factors evaluated had %CV values <3.0% as shown in Table 8. The High Negative panel

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member is 10x below the assay LoD and the %CV for this panel is expected to be higher than the others. The highest source of variability for this panel was within-run variability.

Table 8: kRLU Signal Variability of the Aptima SARS-CoV-2 Assay by Panel Member

Panel	Between Days		Between Instruments		Between Operators		Between Lots		Between Runs		Within Runs		Total
	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)
Negative	0.91	0.31	4.97	1.72	0.0	0.0	4.04	1.40	0.0	0.0	6.75	2.33	9.35	3.23
High Negative*	30.45	4.85	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	244.08	38.91	245.97	39.21
Low Positive	6.46	0.57	6.74	0.60	0.0	0.0	28.10	2.48	0.0	0.0	31.77	2.81	43.43	3.84
Moderate Positive	8.53	0.74	5.59	0.49	0.0	0.0	22.98	2.00	11.06	0.96	15.59	1.36	31.59	2.75

*Panel was built to 10x below the assay LoD. Higher variability is expected in this panel.

vent that variability from son

Collection Device Equivalency

Equivalence between NP specimens collected into VTM/UTM and NP and AN swab specimens collected in RespDirect (eSTM) was evaluated by testing individual negative specimens and contrived positive panels prepared from paired negative clinical samples collected from patients with symptoms of COVID-19. Contrived panels were prepared by spiking individual donor paired NP specimens, and AN swab specimens for RespDirect only, with SARS-CoV-2 to 2X and 5X LoD. The results of the negative and contrived panels demonstrated similar agreement comparable between the two collection devices (Table 9).

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Table 9: Results of negative and contrived panels composed of paired individual donor clinical specimens (NP for VTM/UTM and NP/nasal swab for RespDirect), collected with each collection device spiked with SARSCoV-2

Analyte	SampleConcentration	N per CollectionDevice	VTM/UTM-NP% Positive	RespDirect-NP% Positive	RespDirect-nasalswab% Positive
None(Negative Sample)	0	150	0	0	0
SARS-CoV-2	2X LoD	50	100	100	100
	5X LoD	50	100	100	100

Reproducibility

Aptima SARS-CoV-2 Assay reproducibility was evaluated at three US sites using one negative and two positive panel members. Testing was performed using one lot of assay reagents and six operators (two at each site). At each site, testing was performed for at least five days. Each run had three replicates of each panel member.

A negative panel member was created using pooled negative clinical NP swab specimens in VTM/UTM processed into STM (i.e., negative matrix). Positive panel members were created by spiking 1-2X LoD (low positive) or 3-5X LoD (moderate positive) concentrations of SARS-CoV-2 inactivated virus into the negative matrix. The agreement with expected results was 100% for all panel members. The total SARS-CoV-2 signal variability, measured as %CV, was ≤7.93% (SD less than or equal to 91.35) for all positive panel members (Table 10).

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Table 10: kRLU Signal Variability of the Aptima SARS-CoV-2 Assay by Panel Member

			Between Sites		BetweenOperators/Runs1		Between Days		Within Runs		Total
PanelDescription	N	MeankRLU	SD	CV(%)	SD	CV (%)	SD	CV(%)	SD	CV (%)	SD	CV(%)
Negative	90	286.0	27.04	9.45	25.42	8.89	0.45	0.16	6.55	2.29	37.69	13.18
SARS-CoV-2Low Pos	90	1152.2	67.79	5.88	15.16	1.32	25.06	2.18	53.77	4.67	91.35	7.93
SARS-CoV-2Mod Pos	90	1163.7	77.30	6.64	36.60	3.15	4.10	0.35	26.67	2.29	89.68	7.71

CV = coefficient of variation, Mod = moderate, Pos = positive, kRLU = relative light unit x 1000, SD = standard deviation.

¹ Between Operator may be confounded with Between Run; therefore, Between Operator and Between Run estimates are combined in Between Operator/Run.

Brief Description of Clinical Studies

Two clinical studies were performed. Aptima SARS-CoV-2 Assay clinical performance was estimated in prospectively collected NP specimens in Clinical Study 1 and in prospectively collected AN swab specimens in Clinical Study 2.

Clinical Study 1: Prospective Clinical Study - Nasopharyngeal Swab Specimens in UTM/VTM

This study was performed to demonstrate clinical performance characteristics for the Aptima SARS-CoV-2 Assay in NP swab specimens. A prospective multicenter study was conducted using remnant NP swab specimens from male and female individuals of all ages exhibiting signs and/or symptoms of respiratory infection consistent with COVID-19. Four participating US pediatric/adolescent, private and/or university hospitals prospectively provided remnant NP swab specimens stored in viral transport medium (VTM). These specimens were tested at three US sites with the Aptima SARS-CoV-2 Assay. The Aptima SARS-CoV-2 Assay was evaluated for SARS-CoV-2 performance by comparing its results from NP swab specimens in UTM/VTM to a composite comparator algorithm (CCA) consisting of two highly sensitive US FDA EUA SARS-

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CoV-2 molecular tests and a validated PCR followed by bi-directional sequencing (PCR/BDS) assay. A final CCA result was assigned when two of the three comparator assay results were in concordance. Of the 1646 specimens enrolled during the study, 300 were collected between June 2020 and July 2020, while the remaining 1346 were collected between January 2023 and April 2023. A total of 1646 NP swab specimens were tested in valid Aptima SARS-CoV-2 Assay runs, including 9 (0.5%) with initial invalid results. Upon retest, all 1646 specimens yielded final valid results. The final data set consisted of 1495 evaluable NP swab specimens, including 1195 (79.9%) tested fresh and 300 (20.1%) tested after freezing; 149 NP swab specimens were excluded from analysis due to mishandling at the sites. Demographic information for the 1495 evaluable individuals is provided in Table 11.

Total		1495
Sex	Female	842 (56.3%)
	Male	651 (43.5%)
	Unknown	2 (0.1%)
	Mean	33.3
	Median	29.0
	Range	0 - 98
Age (years)	<5	270 (18.1%)
	5-21	373 (24.9%)
	22-59	499 (33.4%)
	≥60	353 (23.6%)

Table 11: Summary of Subject Demographics for Evaluable Prospectively Collected NP Swab Specimens

The performance of the Aptima SARS-CoV-2 Assay with prospective NP swab specimens is summarized in Table 12. Positive Percent Agreement (PPA) was calculated as 100% × (TP / (TP + FN)). True positive (TP) indicates that both the Aptima SARS-CoV-2 assay and the CCA had a positive result for SARS-CoV-2, and false negative (FN) indicates that the Aptima SARS-CoV-2 Assay result was negative while the CCA was positive. Negative Percent Agreement

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(NPA) was calculated as 100% × (TN / (TN + FP)). True negative (TN) indicates that both the Aptima SARS-CoV-2 assay and the CCA had negative results, and false positive (FP) indicates that the Aptima SARS-CoV-2 Assay result was positive while the CCA was negative. NP specimens that obtained discordant results underwent additional testing with a US FDA EUA SARS-CoV-2 molecular test, volume permitting.

Table 12: Aptima SARS-CoV-2 Assay Performance with NP Swab Specimens

NP SwabSpecimen Type		Positive Percent Agreement			Negative Percent Agreement
	TP/(TP+FN)	%	95% CI1	TN/(FP+TN)	%	95% CI1
Fresh2	80/82	97.6	91.5-99.3	1107/1113	99.5	98.8-99.8
Frozen2	44/48	91.7	80.4-96.7	251/252	99.6	97.8-99.9
Overall	124/1303	95.4	90.3-97.9	1358/13654	99.5	98.9-99.8

CI = confidence interval, FN = false negative, FP = false positive, TN = true negative, TP = true positive.

¹ Score Cl.

2 All fresh samples were collected in 202

3 One (1) specimen with a false negative result tested negative the test, while 4 tested positive and 1 had an inconclusive result usin

4 One (1) specimen with a false positive result tested positive for SARS while 5 tested negative and 1 had no result using the same

Clinical Study 2: Prospective Clinical Study – Anterior Nasal Swab Specimens in UTM/VTM and eSTM (RespDirect Collection Kit)

This study was performed to demonstrate clinical performance characteristics for the Aptima SARS-CoV-2 Assay in anterior nasal swab specimens. The clinical performance of the Aptima SARS-CoV-2- 2 Assay was evaluated using anterior nasal swab specimens collected in a prospective, multicenter clinical study. Male and female individuals of all ages exhibiting signs and/or symptoms of respiratory infection consistent with COVID-19 were enrolled at nine geographically and ethnically diverse US sites during the 2022-2023 respiratory season. Two anterior nasal swab specimens were prospectively collected from each individual (in a clinical setting): one specimen collected using a synthetic flocked swab by a healthcare professional

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(HCP) and stored in UTM/VTM; one specimen collected by the patient (under HCP supervision) or the HCP using either a synthetic flocked swab and stored in UTM/VTM or using the RespDirect flocked swab and stored in a Direct Capture Tube containing eSTM (RespDirect Collection Kit). The Aptima SARS-CoV-2 Assay was evaluated for SARS-CoV-2 performance by comparing its results from anterior nasal swab specimens in UTM/VTM or in eSTM to a composite comparator algorithm (CCA) consisting of two highly sensitive US FDA EUA SARS-CoV-2 molecular tests and a validated PCR/BDS assay. A final CCA result was assigned when two of the three comparator assay results were in concordance. Of the 2301 enrolled subjects, six did not meet eligibility criteria and were withdrawn. A total of 2241 specimens in UTM/VTM and eSTM from 2295 non-withdrawn subjects were tested in valid Aptima SARS-CoV-2 Assay runs, including 23 (1.0%) with initial invalid results. Upon retest, 13 specimens yielded valid results and 10 yielded final invalid results, for a total of 2231 (99.6%) specimens with final valid results. An additional 118 subjects were not evaluable due to specimen withdrawal, missing/invalid Aptima results, or an unknown CCA result, leaving 2177 individuals evaluable for the performance analyses, including 1159 with evaluable anterior nasal swab specimens in UTM/VTM, and 1018 with evaluable nasal swab specimens in eSTM. Demographic information for the 2177 evaluable individuals is provided in Table 13.

Total		2177
Sex	Female	1287 (59.1%)
	Male	890 (40.9%)
Age (years)	Mean	40.7
	Median	40.0
	Range	0 - 90
COVID-19 vaccination status	Fully vaccinated	1451 (66.7%)
	Partially vaccinated	106 (4.9%)
	Unvaccinated	601 (27.6%)
	Unknown	19 (0.9%)

Table 13: Summary of Subject Demographics for Prospectively Collected Nasal Swab Specimens

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The performance of the Aptima SARS-CoV-2 Assay with prospective anterior nasal swab specimens is summarized in Table 14. PPA and NPA percent agreement were calculated as described for Clinical Study 1.

Table 14: Aptima SARS-CoV-2 Assay Performance with Anterior Nasal Swab Specimens

Nasal SwabSpecimen Type	Positive Percent Agreement			Negative Percent Agreement
	TP/(TP+FN)	%	95% CI1	TN/(FP+TN)	%	95% CI1
UTM/VTM	138/143	96.5	92.1-98.5	992/1016	97.6	96.5-98.4
RespDirect eSTM	108/108	100	96.6-100	892/910	98.0	96.9-98.7

Cl = confidence interval, eSTM = enhanced specimen transport medium, FN = false negative, TN = true negative, TP = true positive, UTMVTM = universal/viral transport medium.

1 Score CI.

VIII. CONCLUSIONS

The analytical and clinical study results demonstrate that the Aptima SARS-CoV-2 Assay on the Panther/Panther Fusion system performs comparably to the predicate device that is currently marketed for the same intended use.

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.