K Number

Device Name

DASH® SARS-CoV-2 & Flu A/B Test

Manufacturer

Nuclein, LLC

Date Cleared

2024-12-20

(196 days)

Product Code

QOF OOF

Regulation Number

866.3981

Intended Use

The DASH® SARS-CoV-2 & Flu A/B Test is a rapid reverse transcription polymerase chain reaction (RT-PCR) assay performed on the DASH Rapid PCR Instrument and is intended for the simultaneous in vitro qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B virus ribonucleic acid (RNA) in anterior nasal swab specimens from patients with signs and symptoms of respiratory tract infection. The test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza B in humans in conjunction with other clinical, epidemiologic and laboratory findings. Positive results of a specific target are indicative of that viral RNA and may not be the definite cause of disease. Positive results do not rule out co-infection with other pathogens. Negative results do not preclude SARS-COV-2, influenza A or influenza B infection and should not be used as the sole basis for patient management decisions.

Device Description

The DASH® SARS-CoV-2 & Flu A/B Test is a rapid, polymerase chain reaction (PCR) assay performed on the DASH Rapid PCR Instrument (DASH Instrument) with the DASH External Controls. The external control materials and DASH Instrument are sold and distributed separately from the DASH SARS-CoV-2 & Flu A/B Test. The DASH SARS-CoV-2 & Flu A/B Test (for use with the DASH Rapid PCR System components) uses reverse transcription polymerase chain reaction (RT-PCR) for rapid qualitative detection and differentiation of SARS-CoV-2, Flu A and Flu B from nasal swabs. The test combines the technologies of sequence specific capture sample preparation and RT-PCR amplification. The DASH SARS-CoV-2 & Flu A/B Test cartridge contains all reagents necessary to perform the test. An anterior nares nasal swab with a 30-mm breakpoint is used to collect a specimen. The nasal swab specimen is added directly to the DASH SARS-CoV-2 & Fly A/B Test cartridge sample chamber. The cartridge is capped and inserted into the DASH Rapid PCR Instrument to initiate the test, and all subsequent test steps are performed automatically by the DASH Instrument.

More Information

Contact

Type

Center

Panel

Date Received

Product Code

Subsequent Product Codes

Applicant Name (Manufacturer)

Device Name

Decision

Street 1

Street 2

City

State

Country Code

ZIP

Postal Code

Class Advise Committee

SSP Indicator

Third Party Reviewed

Expedited Review

Predicate Devices

K230719

Reference Devices

BioFire System and SPOTFIRE RSP Pos & Neg Controls, SPOTFIRE RSP Positive Control, SPOTFIRE RSP Negative Control

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard RT-PCR assay and instrument. There is no mention of AI, ML, or any algorithms beyond basic signal processing for PCR result interpretation. The focus is on the analytical and clinical performance of the molecular test itself.

Therapeutic

No.
The device is an in vitro diagnostic test intended for the qualitative detection and differentiation of specific viral RNA, aiding in diagnosis rather than providing treatment or therapy.

Diagnostic

Yes
The "Intended Use / Indications for Use" section explicitly states that the device is "intended for the simultaneous in vitro qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B virus ribonucleic acid (RNA)... The test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza B in humans in conjunction with other clinical, epidemiologic and laboratory findings." This clearly indicates its role in diagnosing diseases.

SaMD (Software as a Medical Device)

The device is described as a rapid reverse transcription polymerase chain reaction (RT-PCR) assay performed on a specific instrument (DASH Rapid PCR Instrument) using a test cartridge containing reagents. This clearly indicates the device includes hardware components (instrument, cartridge) and is not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the test is intended for "simultaneous in vitro qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B virus ribonucleic acid (RNA) in anterior nasal swab specimens". The term "in vitro" is a key indicator of an IVD, meaning it's used to test samples taken from the human body outside of the body.
Device Description: The description details a test performed on a specific instrument using a cartridge containing reagents. This is consistent with the format of an IVD test kit.
Sample Type: The test uses "anterior nasal swab specimens," which are biological samples taken from a patient.
Purpose: The test is intended to "aid in the differential diagnosis" of specific infections, which is a diagnostic purpose.
Performance Studies: The document includes detailed descriptions of analytical and clinical performance studies, which are required for the validation and regulatory approval of IVDs.
Predicate Device: The mention of a "Predicate Device" (K230719: BioFire SPOTFIRE Respiratory Panel Mini) is common in regulatory submissions for new IVDs, where the new device is compared to an already cleared IVD.

All of these elements strongly indicate that the DASH® SARS-CoV-2 & Flu A/B Test is an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Positive results of a specific target are indicative of that viral RNA and may not be the definite cause of disease. Positive results do not rule out co-infection with other pathogens. Negative results do not preclude SARS-COV-2, influenza A or influenza B infection and should not be used as the sole basis for patient management decisions.

Product codes (comma separated list FDA assigned to the subject device)

OOF, NSU

Device Description

The test combines the technologies of sequence specific capture sample preparation and RT-PCR amplification. The DASH SARS-CoV-2 & Flu A/B Test cartridge contains all reagents necessary to perform the test. An anterior nares nasal swab with a 30-mm breakpoint is used to collect a specimen. The nasal swab specimen is added directly to the DASH SARS-CoV-2 & Fly A/B Test cartridge sample chamber. The cartridge is capped and inserted into the DASH Rapid PCR Instrument to initiate the test, and all subsequent test steps are performed automatically by the DASH Instrument.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

anterior nasal swab specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

CLIA-waived point-of-care (POC) use. Untrained operators.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

A multi-site reproducibility study was conducted using contrived nasal swabs and a 3-member panel consisting of a true negative (no analyte), low positive (2X LoD), and a moderate positive (5X LoD) panel member. The study was conducted using three (3) lots of DASH SARS-CoV-2 & Flu A/B Test cartridges by nine (9) untrained operators across three (3) sites, and performed over five (5) non-consecutive days, at two (2) runs per day, with three (3) replicates per panel member, totaling over 810 samples (270 replicates per panel member) tested throughout the course of the study.

Positive samples were contrived using co-spiked inactivated SARS-Cov-2, Isolate hCoV- 19/USA/GA-EHC-2811C/2021, Lineage B.1.1.529; Omicron Variant, cultured Flu A H1N1 Victorial/2570/19, and Flu B Utah/9/14 - Yamagata Lineage.

Clinical Performance:
795 evaluable subjects for SARS-CoV-2 and 792 evaluable subjects for Flu A and Flu B.
Source: 7 geographical locations within the United States, prospective collection of specimens from individuals showing signs and symptoms of respiratory infection from January to March 2024.
Specimen type: anterior nares swabs from both sides of the nose were either healthcare provider-collected or adult-collected from individuals below 14 years, or self-collected from individuals aged 14 years or older.
Comparator: FDA cleared RT-PCR test for SARS-CoV-2, and a second FDA cleared test for Flu A and Flu B.
Discordant results were investigated using a third highly sensitive FDA cleared test.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance:

Analytical Sensitivity (Limit of Detection): LoD was determined for the DASH® SARS-CoV-2 & Flu A/B Test utilizing five (5) viral strains. All samples were prepared in pooled nasal matrix (PNM).
- SARS-CoV-2 (Omicron Variant): 1200 copies/swab
- Flu A (H1N1 Victoria/2570/19): 1.35 TCID50/swab
- Flu A (H3N2 /Darwin/9/21): 0.225 TCID50/swab
- Flu A (Washington/02/19 – Victoria Lineage): 0.10 TCID50/swab
- Flu B (Utah/9/14 - Yamagata Lineage): 0.675 TCID50/swab
Within-Laboratory Precision: 96 replicates per concentration of SARS-CoV-2, Flu A, and Flu B targets tested over a 12-day period by two (2) operators at a single site. Triple positive samples (SARS-CoV-2, Flu A, and Flu B) were prepared in simulated clinical nasal matrix (SCNM) at 2X and 5X LoD concentrations, and negative samples. Achieved 100% positivity for each target at 2X and 5X LoD, and 0% positivity for negative samples.
Reproducibility: A multi-site study with over 810 samples (270 replicates per panel member) tested (true negative, low positive (2X LoD), and moderate positive (5X LoD)) using three (3) lots of cartridges by nine (9) untrained operators across three (3) sites, over five (5) non-consecutive days with two (2) runs per day.
- All analytes: low positive: 99.3%-100% agreement with expected results; moderate positive: 99.6%-100% agreement with expected results; negative: 100% agreement with expected results.
Analytical Specificity (Cross-Reactivity & Microbial Interference):
- In silico Analysis: Completed on 1,217 non-target organisms.
- Cross-Reactivity Wet Testing: 50 different viruses, bacteria, and fungi tested individually in SCNM. None demonstrated cross-reactivity.
- Microbial Interference: Over 50 different viruses, bacteria, and fungi tested individually or in pooled groups in SCNM in the presence of test viral targets at 3X LoD. None demonstrated interference.
Competitive Interference: Evaluated by testing one viral target at high concentration while others were at 3xLoD. Demonstrated that high concentrations of one virus can inhibit detection of others at 3xLoD.
Exogenous/Endogenous Interfering Substances: Evaluated medical and non-medical substances in SCNM with test targets at 3X LoD. Interference observed with Biotin (4.58 µg/mL) and Flonase® (5% v/v), but not at lower concentrations. No interference observed for other substances tested.
Analytical Reactivity (Inclusivity): Wet testing of 7 strains of SARS-CoV-2, 21 strains of Flu A, and 10 strains of Flu B at concentrations near LoD. 100% positive call rate for 3 replicates of each strain. In silico analysis performed on SARS-CoV-2, Flu A, and Flu B sequences from NCBI databases showed high prediction of detection (Influenza A: >=99.97%; Influenza B: >=98.27%; SARS-CoV-2: >=99.99%).

Comparison Studies:

Matrix Equivalency: Compared clinical pooled nasal matrix (PNM) and simulated clinical nasal matrix (SCNM) using co-spiked samples at 2x and 5x LoD. Positive rates were 100% at 5x LoD and >=95% at 2x LoD for both matrices. All negative samples were negative.
Fresh vs. Frozen Contrived Samples: Established equivalency between freshly prepared samples and those subjected to two (2) freeze/thaw cycles for co-spiked samples at 2x and 5x LoD in SCNM. Positive rates were 100% at 5x LoD and >=95% at 2x LoD. All negative samples were negative.

Clinical Performance:

Accuracy and Ease of Use Study: Evaluated during the 2023-2024 respiratory season at 7 CLIA-waived POC sites in the US by untrained operators.
- Subject demographics: N=795, Mean age: 37.3 years, range 2-87. 58.5% Female, 41.5% Male.
- Comparison of results with FDA cleared RT-PCR tests.
- PPA and NPA calculated for each target.
- Invalid results: 24 instances (2.9%) out of 833 samples.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Clinical Performance Results:

SARS-CoV-2:
- Positive Percent Agreement (PPA): 160/168 (95.2%, 95% CI: 90.9% - 97.6%)
- Negative Percent Agreement (NPA): 624/627 (99.5%, 95% CI: 98.6% - 99.8%)
Flu A:
- Positive Percent Agreement (PPA): 50/53 (94.3%, 95% CI: 84.6% - 98.1%)
- Negative Percent Agreement (NPA): 725/739 (98.1%, 95% CI: 96.8% - 98.9%)
Flu B:
- Positive Percent Agreement (PPA): 36/37 (97.3%, 95% CI: 86.2% - 99.9%)
- Negative Percent Agreement (NPA): 749/755 (99.2%, 95% CI: 98.3% - 99.6%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K230719: BioFire SPOTFIRE Respiratory Panel Mini

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

BioFire System and SPOTFIRE RSP Pos & Neg Controls, SPOTFIRE RSP Positive Control, SPOTFIRE RSP Negative Control

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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.

Summary

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.

Nuclein, LLC Chelynne Lee Associate Director Design Assurance and Regulatory Affairs 8305 Cross Park Dr. Austin, Texas 78754

Re: K241652

Trade/Device Name: DASH SARS-CoV-2 & Flu A/B Test 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: OOF Dated: September 25, 2024 Received: September 25, 2024

Dear Chelynne Lee:

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.

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 (QS) 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 OS 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 (QS) 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 Rue"). 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-devices/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 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

Image /page/2/Picture/3 description: The image contains the letters FDA in a stylized font on the left side of the image. To the right of the letters, the name "Stefanie Akselrod -S" is written in a simple, sans-serif font. The name is written in two lines, with "Stefanie" on the top line and "Akselrod -S" on the bottom line. The letters FDA are in a light blue color, while the name is in black.

For

Himani Bisht, Ph.D. Assistant Director 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

Indications for Use

510(k) Number (if known) K241652

Device Name DASH® SARS-CoV-2 & Flu A/B Test

Indications for Use (Describe)

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

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

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510(k) Summary [21 CFR 807.95]

807.92(a)(1), (2), (3)
Date Prepared:	December XX, 2024
Submitter's Name:	Chelynne Lee
Nuclein, LLC.
8305 Cross Park Dr. Austin,
Tx 78754
+1 512-720-4935
Official Correspondent:	Chelynne Lee
Associate Director Design Assurance and Regulatory Affairs
+1 512-720-4935
Alternate Correspondent:	Ben Treece
VP, Quality Assurance and Regulatory Affairs
+1 512-649-3033
Device Trade Name:	DASH® SARS-CoV-2 & Flu A/B Test
Common Name:	DASH SARS-CoV-2 & Flu A/B Test
Review Panel:	Microbiology
Classification Name:	Multi-Target Respiratory Specimen Nucleic Acid Test Including Sars- Cov-
2 And Other Microbial Agents
Classification Code:	QOF: Multi-Target Respiratory Specimen Nucleic Acid Test Including
Sars-Cov-2 And Other Microbial Agents
NSU: Instrumentation For Clinical Multiplex Test Systems
Predicate Device:	K230719: BioFire SPOTFIRE Respiratory Panel Mini
Classification Code: QOF
Regulation: 866.3981

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Device Description 807.92(a)(4):

Intended Use 807.92(a)(5):

The DASH SARS-CoV-2 & Flu A/B Test is a rapid reverse transcription polymerase chain reaction (RT-PCR) assay performed on the DASH Rapid PCR Instrument and is intended for the simultaneous in vitro qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B virus ribonucleic acid (RNA) in anterior nasal swab specimens from patients with signs and symptoms of respiratory tract infection. The test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, and influenza B in humans in conjunction with other clinical, epidemiologic and laboratory findings.

Positive results of a specific target are indicative of that viral RNA and may not be the definite cause of disease. Positive results do not rule out co-infection with other pathoqens. Neqative results do not preclude SARS-CoV-2, influenza A or influenza B infection and should not be used as the sole basis for patient management decisions.

Technological Characteristics 807.92(a)(6):

The DASH SARS-CoV-2 & Flu A/B Test combines the technologies of sequence-specific capture and RT-PCR amplification. The test cartridge contains all the reagents necessary to perform the DASH SARS-CoV-2 & Flu A/B Test. The assay reagents are lyophilized and rehydrated with buffer as the test is performed by the DASH instrument. There are no usermediated sample preparation steps; an anterior nares swab (ANS) is placed directly into the cartridge sample chamber containing lysis buffer that is then closed. The cartridge is then placed in the DASH Instrument which performs all subsequent steps automatically.

The target RNA molecules are isolated from specimens using biotinylated target- specific capture oligomers which hybridize to specific regions on the target nucleic acids. After hybridization to target RNA, streptavidin-coated paramagnetic particles bind the biotinylated capture oligomers. The paramagnetic particles, including the bound target nucleic acids, are washed to remove residual specimen matrix that may contain amplification reaction inhibitors. After the target capture steps are completed, the target RNA is amplified. 40 PCR temperature cvcles are performed. During the 40 cycles, fluorescence is measured in a single chamber with a four-channel LED fluorometer. A software algorithm determines whether any of the targets are positive. The alqorithm includes the requirement for the internal control to have performed

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as intended, with its purpose to ensure adequate processing of the target virus through steps of sample purification, nucleic acid amplification, and monitoring for the presence of inhibitors in the RT-PCR test. External controls are sold separately and recommended to be used to confirm that the test cartridge and instrument are functioning as intended. Each control (positive or negative) is run as an individual sample in the exact method and process as a DASH® SARS-CoV-2 & Flu A/B Test patient sample.

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Predicate Comparison:

Predicate Comparison	Device & Predicate Device(s):	K241652	K230719
Device Trade Name	DASH® SARS-CoV-2 & Flu A/B Test	BIOFIRE® SPOTFIRE® Respiratory Panel Mini
Regulation Number and Name	SAME	21 CFR 866.3981; Devices to detect and identify nucleic acid targets in respiratory samples from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-analyte test.
Product Code	SAME		QOF
Technology/Detection	SAME, with exception of a single reaction chamber vs array/spot		PCR
Magnetic bead purification technology
Array/spot format
Intended Use	The DASH® SARS-CoV-2 & Flu A/B Test is a rapid reverse transcription polymerase chain reaction (RT-PCR) assay performed on the DASH Rapid PCR Instrument and is intended for the simultaneous in vitro qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B virus ribonucleic acid (RNA) in anterior nasal swab specimens from patients with signs and symptoms of respiratory tract infection. The test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, and influenza B in humans in conjunction with other clinical, epidemiologic and laboratory findings. Positive results of a specific target are indicative of the presence of that viral RNA and may not be the definite cause of disease. Positive results do not rule out co-infection with other pathogens. Negative results do not preclude SARS-CoV-2, influenza A or influenza B infection and should not be used as the sole basis for patient management decisions.		The BIOFIRE® SPOTFIRE® Respiratory ® Panel Mini (SPOTFIRE R Panel Mini) is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE® SPOTFIRE® System for the simultaneous, qualitative detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19. The following organism types are identified and differentiated using the SPOTFIRE R Panel Mini:
• Coronavirus SARS-CoV-2
• Human rhinovirus
• Influenza A virus
• Influenza B virus
• Respiratory syncytial virus
Nucleic acids from the viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection are indicative of the presence of the identified microorganism and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the SPOTFIRE R Panel Mini may not be the definite cause of disease. Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
Test Format	SAME		Single use
Automated Test Processes	SAME, but also includes sample extraction		Amplification and detection
Results Reported	SAME		Qualitative
Specimen Type	Anterior Nares Swab		Nasopharyngeal swab in transport media
Transport Media	N/A		VTM
Controls	SAME, with the exception of there being a single internal control		Two internal controls:
1 RNA Process control
2 PCR2 control
External Controls are provided separately (see K230868)
Instrument Systems	DASH Rapid PCR Instrument		SPOTFIRE System

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Summary of Non-Clinical Test Performance and Safety Testing 807,92(b)(1);

The DASH® Rapid PCR System has met the special controls established for product codes QOF and NSU.

The DASH Rapid PCR Instrument was evaluated for performance and found to conform with the following standards:

. IEC 60601-1-2 Ed. 4.1:2020 Medical electrical equipment - Part 1-2: General requirements for basic safety and essential performance - Collateral Standard: Electromagnetic disturbances - Requirements and tests
IEC 61010-1, 3rd edition, May 11, 2012, revision 2023-06-06 Safety requirements for ● electrical equipment for measurement, control, and laboratory use - Part 1: general requirements
61010-2-010:2019 Safety requirements for electrical equipment for . IEC measurement, control, and laboratory use - Part 2-010: Particular requirements for laboratory equipment for the heating of materials
. 61010-2-101:2018 Safety requirements for electrical equipment for IEC measurement, control, and laboratory use - Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipment

The DASH SARS-CoV-2 & Flu A/B Test was evaluated for performance and found to conform with the following FDA recognized standards:

CLSI EP12 3rd Edition, Evaluation of Qualitative, Binary Output Examination Performance
. CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition.
CLSI MM03-3rd Edition, Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline
CLSI EP07 3rd Edition, Interference Testing in Clinical Chemistry
. CLSI EP37 1st Edition. Supplemental Tables for Interference Testing in Clinical Chemistrv
. CLSI EP25 2nd Edition, Evaluation of Stability of In Vitro Medical Laboratory Test Reagents
. CLSI EP25-A. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline

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Performance Characteristics

Analytical Performance:

Analytical Sensitivity (Limit of Detection)

Limit of detection (LoD) was determined for the DASH® SARS-CoV-2 & Flu A/B Test on the DASH Rapid PCR Instrument utilizing five (5) viral strains representing the test targets. All samples were prepared in pooled nasal matrix (PNM) and evaluated to determine the lowest concentration at which ≥95% of replicates yield a positive result. The final LoD results are summarized in the table below:

Limit of Detection Results

| Viral Target | Strain | Source/
Product Type | LoD*
(copies/swab) | LoD*
(copies/mL) |
|--------------|------------------------------------------------------------------------------------|------------------------------------|-----------------------|-----------------------|
| SARS-CoV-2 | Isolate hCoV- 19/USA/GA-EHC-
2811C/2021
(Lineage B.1.1.529; Omicron Variant) | BEI Resources/
Gamma-Irradiated | 1200
copies/swab | 3.00E+04
copies/mL |
| Flu A | H1N1 Victoria/2570/19 | Zeptometrics/
Culture Fluid | 1.35
TCID50/swab | 33.75
TCID50/mL |
| | H3N2 /Darwin/9/21 | Zeptometrics/
Culture Fluid | 0.225
TCID50/swab | 5.63 TCID50/mL |
| | Washington/02/19 – Victoria Lineage | Zeptometrics/
Culture Fluid | 0.10
TCID50/swab | 2.50 TCID50/mL |
| Flu B | Utah/9/14 - Yamagata Lineage | Zeptometrics/
Culture Fluid | 0.675
TCID50/swab | 16.88
TCID50/mL |

The concentration stated per swab represents microbial material prepared in 40uL pooled nasal matrix and applied to the swab. The per mL concentration is therefore represented as 25x the stated amount per swab.

Within-Laboratory Precision

Precision of the DASH SARS-CoV-2 & Flu A/B Test was established and executed over a 12-day period, conducted by two (2) operators at a single site, and resulted in 96 replicates per concentration of the SARS-CoV-2, Flu A and Flu B targets.

Triple positive samples (SARS-CoV-2, Flu A. and Flu B) were prepared in simulated clinical nasal matrix (SCNM) at 2X and 5X LoD concentrations, and negative (no analyte) samples were SCNM without viral targets. Results of replicate samples tested by each operator at 2X and 5X LoD achieved 100% positivity for each target. Negative samples also showed the expected result of 0% positivity for each target. A percent positive agreement of 100% was achieved between operators as well as between testing days for each target at 2X and 5X LoD. Data analysis included a statistical two-way nested ANOVA for each target across different sources of variability (e.g., operator, day, run). The following tables provide a summary of results:

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Summary of Precision Study Results

| Viral Strain | Concentration | Results
(Call Accuracy/Agreement) |
|---------------------------------------------------|---------------|--------------------------------------|
| SARS Cov-2 19/USA/GA-
EHC-2811C/2021 (Omicron) | 2x LoD | 96/96
(100%) |
| SARS Cov-2 19/USA/GA-
EHC-2811C/2021 (Omicron) | 5x LoD | 96/96
(100%) |
| Flu A H1N1 Victoria /2570/19 | 2x LoD | 96/96
(100%) |
| Flu A H1N1 Victoria /2570/19 | 5x LoD | 96/96
(100%) |
| Flu B Yamagata (Utah/09/14) | 2x LoD | 96/96
(100%) |
| Flu B Yamagata (Utah/09/14) | 5x LoD | 96/96
(100%) |
| Negative | N/A | 96/96
(100%) |

Mean, standard deviation (SD), and percent coefficient of variation (%CV) for Cq*

| Sample | Sample
Type | N | Mean
Cq* | Between
Operator | | Between Day
(Within
Operator) | | Between Runs
(Within Day) | | Within Run/
Repeatability | | Total | |
|----------------|----------------------|----|-------------|---------------------|-----|-------------------------------------|-----|------------------------------|-----|------------------------------|-----|-------|-----|
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Flu A | Low
Positive | 96 | 24.0 | 0.4 | 1.5 | 0.3 | 1.3 | 0.0 | 0.0 | 1.2 | 5.2 | 1.3 | 5.5 |
| | Moderate
Positive | 96 | 22.7 | 0.3 | 1.2 | 0.3 | 1.1 | 0.2 | 0.9 | 0.5 | 2.3 | 0.7 | 3.0 |
| Flu B | Low
Positive | 96 | 25.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.9 | 3.6 | 0.9 | 3.6 |
| | Moderate
Positive | 96 | 24.8 | 0.0 | 0.0 | 0.4 | 1.5 | 0.0 | 0.0 | 0.6 | 2.2 | 0.7 | 2.7 |
| SARS-
CoV-2 | Low
Positive | 96 | 29.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.8 | 2.7 | 0.8 | 2.7 |
| | Moderate
Positive | 96 | 28.2 | 0.0 | 0.0 | 0.2 | 0.9 | 0.2 | 0.6 | 0.5 | 1.8 | 0.6 | 2.0 |

Note: SD and CV% were estimated using ANOVA variance component analysis using the R Package VCA.

*DASH software identifies the cycle at which an amplification curve meets a predefined mathematical criterion as Cq.

Reproducibility

A multi-site study was conducted to demonstrate the reproducibility of the DASH® SARS-CoV-2 & Flu A/B Test with analyte concentrations near the detection limit of the assay, using contrived nasal swabs and a 3-member panel consisting of a true negative (no analyte), low positive (2X LoD), and a moderate positive (5X LoD) panel member. The study was conducted using three (3) lots of DASH SARS-CoV-2 & Flu A/B Test cartridges by nine (9) untrained operators across three (3) sites, and performed over five (5) non-consecutive days, at two (2) runs per day, with three (3) replicates per panel member, totaling over 810 samples (270 replicates per panel member) tested throughout the course of the study.

The DASH SARS-CoV-2 & Flu A/B Test reported the expected positive results for panel members in 97.8%-100% of the samples and the expected negative results in 100% of samples. A summary of the results (percent (%) agreement with the expected positive result) by site for each panel member and a summary by analyte are provided below:

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| Analyte* | Sample
Concentration | % Agreement with Expected Results (n Agreement/N Tested) (95% CI)# | Site 1 | Site 2 | Site 3 | Overall |
|------------|-------------------------|--------------------------------------------------------------------|------------------------------------|-----------------------------------|-------------------------------------|---------|
| SARS-CoV-2 | Low Positive | 100.0% (90/90)
(95.9% -100.0%) | 97.8% (88/90)
(92.3% - 99.4%) | 100.0% (90/90)
(95.9% -100.0%) | 99.3% (268/270)
(97.3% -99.8%) | |
| | Moderate Positive | 98.9% (89/90)
(94.0% - 99.9%) | 100.0% (89/89)^
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 99.6% (268/269)
(97.9% -100.0%) | |
| | Negative | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (270/270)
(98.6% -100.0%) | |
| Flu A | Low Positive | 100.0% (90/90)
(95.9% -100.0%) | 97.8% (88/90)
(92.3% - 99.4%) | 100.0% (90/90)
(95.9% -100.0%) | 99.3% (268/270)
(97.3% -99.8%) | |
| | Moderate Positive | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (89/89)^
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (269/269)
(98.6% -100.0%) | |
| | Negative | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (270/270)
(98.6% -100.0%) | |
| Flu B | Low Positive | 100.0% (90/90)
(95.9% -100.0%) | 97.8% (88/90)
(92.3% - 99.4%) | 100.0% (90/90)
(95.9% -100.0%) | 99.3% (268/270)
(97.3% -99.8%) | |
| | Moderate Positive | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (89/89)^
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (269/269)
(98.6% -100.0%) | |
| | Negative | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (90/90)
(95.9% -100.0%) | 100.0% (270/270)
(98.6% -100.0%) | |

Qualitative Results by Analyte Concentration and Site

*All samples were co-spiked with all analytes, so results are presented three times, including the Negative results.

A total of 15 samples generated invalid results on the first attempt; 14 samples generated valid results upon repeat and were included in the data analysis.

^One Moderate Positive sample produced an error/no result and was excluded from the analysis.

For each panel member, statistical analysis of mean Ct, standard deviation (SD), and percent coefficient of variation (%CV) was evaluated for between sites, between day, between operator, between run, and within run, as presented in the table below:

| Analyte | Sample
Type | n/N | Mean
Cq* | Between
Site | | Between Lot | | Between
Day | | Between
Operator | | Between
Run | | Within-Run | | Total | |
|----------------|----------------------|---------|-------------|-----------------|-----|-------------|-----|----------------|-----|---------------------|-----|----------------|-----|------------|-----|-------|-----|
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| SARS-
CoV-2 | Low
Positive | 268/270 | 29.6 | 0.2 | 0.7 | 0.6 | 2.1 | 0 | 0 | 0.0 | 0.0 | 0.0 | 0.0 | 2.7 | 9.2 | 2.8 | 9.4 |
| | Moderate
Positive | 268/269 | 28.4 | 0.0 | 0.0 | 0.5 | 1.7 | 0 | 0 | 0.4 | 1.5 | 0.0 | 0.0 | 1.9 | 6.5 | 2.0 | 6.9 |
| Flu A | Low
Positive | 268/270 | 24.2 | 0.0 | 0.2 | 0.0 | 0.0 | 0 | 0 | 0.0 | 0.0 | 0.4 | 1.6 | 2.3 | 9.5 | 2.3 | 9.6 |
| | Moderate
Positive | 269/269 | 23.1 | 0.1 | 0.3 | 0.1 | 0.6 | 0 | 0 | 0.3 | 1.3 | 0.0 | 0.0 | 0.9 | 3.7 | 0.9 | 4.0 |
| Flu B | Low
Positive | 268/270 | 26.1 | 0.0 | 0.0 | 0.3 | 1.1 | 0 | 0 | 0.3 | 1.2 | 0.0 | 0.0 | 2.3 | 8.9 | 2.4 | 9.1 |
| | Moderate
Positive | 269/269 | 25.3 | 0.2 | 0.8 | 0.2 | 0.7 | 0 | 0 | 0.2 | 0.7 | 0.2 | 0.8 | 0.7 | 2.9 | 0.8 | 3.3 |

Cq Variability Analysis Results

Note: SD and CV% were estimated using REML variance component analysis using the R Package VCA. *DASH software identifies the cycle at which an amplification curve meets a predefined mathematical criterion as Cq.

Analytical Specificity (Cross-Reactivity & Microbial Interference)

In silico Analysis

In silico analysis was completed on 1,217 non-target organisms. Any microorganism showing >80% homology to a single primer, probe, or capture oligonucleotide was further evaluated for homology for the other target primers and probes for amplification.

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DASH® SARS-CoV-2 & Flu A/B Test primers for the SARS-CoV-2 targets exhibited >80% homology to SARS-coronavirus isolate Tor2 and although amplification may be possible, it will not be detected by the DASH SARS-CoV-2 & Flu A/B Test. Performance of DASH SARS-CoV-2 & Flu A/B Test with SARS-coronavirus Tor2 has not been confirmed by wet testing analysis.

Cross-Reactivity Wet Testing

Cross-Reactivity was established for the DASH SARS-CoV-2 & Flu A/B Test on the DASH Instrument by wet testing of 50 different viruses, bacteria, and fungi that are common causes of respiratory infections. All samples were prepared in simulated clinical nasal matrix (SCNM) and tested individually, without the presence of the test viral targets. Cross-reactivity was not present if the target had a 0% positive call rate for all three (3) replicates tested.

The table that follows provides a summary of the wet tested results. None of the evaluated organisms demonstrated cross-reactivity with the assay at the tested concentrations as shown in the table below:

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Cross-reactivity Viral Wet Testing Results Summary

Viruses/Viral RNA	Concentration per swab	Concentration per mL
Adenovirus type C1	1.44E+04
TCID50/swab	3.60E+05
TCID50/mL
Cytomegalovirus (also known as
HHV-5)	2.82E+03
TCID50/swab	7.05E+04
TCID50/mL
Epstein-Barr virus (also known as
HHV-4)	1.51E+04
TCID50/swab	3.78E+05
TCID50/mL
Human coronavirus 229E	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
Human coronavirus OC43	4.75E+03
TCID50/swab	2.19E+05
TCID50/mL
Human coronavirus HKU1	1.10E+04
Copies/swab	2.75E+05
Copies/mL
Human coronavirus NL63	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
Human Metapneumovirus	1.19E+04
TCID50/swab	2.98E+05
TCID50/mL
Measles virus	1.54E+04
TCID50/swab	3.85E+05
TCID50/mL
Middle East Respiratory
Syndrome coronavirus (MERS-CoV)	1N/A
Genomic RNA Middle East
Respiratory Syndrome coronavirus
(MERS-CoV)	1.35 ng RNA/swab	33.8 ng RNA/mL
Mumps virus	1.19E+04
TCID50/swab	2.98E+05
TCID50/mL
Parainfluenza virus 1	4.75E+03
TCID50/swab	1.19E+05
TCID50/mL
Parainfluenza virus 2	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
Parainfluenza virus 3	1.44E+04
TCID50/swab	3.60E+05
TCID50/mL
Parainfluenza virus 4	2.82E+03
TCID50/swab	7.05E+04
TCID50/mL
Respiratory syncytial virus	2.82E+03
TCID50/swab	7.05E+04
TCID50/mL
Rhinovirus type 1A	2.82E+03
TCID50/swab	7.05E+04
TCID50/mL
Enterovirus type 68	3.40E+03
TCID50/swab	8.50E+04
TCID50/mL
Severe Acute Respiratory
Syndrome coronavirus (2003 RNA)	9.80E+03
Copies/swab	2.45E+05
Copies/mL
Bacteria	Concentration per swab	Concentration per mL
Bordetella pertussis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Bordetella parapertussis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Chlamydia pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Corynebacterium diphtheriae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Escherichia coli	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Haemophilus influenzae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Fusobacterium necrophorum	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Lactobacillus acidophilus	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Legionella pneumophila	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Moraxella catarrhalis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycobacterium tuberculosis
(heat-inactivated)	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycoplasma pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycoplasma genitalium	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria meningitidis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria elongata subsp.
glycolytica	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria mucosa	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Nocardia asteroides	1.74E+05
CFU/swab	4.35E+06
CFU/mL
Pseudomonas aeruginosa	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Staphylococcus aureus	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Staphylococcus epidermidis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus pyogenes	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus salivarius	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus mitis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus mutans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Fungi	Concentration per swab	Concentration per mL
Aspergillus niger	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Candida albicans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Cryptococcus neoformans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Pneumocystis jirovecii	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Eikenella corrodens	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Microorganism	Concentration per swab	Concentration per mL
Adenovirus C1	1.44E+04
TCID50/swab	3.60E+05
TCID50/mL
Cytomegalovirus (also known as
HHV-5)	2.48E+03
TCID50/swab	6.20E+04
TCID50/mL
Epstein-Barr virus (also known as
HHV-4)	1.51E+04
TCID50/swab	3.78E+05
TCID50/mL
Human coronavirus 229E	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
Human coronavirus OC43	4.75E+03
TCID50/swab	2.19E+05
TCID50/mL
Human coronavirus HKU1	9.68E+03
Copies/swab	2.41E+05
Copies/mL
1Human coronavirus NL63	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
1Measles virus	1.54E+04
TCID50/swab	3.85E+05
TCID50/mL
Genomic RNA Middle East
Respiratory Syndrome coronavirus
(MERS-CoV)	1.35
ng RNA/swab	33.8
ng RNA/mL
Middle East Respiratory
Syndrome coronavirus (MERS-CoV)	2N/A
Mumps virus	1.19E+04
TCID50/swab	2.98E+05
TCID50/mL
Human Metapneumovirus	1.19E+04
TCID50/swab	2.98E+05
TCID50/mL
Parainfluenza virus 1	4.75E+03
TCID50/swab	1.19E+05
TCID50/mL
Parainfluenza virus 2	4.00E+03
TCID50/swab	1.00E+05
TCID50/mL
Parainfluenza virus 3	1.44E+04
TCID50/swab	3.60E+05
TCID50/mL
Parainfluenza virus 4	2.48E+03
TCID50/swab	6.20E+04
TCID50/mL
Respiratory syncytial virus	2.48E+03
TCID50/swab	6.20E+04
TCID50/mL
Rhinovirus type 1A	2.48E+03
TCID50/swab	6.20E+04
TCID50/mL
Enterovirus type 68	2.99E+03
TCID50/swab	7.48E+04
TCID50/mL
Severe Acute Respiratory
Syndrome coronavirus (SARS-CoV)	8.62E+03
Copies/swab	2.16E+05
Copies/mL
Bordetella pertussis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Bordetella parapertussis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Candida albicans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Chlamydia pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Corynebacterium diphtheriae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Escherichia coli	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Haemophilus influenzae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Fusobacterium necrophorum	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Microorganism	Concentration per swab	Concentration per mL
Lactobacillus acidophilus	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Legionella pneumophila	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Moraxella catarrhalis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycobacterium tuberculosis (heat-
inactivated)	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycoplasma pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Mycoplasma genitalium	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria meningitidis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria elongata subsp.
glycolytica	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Pseudomonas aeruginosa	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Staphylococcus aureus	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Staphylococcus epidermidis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus pneumoniae	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus pyogenes	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus salivarius	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Aspergillus niger	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Cryptococcus neoformans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Pneumocystis jirovecii	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Eikenella corrodens	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Neisseria mucosa	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus mitis	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Streptococcus mutans	5.00E+04
CFU/swab	1.25E+06
CFU/mL
Nocardia asteroides	1.74E+05
CFU/swab	4.35E+06
CFU/mL

1 Concentration not available from vendor, sample is qualitative non-infectious purified intact viral particles.

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Cross-reactivity Bacterial Wet Testing Results Summary

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Cross-reactivity Fungal Wet Testing Results Summary

Microbial Interference

Microbial Interference was evaluated for the DASH® SARS-CoV-2 & Flu A/B Test on the DASH Instrument by wet testing over 50 different viruses, bacteria, and fungi that are common causes of respiratory infections. All samples were prepared in simulated clinical nasal matrix (SCNM) and tested with individual microorganisms or with pooled groups of up to four (4) microorganisms in the presence of the test viral targets at 3X their individual LoDs. Microbial non-interference was determined if the target had a 100% positive call rate for all three (3) replicates tested.

The following tables provide a summary of the wet tested results. None of the evaluated microorganisms demonstrated interference with the assay at the tested concentrations as shown in the table below:

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Microbial Interference Wet Testing Results Summary

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Note: Thicker outer border indicates groups of pooled microorganisms that were tested.

¹Tested individually as interference was observed when tested as a group.

1Tested individually as interference was observed when tested as a group.
²Concentration not available from vendor, sample is qualitative non-infectious purified intact vir

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Competitive Interference

Competitive interference can occur when there is a co-infection and one viral target concentration is near the LoD while another viral target is present at high concentration.

Competitive Interference was evaluated for the DASH® SARS-CoV-2 & Flu A/B Test on the DASH Rapid PCR Instrument. One (1) viral target was tested at a high concentration while the other two viral targets were held at 3xLOD. Replicates of three (3) were tested. If results reported less than 3/3 positive results for all targets, the concentration of the competing virus was reduced until positive results were achieved for all targets.

Study results demonstrate that SARS-CoV-2, at concentrations >1.41E+06 copies/mL, inhibit detection of Flu A at 3xLoD, and at SARS-CoV-2 concentrations of >5.65E+06 copies/mL, detection of Flu B at 3xLoD is inhibited. Flu A at concentrations of >1.38E+06 TCIDsolmL inhibited detection of Flu B at 3xLoD. In addition, Flu B at concentrations >1.01E+03 TCIDso/mL inhibited detection of both SARS-CoV-2 and Flu A at 3xLoD. The highest coinfection concentration of the competing virus, where the remaining targets are detected for three (3) replicates at 3xLOD, are reported in the table below:

Competitive Interference Summary

	Concentration* at which All Three (3) Targets Achieved Positive Calls
Competing
Virus	SARS-CoV-2
SARS-Cov-2, Isolate
hCoV- 19/USA/GA-EHC-
2811C/2021 (Lineage
B.1.1.529; Omicron
Variant)	Flu A
Flu A H1N1
A/Victoria/2570/2019	Flu B
Flu B Utah/9/14 -
Yamagata Lineage
SARS-CoV-2	5.65E+04 copies/swab
1.41E+06 copies/mL
~47x LoD	3x LoD	3x LoD
Flu A	3x LoD	5.52E+04 TCID50/swab
1.38E+06 TCID50/mL
~3405x LoD	3x LoD
Flu B	3x LoD	3x LoD	40.40 TCID50/swab
1.01E+03 TCID50/mL
~60x LoD

The concentration stated per swab represents microbial material prepared in 40µL of simulated clinical nasal matrix and applied to the swab. The per mL concentration is therefore represented as 25x the stated amount per swab.

Exogenous/Endogenous Interfering Substances

Medically relevant substances found within clinical respiratory specimens (known as endogenous materials) and substances not commonly found in clinical respiratory specimens (exogenous substances) were evaluated for their potential impact on the DASH SARS-CoV-2 & Flu A/B Test, and its ability to detect SARS-CoV-2, Flu A, and Flu B.

All samples were prepared in simulated clinical nasal matrix (SCNM). Each positive sample was composed of a single exogenous interfering substance and combined with a contrived co-spiked sample of SARS-CoV-2, Flu A, and Flu B at 3X LoD concentrations. Negative (no analyte) samples were prepared with simulated clinical nasal matrix (SCNM). Each interferent was tested in triplicate.

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Interference with the SARS-CoV-2 assay was observed in the presence of Biotin (vitamin B- 7) at 4.58 µg/mL. When diluted by 2-fold, no further interference was observed at 2.29 µg/mL for each of the three (3) replicate samples.

Interference with the SARS-CoV-2 assay was observed in the presence of Flonase® (5% v/v, active ingredient Fluticasone Propionate). Subsequent confirmatory testing was performed at a lower Flonase concentration (2.5% v/v) and provided objective evidence of no observed interference, wherein 3/3 replicates resulted in positive results for all viral targets.

No interference was observed for any of the substances tested at the concentrations noted in the table below:

Interfering Substance	Concentration
Afrin® Original Nasal Spray	15% v/v
Chloroseptic® Sore Throat spray	20% v/v
Flonase®	2.5% v/v*
Relenza®	0.3 mg/ml
Tobramycin	4 µg/mL
Mupirocin	10 mg/mL
Mucin	0.1% w/v
Blood	2% v/v
Biotin	2.29 µg/mL*
Viral Transport Medium	50% v/v
Neo-Synephrine® Nasal Spray	15% v/v
Walgreens Saline Nasal Spray	15% v/v
Beclomethasone Dipropionate, USP
(Micronized)	0.068 mg/mL
Flunisolide Nasal Solution, USP 0.025%	0.04 mg/mL
Nasacort® Nasal Spray	0.04 mg/mL
Walgreens 24 Hour Budesonide Nasal
Spray	0.051 mg/mL
Nasonex	0.04 mg/mL
Zicam® Allergy Relief Nasal Spray	20% v/v
Snuff	0.1% (w/v)
Latex glove powder	1% (w/v)
Mucinex® Insta Sooth Kickstart Sore
Throat	1.7 mg/mL

Exogenous/Endogenous Interfering Substances Study Results Summary

*Interference potential was observed at 2x higher concentrations.

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Analytical Reactivity (Inclusivity)

Inclusivity was established for the DASH® SARS-CoV-2 & Flu A/B Test on the DASH Instrument through wet testing and in silico analysis. Wet testing was performed by evaluating seven (7) strains of SARS-CoV-2, 21 strains of Flu A, and 10 strains of Flu B. Each strain was evaluated at concentrations near LoD, prepared in pooled nasal matrix (PNM), and tested individually, with three (3) replicates evaluated per strain. Testing was executed in a blinded fashion with random negatives. Inclusivity was determined if the target had a 100% positive call rate for all three (3) replicates tested. The table that follows summarizes the wet tested analytical reactivity results:

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Inclusivity Wet Test Results Summary

The concentration stated per swab represents microbial material prepared in 40μL of pooled nasal matrix and applied to the swab. The per mL concentration is therefore represented as 25x the stated amount per swab.

'Testing at a lower concentration (i.e., 3x LoD) failed to yield 100% detection that yielded 100% detection was 6x LoD. ?Testing at a lower concentration (i.e., 3x LoD) failed to yield 100% detection. The lowest concentration that yielded 100% detection was 12x LoD. 1nfluenza A H1N1 (Singapore/63/04) was detected in 2/3 replicates at 3x LoD. The strain was reprepared at 3x LoD and tested in 7 replicates for 100% detection.

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In Silico Analysis for SARS-CoV-2, Flu A, and Flu B:

The inclusivity of the DASH® SARS-CoV-2 & Flu A/B Test was also evaluated using in silico analysis of the sample preparation capture oligos and the PCR primers and probes for SARS-CoV-2 and influenza A and B in relation to sequences available in the NCBI SARS-CoV-2 Data Hub and the NCBI Influenza Virus Database.

Genomic sequences for SARS-CoV-2 were retrieved from the NCBI SARS-CoV2 Data Hub on May 8, 2024, and included all lineages and variants of concern (VOC) or variants of interest (VOI). The influenza genomic sequences were retrieved from the NCBI Influenza Virus Database on May 13, 2024. Influenza A sequences were limited to areas of interest, including subtype H1N1 (excluding Swine flu) or H3N2, based upon completeness. Influenza B sequences were required to be full-length, with no subtype limitations applied. All genomic sequences containing ambiguous nucleotides were excluded from the analysis.

The query oligos for the specific virus were aligned to the filtered sets of viral genome sequences using the Burrows-Wheeler Aligner (BWA) program, invoking parameters that guarantee any difference would be a mismatch or an insertion/deletion.

The genomic sequences were ranked according to the number of capture oligos and PCR oligos and probes containing zero (0), one (1), and two (2) or more mismatches. Melting temperature (Tm) analysis was performed for any oligo:genome pairing that contained a mismatch, and the pairings that passed the Tm analysis criteria were combined with pairings containing no mismatches, then grouped into amplifiable sets. The resulting oligo:genome pairings were counted to determine the number of sequences predicted to be captured and, if captured, predicted to be detected. The results of this predicted-to-detect analysis are shown in the table below:

| Target | Sequences Predicted to be
Detected |
|-------------|---------------------------------------|
| Influenza A | ≥99.97% (18112 of 18117) |
| Influenza B | ≥98.27% (8136 of 8279) |
| SARS-CoV-2 | ≥99.99% (994778 of 994846) |

Sequences Predicted to Be Detected by PCR According to in silico Analysis

DASH SARS-CoV-2 & Flu A/B Test in silico inclusivity analysis for Flu A subtypes H7N7 and H5N3 was also performed. As influenza A H7N7 and H5N3 did not have reference genomes available for analysis, an alternative method was used for assessment involving a GISAID (Global Initiative on Sharing All Influenza Data) database query to compare oligonucleotide sequences of the detection limiting region of the DASH SARS-CoV-2 & Flu A/B Test. The resulting data, placed through a BLAST analysis, demonstrated that the subtype H5N3 and H7N7 of Flu A are expected to be detected by DASH SARS-CoV-2 & Flu A/B Test. Wet testing for H5N3 strain of Flu A was also performed, confirming inclusivity of DASH SARS-CoV-2 & Flu A/B Test with the H5N3 strain of Flu A (see Inclusivity Wet Test Results Summary table above). The Flu A H7N7 strain was not available for wet testing confirmation.

Additional influenza A, in silico inclusivity analysis was performed for all human host influenza A subtypes (H5N1, H5N6, H9N2, H10N3, and H10N5) available from the GISAID database, and collected from November 5, 2023 to November 5, 2024, wherein all possible combinations of hemagglutinin and neuraminidase were considered. Each sequence of the limiting region was required

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to be of full-length, with no subtype limitations applied, and all sequences containing ambiguous nucleotides were excluded from the analysis. The resulting oligo:genome pairings were counted to determine the number of sequences predicted to be captured and, if captured, predicted to be detected. Based on in silico inclusivity analysis of the influenza A oligos, it is predicted that the test will detect ~97.3% (n = 37) of all human host influenza A sequences collected between November 5, 2023 and November 5 2024. The performance characteristics of DASH® SARS-CoV-2 & Flu AJB Test with these Flu A subtypes have not been confirmed by wet testing analysis.

Comparison Studies:

Matrix Equivalency

Equivalency was established between the following matrix materials that were utilized in the evaluation of the DASH SARS-CoV-2 & Flu A/B Test performance:

· Clinical pooled nasal matrix (PNM)
Simulated clinical nasal matrix (SCNM) .

SCNM is formulated in PBS with glycerol and mucins (i.e., 1X PBS, ~1% NaCl, 2.5% Porcine Mucin, 15% dlycerol) added to mimic the nasal cavity fluid. The study was performed using co-spiked samples consisting of one SARS-CoV-2 strain (19/USA/GA-EHC-2811C/2021 - Omicron), one influenza A strain (H1N1 Victoria /2570/19), and one influenza B strain (Yamagata - Utah/09/14). Negative (nonanalyte) samples were evaluated with only the matrix material.

Co-spiked samples with all viral targets were prepared in both matrices at 2x and 5x LoD. For both matrices, all replicates tested at 5x LoD were positive at a 100% rate and, as expected, replicates tested at 2x LoD varied and were positive at a rate of ≥95%. All negative samples were negative for both matrices. For both matrices, 10 replicates of the negative samples, 40 replicates of the 2x LOD positive samples and 10 replicates of the 5x LOD positive samples were tested.

Results provide evidence that performance of the DASH SARS-CoV-2 & Flu A/B Test is equivalent with viral materials prepared in either clinical pooled nasal matrix (PNM) or in simulated clinical nasal matrix, and further supports the use of SCNM in select analytical studies.

Fresh vs. Frozen Contrived Samples

This study was performed to establish equivalency between freshly prepared samples and those that were subjected to two (2) successive freeze/thaw cycles for materials used in the evaluation of the DASH SARS-CoV-2 & Flu A/B Test performance. Co-spiked samples were prepared in SCNM at 2x and 5x LoD concentrations containing the following viral materials: one SARS-Cov-2 strain (19/USA/GA-EHC-2811C/2021 - Omicron), one influenza A strain (H1N1 Victorial2570/19), and one influenza B strain (Yamagata - Utah/9/14). Negative (no analyte) samples prepared with only the matrix material were also evaluated.

For both conditions evaluated, all replicates tested at 5x LoD were positive at a 100% rate and, as expected, replicates tested at 2x LoD varied and were positive at a rate of ≥95%. All negative samples were negative for both conditions. Positive samples co-spiked at 5x LoD and negative samples were both tested in replicates of 10 for each condition, and positive samples prepared at 2x LoD were tested in replicates of 40 for each condition.

Results demonstrate equivalency between fresh and frozen contrived sample performance on the DASH SARS-CoV-2 & Flu A/B Test, and supports the use of frozen contrived samples within select analytical studies.

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Summary of Clinical Tests 807.92(b)(2):

Clinical Performance:

Clinical performance characteristics of the DASH® SARS-CoV-2 & Flu A/B Test were evaluated for both accuracy and ease of use during the 2023-2024 respiratory season. The predominant subtypes during this season were (H1N1)odm09 and H3N2 for influenza A. and Victoria lineage for influenza B according to the Weekly US Influenza Surveillance Report from May 17, 2024. Seven (7) geographical locations within the United States, participated in the prospective collection of specimens from individuals showing signs and symptoms of respiratory infection from January to March 2024. Each of these sites were representative of the intended use setting, point-of-care (POC) use, and were CLIA-waived. All testing was performed by untrained operators without any prior hands-on device experience or laboratory training. Operators used only the written test procedure provided with the DASH SARS-CoV-2 & Flu A/B Test (i.e. quick reference guide) and the DASH Instrument. For testing with the DASH SARS-CoV-2 & Flu A/B Test, anterior nares swabs from both sides of the nose were either healthcare providercollected or adult-collected from individuals below 14 years, or self-collected from individuals aged 14 years or older. The table below provides a summary of the subject demographics associated with the specimens collected within the study:

Subject Demographics	Overall (N=795)
Age: Mean (SD)	37.3 (18.3)
Age: Median [Min, Max]	36 [2, 87]
Age Group
2 - 24 - 64 years of age	530 (66.7%)
≥65 years of age	64 (8.1%)
Sex at Birth
Female	465 (58.5%)
Male	330 (41.5%)
Ethnicity
Hispanic/Latino	303 (38.1%)
Not Hispanic/Latino	492 (61.9%)
Race
American Indian or Alaskan Native	3 (0.4%)
Asian	4 (0.5%)
Black or African American	111 (14.0%)
Native Hawaiian/Pacific Islander	4 (0.5%)
White	661 (83.1%)
Unknown/Prefer not to answer	6 (0.8%)
Other (Mixed race/biracial)	6 (0.8%)
Total	795 (100%)

Summary of Subject Demographics

Performance of the DASH SARS-CoV-2 & Flu A/B Test was assessed by a comparison of results with an FDA cleared RT-PCR test for SARS-CoV-2, and a second FDA cleared test for Flu A and Flu B.

Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA), including 95%

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confidence intervals, were calculated for each target nucleic acid in comparison with the designated comparator test. All discordant results between the DASH® SARS-CoV-2 & Flu A/B Test and the comparator were investigated using a third highly sensitive FDA cleared test and are footnoted under the Performance table that shortly follows.

Of the 817 subjects enrolled in the study, 795 subjects were evaluable for at least one (1) analyte/target and were included in the data analysis. There were 795 evaluable subjects for SARS-CoV-2 and 792 evaluable subjects for Flu A and Flu B. The most common reason for exclusion was due to the comparator sample being unable to be tested within the stability window of the test IFU. The following table provides a summary of the study results:

Test Targets	Positive Percent Agreement
	$TP/(TP+FN)$	PPA
(95% CI)	$TN/(TN+FP)$	NPA
(95% CI)
¹SARS-CoV-2	160/168	95.2%
(90.9% - 97.6%)	624/627	99.5%
(98.6% - 99.8%)
²Flu A	50/53	94.3%
(84.6% - 98.1%)	725/739	98.1%
(96.8% - 98.9%)
³Flu B	36/37	97.3%
(86.2% - 99.9%)	749/755	99.2%
(98.3% - 99.6%)

Clinical Performance Results

1 SARS-CoV-2 (PPA): One (1) sample was negative by another FDA cleared test, agreeing with the DASH. 2 Flu A PPA: Two (2) samples were negative by another FDA cleared test, agreeing with the DASH.

Flu A NPA: 13 samples were positive by another FDA cleared test, agreeing with the DASH.

3 Flu B PPA: One (1) sample was negative by another FDA cleared test, agreeing with the DASH.

Flu B NPA: Four (4) samples were positive by another FDA cleared test, agreeing with the DASH.

Of the 833 samples tested on DASH SARS-CoV-2 & Flu A/B Test, including 16 repeat samples, there were 24 instances of invalid results (2.9%; 95% Cl: 1.9% - 4.3%).

Conclusion 807.92(b)(3):

The DASH SARS-CoV-2 & Flu A/B Test; DASH Rapid PCR Instrument; DASH SARS-CoV-2 & Flu AlB Positive Control and DASH Negative Control. as part of the DASH Rapid PCR System, are substantially equivalent to the predicate and reference devices (BioFire SPOTFIRE Respiratory Panel Mini with SPOTFIRE System and SPOTFIRE RSP Pos & Neg Controls external controls). The subject device meets requirements as described in FDA Guidance Class II Special Controls established for product codes QOF and NSU.

The subject device and the predicates all achieve their intended use as a system; assay, instrument, external controls. They share the same intended use, patient population, fundamental technology, and are both for the qualitative detection and differentiation of SARS-CoV-2, influenza A and influenza B in upper respiratory swab samples. The subject device and primary predicate utilize similar reagents, have an internal control, and provide external QC controls that are sold separately.

The risks identified in the special controls have been mitigated by the subject device through

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design control and evidence is demonstrated by performance studies and in the labeling. Both the subject device and primary predicate device completed testing as described in the special controls guidance including sensitivity, specificity, interfering substances testing, and method comparison. Demonstration that the risks are mitigated by the subject device through the completion of performance studies and through labeling.

The results of performance testing demonstrate that the device system functions as intended, and differences between DASH® SARS-CoV-2 & Flu A/B Test (for use with the DASH Rapid PCR System components) and the predicate(s) do not raise new or different concerns of safety or effectiveness when used as intended. The DASH SARS- CoV-2 & Flu A/B Test, as part of the DASH Rapid PCR System, is substantially equivalent to the primary predicate, BIOFIRE® SPOTFIRE® Respiratory (R) Panel Mini and the reference devices, BioFire System and SPOTFIRE RSP Pos & Neg Controls, SPOTFIRE RSP Positive Control, SPOTFIRE RSP Negative Control.