VELO Respiratory Test

K251742 · Lex Diagnostics Limited · QOF · Feb 13, 2026 · Microbiology

Device Facts

• Record ID: K251742
• Device Name: VELO Respiratory Test
• Applicant: Lex Diagnostics Limited
• Product Code: QOF · Microbiology
• Decision Date: Feb 13, 2026
• Decision: SESE
• Submission Type: Dual Track
• Regulation: 21 CFR 866.3981
• Device Class: Class 2

Indications for Use

The VELO Respiratory Test is an automated rapid multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) test performed on the VELO Instrument and is intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B virus nucleic acid in anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2 and influenza can be similar. The VELO Respiratory Test is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, and/or influenza B infection if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A and influenza B viral nucleic acid are generally detectable in ANS specimens during the acute phase of infection. Positive results do not rule out co-infection with other organisms. The agent(s) detected by the VELO Respiratory Test may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2, influenza A, and/or influenza B infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Device Story

VELO Respiratory Test is an automated, rapid, multiplex real-time RT-PCR assay for qualitative detection of SARS-CoV-2, influenza A, and influenza B RNA. Input: anterior nasal swab (ANS) specimen collected directly into a single-use Test Cartridge. Operation: Cartridge inserted into reusable VELO Instrument; instrument performs automated elution, thermal lysis, and real-time RT-PCR amplification. Output: 'Detected' or 'Not Detected' results reported on instrument screen in under 10 minutes. Used in clinical settings (physician offices, urgent care) by healthcare personnel. Provides rapid differential diagnosis to aid clinical decision-making; helps distinguish between respiratory infections with similar symptoms. Benefits patient through timely diagnosis and management.

Clinical Evidence

Clinical performance was evaluated in a prospective study of 1,718 symptomatic subjects across nine CLIA-waived sites. Compared to an FDA-cleared comparator, the test demonstrated: Influenza A (PPA 92.4%, NPA 99.1%), Influenza B (PPA 93.3%, NPA 99.8%), and SARS-CoV-2 (PPA 96.2%, NPA 99.5%). Retrospective testing supplemented Influenza B data (PPA 93.8%, NPA 100%). Analytical studies confirmed LoD, inclusivity, and lack of cross-reactivity/interference with 52 common respiratory pathogens.

Technological Characteristics

Multiplexed RT-PCR assay using hydrolysis probes. Features ultra-fast thermal cycling via high surface-area-to-volume ratio reaction chambers. Employs thermal lysis for nucleic acid release. Endogenous RNaseP sample process control. Standalone benchtop instrument with integrated screen. Software-controlled automated workflow. No external reagent preparation or pipetting required.

Indications for Use

Indicated for individuals with signs and symptoms of respiratory tract infection to detect and differentiate SARS-CoV-2, influenza A, and influenza B nucleic acid in anterior nasal swab specimens. Intended for use by non-laboratory trained healthcare professionals in CLIA-waived settings.

Regulatory Classification

Identification

A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.

Special Controls

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

Predicate Devices

• cobas® SARS-CoV-2 & Influenza A/B for use on the cobas Liat System (K223591)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K251742

B Applicant

LEX Diagnostics Limited

C Proprietary and Established Names

VELO Respiratory Test

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The Sars-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology |

## II Submission/Device Overview:

### A Purpose for Submission:

To demonstrate that the performance of the VELO Respiratory Test is substantially equivalent to the cobas SARS-CoV-2 & Influenza A/B for use on the cobas Liat System (K223591) and to obtain clearance for the VELO Respiratory Test.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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B Measurand:
- Influenza A RNA
- Influenza B RNA
- Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA

C Type of Test:
Qualitative reverse transcriptase polymerase chain reaction (RT-PCR)

III Intended Use/Indications for Use:

A Intended Use(s):
See Indications for Use below.

B Indication(s) for Use:
The VELO Respiratory Test is an automated rapid multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) test performed on the VELO Instrument and is intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B virus nucleic acid in anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2 and influenza can be similar.

The VELO Respiratory Test is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, and/or influenza B infection if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A and influenza B viral nucleic acid are generally detectable in ANS specimens during the acute phase of infection.

Positive results do not rule out co-infection with other organisms. The agent(s) detected by the VELO Respiratory Test may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza A, and/or influenza B infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For In Vitro Diagnostic Use Only

D Special Instrument Requirements:
For use with the VELO Instrument

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IV Device/System Characteristics:

A Device Description:

The VELO Respiratory Test is an automated rapid multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) test performed on the VELO Instrument and is intended for the simultaneous qualitative detection and differentiation of SARS-CoV-2, influenza A, and influenza B virus RNA in ANS specimens from individuals with signs and symptoms of respiratory tract infection. The VELO System is comprised of a single-use VELO Respiratory Test, and a reusable VELO Instrument. To perform the test, an ANS specimen is collected using the provided ANS swab and the swab is inserted directly into the VELO Respiratory Test Cartridge sample port. The Test Cartridge contains all necessary reagents for the detection of influenza A, influenza B and SARS-CoV-2 viral RNA and the endogenous sample and process control (SPC). The Test Cartridge is then capped and inserted into the VELO Instrument to initiate the test, and all subsequent test steps, including sample processing, target amplification by real-time RT-PCR, fluorescence detection, result interpretation and result reporting are performed automatically by the VELO Instrument.

B Principle of Operation:

The VELO Respiratory Test is performed on the VELO Instrument which automates and integrates sample processing, target amplification by real-time RT-PCR, fluorescence detection, result interpretation and result reporting. Each VELO Respiratory Test Cartridge contains prepackaged reagents for the detection of influenza A, influenza B and SARS-CoV-2 viral RNA from anterior nasal swab specimens. RT-PCR is used with specific primers and probes within the Test Cartridge to amplify and detect sequences unique to each target pathogen. Specifically, the Matrix protein gene of influenza A, the Non-structural gene of influenza B and the ORF 1a/b non-structural region and Membrane gene of SARS-CoV-2. Each Test Cartridge also contains an endogenous SPC that serves as an Internal Control (IC) to ensure adequate sample collection and processing. The SPC monitors the overall performance of the RT-PCR reaction for potential sample mediated inhibition or failure of the reagents. Additionally, the SPC verifies that the RT-PCR reaction conditions (temperature and duration) are optimal for the amplification process.

Once collected, the anterior nasal swab specimen is directly inserted into the Test Cartridge sample port with the swab shaft then removed at a pre-specified breakpoint. All further operational steps are then automatically executed by the VELO Instrument. Sample processing includes elution and a thermal lysis step that then directly rehydrates the lyophilized RT-PCR reagents, with each target RT-PCR amplification reaction proceeding in an independent PCR chamber.

In the event of amplification, a fluorescence signal is generated through the degradation of oligonucleotide probes modified with 5' fluorophores and 3' quenchers. Fluorescence is monitored by the VELO Instrument with every thermal cycle and reports as "Detected" once meeting pre-determined criteria. Test outcomes are reported to the operator in real-time via the Instrument view screen with 'Not Detected' results available in under 10 minutes with the completion of all cycles. When the test ends, all results can be viewed via the instrument view screen, and the Test Cartridge may be removed for disposal.

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C Instrument Description Information:

1. Instrument Name:
VELO Instrument, software version v7.2.1 or higher.

2. Specimen Identification:
Specimen identification is either entered manually or scanned via a barcode scanner.

3. Specimen Sampling and Handling:
Direct anterior nasal swabs. The anterior nasal swab specimen is collected and added directly to the VELO Respiratory Test Cartridge sample port. No collection media is used to store the swab. Each specimen is processed individually.

4. Calibration:
Not applicable.

5. Quality Control:

Internal Controls
The assay contains an endogenous sample and process control (SPC) that serves as an Internal Control (IC) to ensure adequate sample collection and processing. The SPC monitors the overall performance of the RT-PCR reaction for potential sample mediated inhibition or failure of the reagents. Additionally, the SPC verifies that the RT-PCR reaction conditions (temperature and duration) are optimal for the amplification process. Detection of the internal control is not required for a valid result when target RNA is detected.

External Controls
External Positive and Negative controls are not provided, but commercially available, inactivated virus controls such as the NATtrol Flu/RSV/SARS-CoV-2 from ZeptoMetrix are recommended. External Controls (EC) may be performed to conform with internal Quality Control (QC) procedures, or with local, state, or federal regulations. The use of external controls may be appropriate when receiving new device shipments, training new operators, or when problems with testing are suspected or identified. The NATtrol Flu/RSV/SARS-CoV-2 (ZeptoMetrix) external controls were evaluated in the analytical and clinical studies.

V Substantial Equivalence Information:

A Predicate Device Name(s):
cobas SARS-CoV-2 & Influenza A/B Nucleic acid test for use on the cobas Liat System

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B Predicate 510(k) Number(s):

K223591

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K251742 | K223591 |
| --- | --- | --- |
| Device Trade Name | VELO Respiratory Test | Cobas SARS-CoV-2 & Influenza A/B for use on the cobas Liat System |
| General Device Characteristic Similarities | | |
| Regulation Number and Name | Same | 21 CFR 866.3981; Multi-Target Respiratory Specimen Nucleic Acid Test Including SARS-CoV-2 And Other Microbial Agents |
| Product Code | Same | QOF |
| Prescription Use Only | Same | Yes |
| Intended Use/Indications For Use | The VELO Respiratory Test is an automated rapid multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) test performed on the VELO Instrument and is intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B virus nucleic acid in anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2 and influenza can be similar. The VELO Respiratory Test is intended for use as an aid | The cobas SARS-CoV-2 & Influenza A/B nucleic acid test for use on the cobas Liat System (cobas SARS-CoV-2 & Influenza A/B) is an automated rapid multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and/or influenza B virus nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2 and influenza can be similar. cobas SARS-CoV-2 & Influenza A/B is intended for |

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| | in the differential diagnosis of SARS-CoV-2, influenza A, and/or influenza B infection if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A and influenza B viral nucleic acid are generally detectable in ANS specimens during the acute phase of infection.
Positive results do not rule out co-infection with other organisms. The agent(s) detected by the VELO Respiratory Test may not be the definite cause of disease.
Negative results do not preclude SARS-CoV-2, influenza A, and/or influenza B infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. | use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, and/or influenza B infection if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, and influenza B viral nucleic acid are generally detectable in NPS and ANS specimens during the acute phase of infection.
Positive results do not rule out co-infection with other organisms. The agent(s) detected by the cobas SARS-CoV-2 & Influenza A/B may not be the definitive cause of disease.
Negative results do not preclude SARS-CoV-2, influenza A, and/or influenza B infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. |
| --- | --- | --- |
| Test Technology | Same | Multiplexed Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) |
| Test Processes | Same | Automated |
| External Controls | Yes – commercially available | Yes – sold separately as the cobas SARS-CoV-2 & Influenza A/B Quality Control Kit |
| Reagents / Kit Components | Included in VELO Respiratory Test Cartridge, with no user involvement required | Included in Liat assay tube, with no user involvement required |
| Detection | Fluorescence detected automatically by the instrument (VELO Instrument), not reliant on user judgement | Fluorescence detected automatically by the instrument (cobas Liat System), not reliant on user judgement |

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| Test Results | Same | Qualitative |
| --- | --- | --- |
| Test Format | Same | Single use |
| General Device Characteristic Differences | | |
| Specimen Type(s) | Anterior nasal swabs | Nasopharyngeal swabs and anterior nasal swabs |
| Time to Result | In 10 minutes or less | In approximately 20 minutes |
| Sample Processing | Specimen swab loaded directly into VELO Respiratory Test Cartridge where direct elution followed by thermal lysis nucleic acid release before RT-PCR | Specimen swab eluted into transfer media (VTM/UTM) before being loaded into the Liat assay tube, followed by chemical based nucleic acid extraction and purification before RT-PCR |
| Internal Control | Endogenous Sample Process Control | Exogenous Sample Process Control |
| Instrument Systems | VELO Instrument | cobas Liat System |

VI Standards/Guidance Documents Referenced:

1. IEC 61010-1:2016
2. IEC 61010-1 Edition 3.1 2017-01 CONSOLIDATED VERSION 19-34
3. IEC 61010-2-010:2019
4. IEC 61010-2-101:2018
5. IEC 61326-2-6:2021
6. IEC 61000
7. ISO 15223-1 Fourth edition 2021-07
8. ISO 20417 First edition 2021-04 Corrected version 2021-12
9. ISO 14971 Third Edition 2019-12
10. ISO 23640:2015
11. ISO/IEC 14443A
12. IEC 62304 Edition 1.1 2015-06 CONSOLIDATED VERSION
13. ANSI AAMI IEC 62366-1:2015+AMD1:2020 (Consolidated Text)
14. CLSI EP17-A2
15. CLSI EP25 2nd Edition
16. ISTA 3A 2018
17. CLSI EP12 3rd Edition
18. 21 CFR 866.3981 - Special Controls
19. FDA Guidance: "Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses" (July 15, 2011)
20. FDA Guidance: "Respiratory Viral Panel Multiplex Nucleic Acid Assay - Class II Special Controls Guidance for Industry and FDA Staff" (October 9, 2009)
21. FDA Guidance: "Instrumentation for Clinical Multiplex Test Systems - Class II Special Controls Guidance for Industry and FDA Staff" (March 10, 2005)

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22. FDA Guidance: "Design Considerations for Pivotal Clinical Investigations for Medical Devices" (November 7, 2013) - Section 8: Diagnostic Clinical Performance Studies
23. FDA Guidance: "Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable" (April 25, 2006)
24. FDA Guidance: "Significant Risk and Nonsignificant Risk Medical Device Studies" (January 2006)
25. FDA Guidance: "Financial Disclosure by Clinical Investigators" (February 2013)
26. FDA guidance "Reprocessing Medical Devices in Health Care Settings: Validation Methods and Labeling" (March 17, 2015)

## VII Performance Characteristics (if/when applicable):

## A Analytical Performance:

1. Precision/Reproducibility:

a. Precision:

Precision of the VELO Respiratory Test was evaluated over 12 total days, conducted by two operators at a single site. A 3-member panel of contrived nasal swabs was evaluated, consisting of a true negative (no analyte) swab, a low positive (2x LoD of all three targets) swab, and a moderate positive (4x LoD of all three targets) swab. The negative swab samples were contrived using simulated respiratory matrix and the positive swab samples were contrived using simulated respiratory matrix co-spiked with SARS-CoV-2, influenza A and influenza B viruses. The test swab samples were randomized and blinded to the operator running the VELO Instrument. The study was conducted by testing the 3-member panel of contrived nasal swabs using one lot of VELO Respiratory Test cartridges, tested by 2 operators each performing 2 replicates/run and 2 runs per day, for a total of 12 days, resulting in a total of 96 replicates per panel member. Four VELO Instruments were utilized during this study. Results are summarized in Tables 1-3. The results of the study demonstrate acceptable test variability.

Table 1. Summary of % Agreement with expected results (95% CI) by Operator in the Precision Study

| Analyte | Panel member | Operator 1
n/N^{[1]} | Operator 2
n/N^{[1]} | Overall % Agreement
with Expected Results
(n/N^{[1]}) | 95% CI |
| --- | --- | --- | --- | --- | --- |
| SARS-CoV-2 | Negative | 48/48 | 48/48 | 100% (96/96) | 96.2-100% |
| | Low positive
2x LoD | 47/48 | 47/48 | 97.9% (94/96) | 92.7-99.4% |
| | Moderate positive
4x LoD | 47/48 | 48/48 | 99.0% (95/96) | 94.3-99.8% |
| Influenza A | Negative | 48/48 | 48/48 | 100% (96/96) | 96.2-100% |
| | Low positive
2x LoD | 48/48 | 47/48 | 99.0% (95/96) | 94.3-99.8% |
| | Moderate positive
4x LoD | 48/48 | 48/48 | 100% (96/96) | 96.2-100% |

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Table 2. Summary of % Agreement with expected results (95% CI) by VELO Instrument in the Precision Study

| Analyte | Panel member | VELO Instrument n/N[1] | | | | | Overall % Agreement with Expected Results (n/N[1]) | 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | 1 | 2 | 3 | 4 | 5 | | |
| SARS-CoV-2 | Negative | 25/25 | 22/22 | 24/24 | 25/25 | 100% (96/96) | 96.2-100% | |
| | Low positive 2x LoD | 23/24 | 23/24 | 25/25 | 23/23 | 97.9% (94/96) | 92.7-99.4% | |
| | Moderate positive 4x LoD | 23/23 | 23/23 | 25/26 | 24/24 | 99.0% (95/96) | 94.3-99.8% | |
| Influenza A | Negative | 25/25 | 22/22 | 24/24 | 25/25 | 100% (96/96) | 96.2-100% | |
| | Low positive 2x LoD | 23/24 | 24/24 | 25/25 | 23/23 | 99.0% (95/96) | 94.3-99.8% | |
| | Moderate positive 4x LoD | 23/23 | 23/23 | 26/26 | 24/24 | 100% (96/96) | 96.2-100% | |
| Influenza B | Negative | 25/25 | 22/22 | 24/24 | 25/25 | 100% (96/96) | 96.2-100% | |
| | Low positive 2x LoD | 24/24 | 24/24 | 24/25 | 23/23 | 99.0% (95/96) | 94.3-99.8% | |
| | Moderate positive 4x LoD | 23/23 | 23/23 | 26/26 | 24/24 | 100% (96/96) | 96.2-100% | |

[1] n is number of tests with expected results. N is the total number of valid tests

Table 3. Summary of % Agreement with expected results (95% CI) by Day in the Precision Study

| Analyte | Panel member | Day n/N[1] | | | | | | | | | | | | Overall % Agreement with Expected Results (n/N[1]) | 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | | |
| SARS-CoV-2 | Negative | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 100% (96/96) | 96.2-100% |
| | Low positive 2x LoD | 8/8 | 7/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 7/8 | 8/8 | 8/8 | 8/8 | 97.9% (94/96) | 92.7-99.4% |
| | Moderate positive 4x LoD | 8/8 | 8/8 | 8/8 | 7/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 99.0% (95/96) | 94.3-99.8% |
| Influenza A | Negative | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 100% (96/96) | 96.2-100% |
| | Low positive 2x LoD | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 7/8 | 8/8 | 8/8 | 8/8 | 99.0% (95/96) | 94.3-99.8% |
| | Moderate positive 4x LoD | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 100% (96/96) | 96.2-100% |
| Influenza B | Negative | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 100% (96/96) | 96.2-100% |

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| | Low positive 2x LoD | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 7/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 99.0% (95/96) | 94.3-99.8% |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Moderate positive 4x LoD | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 100% (96/96) | 96.2-100% |

[1] n is number of tests with expected results. N is the total number of valid tests

## b. Reproducibility:

The reproducibility of the VELO Respiratory Test was evaluated at three distinct external CLIA-waived sites with a total of nine operators. A 3-member panel of contrived nasal swabs was evaluated consisting of a true negative (no analyte) swab, a low positive (2x LoD of all three targets) swab, and a moderate positive (4x LoD of all three targets) swab. The negative swab samples were contrived using simulated respiratory matrix and the positive swab samples were contrived using simulated respiratory matrix co-spiked with SARS-CoV-2, influenza A and influenza B viruses. The test swab samples were randomized and blinded to the operator running the VELO Instrument. The study was conducted by testing the 3-member panel of contrived nasal swabs using three lots of VELO Respiratory Test cartridges, tested by 9 operators over three sites (3 operators per site), each performing 1 replicate/run and 2 runs per day, over a total of 5 days, resulting in a total of 90 replicates per panel member. Six VELO Instruments (2 instruments per site) were utilized during this study. Results are summarized in Table 4. No significant differences between sites, instruments, lots or operators were observed. The results of the study demonstrate acceptable test reproducibility.

Table 4. Summary of % Agreement with expected results (95% CI) by Site in the Reproducibility Study

| Analyte | Panel member | Site n/N[1] | | | Overall % Agreement with Expected Results (n/N[1]) | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| | | A | B | C | | |
| SARS-CoV-2 | Negative | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| | Low positive 2x LoD | 30/30 | 29/30 | 29/30 | 97.8% (88/90) | 92.3-99.4% |
| | Moderate positive 4x LoD | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| Influenza A | Negative | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| | Low positive 2x LoD | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| | Moderate positive 4x LoD | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| Influenza B | Negative | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| | Low positive 2x LoD | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |
| | Moderate positive 4x LoD | 30/30 | 30/30 | 30/30 | 100% (90/90) | 95.9-100% |

[1] n is number of tests with expected results. N is the total number of valid tests

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The mean and variability analysis between sites, operators, lots, days, runs, within-runs, and overall (total) for Ct values is shown in Table 5. Overall $\% \mathrm{CV}$ was $\leq 4.73\%$ . Overall variability was low, and the study demonstrates assay variability within an acceptable range.

Table 5: Summary of Reproducibility Results (Cq Variability Analysis)

| Analyte | Panel Member Conc. | n/N[1] | Mean Cq[2] | Total | | Between Lot | | Between Site | | Between Operator | | Between Day | | Between Run | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| SARS-CoV-2 | 2x LoD | 88/90 | 29.4 | 0.85 | 2.87 | 0.32 | 1.10 | 0.09 | 0.31 | 0.18 | 0.61 | 0.16 | 0.56 | 0.25 | 0.86 |
| | 4x LoD | 90/90 | 28.7 | 0.78 | 2.72 | 0.04 | 0.14 | 0.33 | 1.16 | 0.52 | 1.81 | 0.18 | 0.64 | 0.25 | 0.87 |
| Flu A | 2x LoD | 90/90 | 28.8 | 0.91 | 3.16 | 0.37 | 1.29 | 0.06 | 0.21 | 0.14 | 0.47 | 0.08 | 0.26 | 0.25 | 0.88 |
| | 4x LoD | 90/90 | 27.2 | 0.89 | 3.21 | 0.34 | 1.22 | 0.16 | 0.59 | 0.33 | 1.20 | 0.14 | 0.51 | 0.24 | 0.87 |
| Flu B | 2x LoD | 90/90 | 30.3 | 1.44 | 4.73 | 0.32 | 1.06 | 0.19 | 0.64 | 0.52 | 1.71 | 0.47 | 1.56 | 0.52 | 1.71 |
| | 4x LoD | 90/90 | 29.2 | 0.98 | 3.35 | 0.10 | 0.34 | 0.31 | 1.07 | 0.47 | 1.60 | 0.28 | 0.95 | 0.31 | 1.08 |

[1]n is number of tests with expected results. N is the total number of valid tests
[2] According to the VELO Instrument detection algorithm, not all targets reported as 'Detected' have a Cq value assigned and recorded in logs. Analysis here is completed for all available Cq values.
$\mathrm{Cq} =$ quantification cycle, $\mathrm{SD} =$ standard deviation, $\% \mathrm{CV} =$ percent coefficient of variation

# 2. Linearity:

Not applicable. This is a qualitative assay.

# 3. Analytical Specificity/Interference:

# a. Analytical Reactivity (Inclusivity):

Wet Testing: The inclusivity of the VELO Respiratory Test was established through wet testing by evaluating ten strains of SARS-CoV-2, ten strains of Influenza A H1N1 (including 5 pandemic 2009 strains), ten strains of Influenza H3N2 and eight strains of Influenza B, as shown in Table 6. These strains are in addition to the SARS-CoV-2, Influenza A H1N1, Influenza H3N2 and Influenza B strains used in the analytical sensitivity study (Table 10). Inactivated strains were used for SARS-CoV-2 and cultured virus were used for influenza strains. Each virus was spiked into pooled negative nasal matrix at analyte concentrations of $3\mathrm{x}$ LoD. This spiked material was then transferred onto a swab and tested on three devices.

Of the 38 viruses tested, 33 were successfully detected in $3/3$ devices for the expected strain (influenza A, influenza B or SARS-CoV-2) on the VELO Respiratory Test at 3x LoD. Influenza A H1N1 was detected in $3/3$ devices at 4x LoD for strains A/NY/01/09 and A/Denver/1/1957, 5x LoD for strain A/Swine/Iowa/15/, and 6x LoD for strains A/California/08/2009 and A/New Jersey/8/1976. The results from this study demonstrate that the VELO Respiratory Test run on the VELO instrument is capable of detecting multiple strains of influenza A, influenza B and SARS-CoV2 at the concentrations tested.

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Table 6. Analytical Reactivity (Inclusivity) Wet-Testing Study Results

| Virus | Strain | Concentration per swab | Number detected/Number Tested (% detected) |
| --- | --- | --- | --- |
| Influenza A H1N1 (including pandemic 2009 strains) | A/California/07/09 | 7.5 TCID_{50} | 3/3 (100%) |
| | A/NY/01/09 | 7.5 TCID_{50} | 5/6 (83%) |
| | | 10 TCID_{50} | 3/3 (100%) |
| | A/Victoria/4897/2022 | 9,000 cp | 3/3 (100%) |
| | A/Wisconsin/67/2022 | 9,000 cp | 3/3 (100%) |
| | A/California/08/2009 | 84 CEID_{50} | 4/6 (67%) |
| | | 140 CEID_{50} | 5/6 (83%) |
| | | 168 CEID_{50} | 3/3 (100%) |
| | A/Denver/1/1957 | 84 CEID_{50} | 5/6 (83%) |
| | | 112 CEID_{50} | 3/3 (100%) |
| | A/New Jersey/8/1976 | 84 CEID_{50} | 0/3 (0%) |
| | | 140 CEID_{50} | 5/6 (83%) |
| | | 168 CEID_{50} | 3/3 (100%) |
| | A/NWS/33 | 84 CEID_{50} | 3/3 (100%) |
| | A/Solomon Island/3/2006 | 84 CEID_{50} | 3/3 (100%) |
| | A/Swine/Iowa/15/30 | 84 CEID_{50} | 3/6 (50%) |
| | | 140 CEID_{50} | 3/3 (100%) |
| Influenza A H3N2 | A/Aichi/2/68 | 236.4 CEID_{50} | 3/3 (100%) |
| | A/Brisbane/10/07 | 236.4 CEID_{50} | 3/3 (100%) |
| | A/California/122/2022 | 9,000 cp | 3/3 (100%) |
| | A/Switzerland/9715293/13 | 7.5 TCID_{50} | 3/3 (100%) |
| | A/Sydney/5/1997 | 236.4 CEID_{50} | 3/3 (100%) |
| | A/Texas/50/12 | 7.5 TCID_{50} | 3/3 (100%) |
| | A/Thailand/8/2022 | 9,000 cp | 3/3 (100%) |
| | A/Uruguay/716/2007 | 236.4 CEID_{50} | 3/3 (100%) |
| | A/Victoria/361/2011 | 236.4 CEID_{50} | 3/3 (100%) |
| | A/Wisconsin/67/05 | 7.5 TCID_{50} | 3/3 (100%) |
| Influenza B | B/Lee/1940 | 26.4 CEID_{50} | 3/3 (100%) |
| Influenza B Victoria Lineage | B/Michigan/1/2021 | 9,000 cp | 3/3 (100%) |
| | B/Brisbane/60/2008 | 26.4 CEID_{50} | 3/3 (100%) |
| | B/Nevada/03/2011 | 26.4 CEID_{50} | 3/3 (100%) |
| | B/Hong Kong/330/2001 | 26.4 CEID_{50} | 3/3 (100%) |
| Influenza B Yamagata Lineage | B/Guangdong/120/00 | 3 TCID_{50} | 3/3 (100%) |
| | B/Massachusetts/2/12 | 3 TCID_{50} | 3/3 (100%) |
| | B/Texas/6/11 | 3 TCID_{50} | 3/3 (100%) |
| SARS-CoV-2 | 2019-nCoV/USA-WA1/2020 | 9,000 cp | 3/3 (100%) |
| | Alpha (B.1.1.7) VOC202012/01 | 9,000 cp | 3/3 (100%) |
| | Beta (B.1.351) VOC202012/02 | 9,000 cp | 3/3 (100%) |
| | Gamma (P.1) | 9,000 cp | 3/3 (100%) |

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| Virus | Strain | Concentration per swab | Number detected/Number Tested (% detected) |
| --- | --- | --- | --- |
| | Delta (B.1.617.2) VOC21APR-02 | 9,000 cp | 3/3 (100%) |
| | BA.2.12.1; USA/NY-Wadsworth-22014351- 01/2022 | 1.5 TCID50 | 3/3 (100%) |
| | BA.4.6; USA/MDHP35538/2022 | 1.5 TCID50 | 3/3 (100%) |
| | BF.7; USA/NY-Wadsworth-22042128-01/2022 | 1.5 TCID50 | 3/3 (100%) |
| | XBB; USA/CA-Stanford-109_S21/2022 | 1.5 TCID50 | 3/3 (100%) |
| | XBB.1.5; USA/NY-Wadsworth-22061020-01/2022 | 1.5 TCID50 | 3/3 (100%) |

In Silico Analysis: The inclusivity of the VELO Respiratory Test was evaluated in May 2025 using in silico analysis of the forward primers, reverse primers, and probes for the SARS-CoV-2 Orf1ab and M targets using sequences available in the NCBI and GISAID gene databases. The in silico analysis of 137,277 SARS-CoV-2 sequences, including variants of concern (VOC), variants under investigation (VUI) and variants under monitoring (VUM), indicate that $100\%$ of SARS-CoV-2 sequences analyzed had no changes within the primer and/or probe binding regions that would be predicted to affect the performance of both assay targets. Therefore, all known SARS-CoV-2 variants are predicted to be detected.

# b. Cross-reactivity:

The analytical specificity (cross-reactivity) of the VELO Respiratory Test was evaluated by testing a cohort of fifty-two (52) non-targeted microorganisms (including viruses, bacteria and fungi) that have a similar genome to influenza A virus, influenza B virus and SARS-CoV-2 or are reasonably likely to be present in the clinical sample (Table 7). Panels were composed of up to three different non-target microorganisms spiked into pooled negative nasal matrix at $\geq 1\times 10^{5}$ units/mL (for viruses) and $\geq 1\times 10^{6}$ units/mL (for bacteria and fungi). Each panel was tested in triplicate. Exclusivity was determined if $0/3$ replicates returned a detected result for each of the three (3) viral targets. None of the evaluated organisms demonstrated cross-reactivity with the VELO Respiratory Test at the tested concentrations listed in Table 7, with each returning $0/3$ replicates detected for each of the three viral targets.

Table 7. Non-targeted Microorganisms Evaluated in the Cross-reactivity and Microbial Interference Wet-Testing Studies

| Microorganism | Concentration (per mL) |
| --- | --- |
| Adenovirus Type 1a | 3.45x106TCID50 |
| Adenovirus Type 7a | 3.37x106TCID50 |
| Adenovirus Type 10a | 9.6x105TCID50 |
| Adenovirus Type 21 | 2x105TCID50 |
| Human Coronavirus OC43 | 2x105TCID50 |

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| Microorganism | Concentration (per mL) |
| --- | --- |
| Human Coronavirus 229E | 2x10^{5} copies |
| Human Coronavirus NL63 | 2.13x10^{5} TCID_{50} |
| Human Coronavirus HKU1^{b} | 2x10^{5} copies |
| MERS-CoV^{c} | N/A^{e} |
| Cytomegalovirus | 2.02x10^{5} TCID_{50} |
| Enterovirus Coxsackievirus CV-A16 | 2x10^{5} TCID_{50} |
| Enterovirus D68 | 2x10^{5} TCID_{50} |
| Enterovirus Type 71 | 2x10^{5} TCID_{50} |
| Epstein Barr Virus | 2x10^{5} TCID_{50} |
| Human parainfluenza Type 1 | 1.6x10^{5} TCID_{50} |
| Human parainfluenza Type 2 | 2x10^{5} TCID_{50} |
| Human parainfluenza Type 3 | 2x10^{5} TCID_{50} |
| Human parainfluenza Type 4 | 2x10^{5} TCID_{50} |
| Measles | 1.9x10^{5} TCID_{50} |
| Human Metapneumovirus Type 1A | 2x10^{5} TCID_{50} |
| Mumps virus | 1.1x10^{5} TCID_{50} |
| Respiratory syncytial virus A1998/3-2 | 2x10^{5} TCID_{50} |
| Respiratory syncytial virus A Long | 2x10^{5} TCID_{50} |
| Respiratory syncytial virus B | 2x10^{5} TCID_{50} |
| Rhinovirus A50, A2 | 1.33x10^{5} TCID_{50} |
| Rhinovirus 20, 15-CV19 | 2x10^{5} TCID_{50} |
| Aspergillus fumigatus | 4.65x10^{5} CFU |
| Aspergillus niger | 2x10^{6} CFU |
| Bordatella parapertussis | 2x10^{6} CFU |
| Bordetella pertussis | 2x10^{6} CFU |
| Candida albicans | 2x10^{6} CFU |
| Chlamydia pneumoniae | 2x10^{6} IFU |
| Corynebacterium xerosis | 2x10^{6} CFU |
| Escherichia coli | 2x10^{6} CFU |
| Fusobacterium necrophorum | 2x10^{6} CFU |
| Hemophilus influenzae | 2x10^{6} CFU |
| Klebsiella pneumoniae | 2x10^{6} CFU |
| Lactobacillus acidophilus | 5.79x10^{6} CFU |
| Legionella pneumophila | 2x10^{6} CFU |
| Moraxella catarrhalis | 2x10^{6} CFU |

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| Microorganism | Concentration (per mL) |
| --- | --- |
| Mycoplasma genitalium^{c} | 2x10^{6} CFU |
| Mycobacterium tuberculosis^{d} | 2x10^{6} copies |
| Mycoplasma pneumoniae | 2x10^{6} CCU |
| Neisseria meningitidis | 2x10^{6} CFU |
| Neisseria mucosa | 2x10^{6} CFU |
| Pneumocystis jirovecii (PJP)^{h} | 2x10^{6} copies |
| Pseudomonas aeruginosa | 2x10^{6} CFU |
| Staphylococcus aureus | 2x10^{6} CFU |
| Staphylococcus epidermis | 2x10^{6} CFU |
| Streptococcus pneumoniae | 2x10^{6} CFU |
| Streptococcus pyogenes | 2x10^{6} CFU |
| Streptococcus salivaris | 2x10^{6} CFU |

a When tested as part of the cross-reactivity study this panel of Adenoviruses (each virus spiked at 2x10⁵ TCID₅₀/mL) resulted in 1/3 replicates detected for one of the VELO Respiratory Test targets. When tested individually at the higher concentrations listed, 0/3 were detected for each of the three viral targets.
b Synthetic nucleic acid.
c Inactivated whole organism.
d Genomic nucleic acid.
e Swabs contrived with 8 μl of NATrol MERS-CoV Stock (Ct 25.7).

c. Microbial Interference:

Microbial interference was evaluated for the VELO Respiratory Test by testing a cohort of fifty-two (52) non-targeted microorganisms (including viruses, bacteria and fungi) that have a similar genome to influenza A virus, influenza B virus and SARS-CoV-2 or are reasonably likely to be present in the clinical sample (Table 7). All samples were prepared in pooled negative nasal matrix and tested with panels of up to three different non-target microorganisms in the presence of the test target analytes, SARS-CoV-2 (BetaCoV/Australia/VIC01/2020), influenza A (A/Hong Kong/8/68) and influenza B (B/Wisconsin/1/2010), co-spiked at 3x LoD. Non-target microorganisms were spiked at ≥1x10⁵ units/mL (for viruses) and ≥1x10⁶ units/mL (for bacteria and fungi. Each panel was tested in triplicate. Absence of microbial interference was determined if 3/3 replicates returned a detected result for each of the three (3) target analytes. None of the evaluated microorganisms demonstrated interference with the VELO Respiratory Test at the tested concentrations listed in Table 7, with each returning 3/3 replicates detected for each of the three viral targets.

d. Competitive Interference:

The impact of competitive interference, caused by co-infections with on-panel analytes, was evaluated for the VELO Respiratory Test by testing contrived samples containing high concentration of SARS-CoV-2, influenza A or influenza B strains in the presence of either one or both of the other target viruses at 3x LoD prepared in pooled negative nasal matrix.

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For this study, competitive interference was assessed using one strain each of SARS-CoV-2 (USA-WA1/2020) and influenza B (B/Wisconsin/1/2010) and two strains of influenza A (A/PR/8/34 - H1N1 strain and A/Hong Kong/8/68 - H3N2 strain). Testing was performed in triplicate. Absence of competitive interference was determined if all replicates for the low concentration (3x LoD) target(s) yielded positive results.

The study showed that influenza B at $1 \times 10^{6}$ copies/swab inhibited detection of influenza A (H1N1) at $3 \times$ LoD, in the presence of SARS-CoV-2 at $3 \times$ LoD (Table 8). Subsequent testing using samples consisting of influenza A (H1N1) at $3 \times$ LoD, in the presence of influenza B at concentrations ranging from $1 \times 10^{5} - 1.5 \times 10^{6}$ copies/swab demonstrated no competitive interference (Table 8). No competitive interference was observed for the other potential co-infections evaluated at the concentrations tested (Table 8).

Table 8. Competitive Interference Results Summary

| Viral Targets in Sample | | | Detection Rate | | |
| --- | --- | --- | --- | --- | --- |
| Influenza A | Influenza B | SARS-CoV-2 | Influenza A | Influenza B | SARS-CoV-2 |
| H3N2 1x106cp/swab | 3xLoD | 3x LoD | 3/3 | 3/3 | 3/3 |
| H1N1 1x106cp/swab | 3xLoD | 3x LoD | 3/3 | 3/3 | 3/3 |
| H3N2 3x LoD | N/A | 1x106cp/swab | 3/3 | 0/3 | 3/3 |
| H1N1 3x LoD | 3x LoD | 1x106cp/swab | 3/3 | 3/3 | 3/3 |
| H3N2 3x LoD | 1x106cp/swab | N/A | 3/3 | 3/3 | 0/3 |
| H1N1 3x LoD | 1x106cp/swab | 3x LoD | 2/3 | 3/3 | 3/3 |
| H1N1 3x LoD | 1x106cp/swab | N/A | 3/3 | 3/3 | 0/3 |
| | 1x105cp/swab | N/A | 3/3 | 3/3 | 0/3 |
| | 9x105cp/swab | N/A | 3/3 | 3/3 | 0/3 |
| | 1.5x106cp/swab | N/A | 3/3 | 3/3 | 0/3 |

*cp/swab = copies/swab

# e. Interfering Substances:

The performance of the VELO Respiratory Test was evaluated in the presence of endogenous and exogenous substances that may be commonly found in ANS specimens. A total of 22 potentially interfering endogenous and exogenous substances (Table 9) were tested at or above clinically relevant levels in pooled negative nasal matrix in the presence and absence of test target analytes. Positive samples were prepared by co-spiking the test target analytes, SARS-CoV-2 (BetaCoV/Australia/VIC01/2020), influenza A (A/Hong Kong/8/68) and influenza B (B/Wisconsin/1/2010) at $3\mathrm{x}$ LoD in pooled negative nasal matrix containing an individual exogenous or endogenous substance. Negative samples consisted of pooled negative nasal matrix containing an individual exogenous or endogenous substance. Each

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sample was tested in triplicate.

Three of the substances tested in the study, blood (15% v/v), Triamcinolone (11 μg/swab) and Otravine Extra Dual Relief Nasal Spray (15% v/v; active ingredients listed in Table 9) gave false negative results with the influenza A target at the initial concentration tested. Mucin (5% w/v) gave a false negative result with the influenza B target and the anti-viral drug Zanamivir (7.5 mg/mL) gave 2 false positive results, one for the SARS-CoV-2 target and one for the influenza B target, and a false negative result for the influenza A target. The FluMist nasal vaccine was not tested as cross-reactivity with the influenza test targets is expected. Additional testing at lower test substance concentrations were performed to determine the concentration where interference is no longer observed. No interference was observed for any of the substances tested at the concentrations noted in Table 9.

Table 9. Exogenous/Endogenous Interfering Substances Study Results

| Potential Interferant | Product | Active Ingredient(s) | Concentration |
| --- | --- | --- | --- |
| Blood (human) | N/A | None Specified | 10% v/v a |
| Leukocytes | N/A | None Specified | 1 x 10^6 cells/swab |
| Mucin: bovine submaxillary gland, type I-S | N/A | Purified mucin protein | 2% w/v b |
| Nasal spray or drops | Zicam Intense Sinus Relief | Oxymetazoline HCl (0.05% w/v) / Menthol | 15% v/v |
| | Phenylephrine | Phenylephrine | 0.03 μg/mL |
| | Calpol saline nasal spray | Sodium chloride (0.9%) with preservatives | 15% v/v |
| Nasal corticosteroids | Pirinase Hayfever once daily spray | Fluticasone propionate (50 μg/spray) | 15% v/v |
| | Boots Adult Hay fever relief | Beclomethasone (50 μg/spray) | 15% v/v |
| | Dexamethasone | Dexamethasone | 12 μg/mL |
| | Flunisolide | Flunisolide | 16 μg/swab |
| | Triamcinolone | Triamcinolone | 10 μg/swab c |
| | Benacort Hayfever Relief Nasal Spray | Budesonide (64 μg/spray) | 15% v/v |
| | Clarinaze Allergy Control 0.05% Nasal Spray | Mometasone furoate (50 μg/spray) | 15% v/v |
| Nasal gel | Zicam, Powerful Allergy Relief | Sulfur / Luffa opperculata / Galphimia glauca / Histaminum hydrochloricum | 15% v/v |
| Sore throat and cough lozenges | Ultra Chloraseptic Spray | Benzocaine (0.71%) | 15% v/v |
| Anti-viral drugs | N/A | Zanamivir | 6.0 mg/mLd |
| | N/A | Oseltamivir phosphate | 0.4 μg/mL |
| Antibiotics | N/A | Mupirocin | 1.5 μg/mL |
| | N/A | Tobramycin | 33 μg/mL |
| Zinc (common ingredient in nasal sprays) | Zinc chloride | Zinc | 0.1 mg/mL |
| Nicotine or Tobacco | Nicorette (Nicotine 0.5 | Nicotine | 15% v/v |
| | Nicorette (Nicotine 0.1 | Nicotine | 10 μg/mL) |
| Nicotine or Tobacco | Nicorette (Nicotine 0.5 | Nicotine | 10 μg/mL) |

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| Potential Interferant | Product | Active Ingredient(s) | Concentration |
| --- | --- | --- | --- |
| | mg/spray) | | |
| Decongestant | Otravine Extra Dual Relief Nasal Spray | Xylometazoline hydrochloride / Ipratropium bromide | 13.5% v/v a |

a Potential interference observed with 15% v/v.
b Potential interference observed with 5 and 2.5% w/v.
c Potential interference observed 11 µg/swab.
d Potential interference observed with 7.5 and 6.75 mg/mL.
Note: FluMist Quadrivalent was not evaluated as cross-reactivity with targets is expected.

4. Assay Reportable Range:

Not applicable.

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

a. Controls – External Control Evaluation:

The assay contains an endogenous sample and process control (SPC) that serves as an Internal Control (IC) and commercially available external positive and negative controls. For more information, see Section IV.C.5. Quality Control, above.

b. In-use Test Cartridge Hold Time - Specimen Stability:

In accordance with the VELO Respiratory Test labeling, patient specimens should be tested immediately after collection for optimal test performance, however, sample storage and handling were evaluated for VELO Respiratory Test to support the following:

- Specimen loaded Test Cartridges are stable up to 30 minutes at room temperature (15-30°C/59-86°F).

c. Kit Shelf-life Stability:

Real-time kit stability data for the VELO Respiratory Test supports the recommended shelf-life stability claim of ambient room temperature storage claim (15-30°C / 59-86°F) for up to 9 months.

d. In-use Test Cartridge Stability – Open Pouch:

In accordance with the VELO Respiratory Test labeling, the sealed VELO Respiratory Test Cartridge foil pouch should be opened at the time of sample loading, however, VELO Respiratory Test Cartridge stability once removed from the foil pouch was evaluated for the VELO Respiratory Test to support the following:

- The VELO Respiratory Test Cartridge is stable for up to one hour after opening the foil pouch at room temperature (15-30°C/59-86°F).

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# 6. Detection Limit:

The limit of detection (LoD) of the VELO Respiratory Test was evaluated for eight viral strains: two Influenza A H1N1 strains (A/PR/8/34 and A/New Cal/20/99), two Influenza A H3N2 strains (A/Hong Kong/8/68 and A/South Australia/55/14), Influenza B Yamagata Lineage (B/Wisconsin/1/2010), Influenza B Victoria Lineage (Malaysia/2506/04), SARS-CoV-2 (Beta CoV/Australia/VIC01/2020), and inactivated Omicron BA.5 (hCoV-19/USA/COR-22-06-3113/2022). Testing used pooled negative nasal matrix with multiple lots of VELO Respiratory Test Cartridges and VELO Instruments.

The preliminary LoD was established as the minimum concentration yielding 3/3 "Detected" results in a limited dilution series for each target viral strain per cartridge lot. The confirmatory LoD was determined by testing the preliminary LoD plus at least one higher and one lower concentration with multiple replicates across $\geq 3$ days. The confirmatory LoD was the lowest concentration consistently detected at $\geq 95\%$ across both cartridge lots. A final LoD validation was performed in which the confirmatory LoD for each strain was tested with a third cartridge lot using 20 replicates. The final confirmatory LoDs (Table 10) represent testing across three cartridge lots with 60 total replicates achieving $\geq 95\%$ detection rates. Co-analyte spiked samples were also evaluated and showed equivalent LoDs to single analyte samples.

Table 10. Confirmed LoD for Influenza A, Influenza B and SARS-CoV-2

| Virus | Strain | Source/Product Type | LoD Copies (cp), IU or TCID_{50} / swab |
| --- | --- | --- | --- |
| SARS-CoV-2 | BetaCoV/Australia/VIC01/2020 | NIBSC Inactivated | 3,000 IU/swab |
| | hCoV-19/USA/COR-22-06-3113/2022 | Zeptometrix Inactivated | 0.5 TCID_{50}/swab |
| Influenza A H1N1 | A/PR/8/34 | ATCC Live | 3,000 cp/swab^{a} |
| | A/New Cal/20/99 | Zeptometrix Live | 2.5 TCID_{50}/swab |
| Influenza A H3N2 | A/Hong Kong/8/68 | ATCC Live | 3,000 cp/swab^{b} |
| | A/South Australia/55/14 | Zeptometrix Live | 2.5 TCID_{50}/swab |
| Influenza B (Yamagata) | B/Wisconsin/1/2010 | ATCC Live | 3,000 cp/swab^{c} |
| Influenza B (Victoria) | Malaysia/2506/04 | Zeptometrix Live | 1.0 TCID_{50}/swab |

$^{a}$ Equivalent to 28.0 CEID$_{50}$/swab
$^{b}$ Equivalent to 78.8 CEID$_{50}$/swab
$^{c}$ Equivalent to 8.8 CEID$_{50}$/swab

# 7. Assay Cut-Off:

For each reaction chamber, the respective target is detected using a detection algorithm which monitors the fluorescence signal generated at each PCR cycle and uses pre-determined

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criteria to establish if amplification has occurred. While 'Detected' targets can be reported before completion of all 42 PCR cycles, if the algorithm criteria have been met (Cq value determined), 'Not Detected' targets can only be reported after all 42 PCR cycles are completed and analyzed.

8. Accuracy (Instrument):

Not Applicable.

9. Carry-Over:

An analytical study was performed to assess potential carryover or cross-contamination in the single-use, self-contained VELO Respiratory Test Cartridge by testing high positive and no template samples in an alternating fashion on the same VELO Instrument. The high positive samples consisted of a single viral target, either SARS-CoV-2 (USA-WA1/2020- ATCC inactivated), influenza A (H1N1 - A/PR/8/34 - live/ H3N2 - A/Hong Kong/8/68 - live) or influenza B (B/Wisconsin/1/2010 - live), prepared by spiking a swab at 1×10⁶ copies/swab in pooled negative nasal matrix. The no template samples were prepared by spiking a swab with nuclease free water only. A no template sample was followed by a positive sample alternating 8 times before running a final no template sample to give 17 runs per VELO instrument. The study was repeated on five VELO Instruments for a total of 40 positive and 45 no template samples. The high positive samples yielded 100% the expected detected result for the target analyte in the sample and not detected for the other two target analytes. The no template samples yielded either 93% the expected invalid result or not detected (7%) for all viral targets. These results demonstrated that there is an acceptable, low likelihood of cross-contamination between samples when the VELO Respiratory Test is performed on the VELO instrument according to the instructions for use.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Not Applicable.

2. Matrix Comparison:

The objective of this study was to establish equivalent performance of the VELO Respiratory Test between the two matrices used in the analytical studies: pooled negative nasal matrix (NNM) and simulated nasal matrix. Simulated nasal matrix was formulated using vials of AmpliRun Negative Respiratory Swab Matrix negative control material (Vircell) rehydrated with a solution of bovine mucin. For this study, SARS-CoV-2 (BetaCoV/Australia/VIC01/2020), influenza A (A/Hong Kong/8/68) and influenza B (B/Wisconsin/1/2010) swabs were co-spiked for both matrices at 2x and 5x LoD. Negative swabs were also prepared for both matrices and included in the evaluation. For both matrices, 10 replicates of the negative samples, 30 replicates of the positive swabs prepared at 2x LoD and 10 replicates of the positive swabs prepared at 5x LoD were tested. The acceptance criteria to demonstrate equivalency was ≥95% detection for samples at 2x LoD, 100% detection for the samples at 5x LoD for each target, and 0% detection of the negative samples. The results obtained in

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this study are summarized in Table 11. The data demonstrated equivalent performance of the test with both the pooled negative nasal matrix and simulated nasal matrix.

Table 11. Matrix Equivalency

| Virus | Concentration | % Detected (Rate) | |
| --- | --- | --- | --- |
| | | NNM | Simulated matrix |
| SARS-CoV-2 | 5x LoD | 100% (10/10) | 100% (10/10) |
| Flu A | 5x LoD | 100% (10/10) | 100% (10/10) |
| Flu B | 5x LoD | 100% (10/10) | 100% (10/10) |
| SARS-CoV-2 | 2x LoD | 97% (29/30) | 100% (30/30) |
| Flu A | 2x LoD | 100% (30/30) | 97% (29/30) |
| Flu B | 2x LoD | 100% (30/30) | 100% (30/30) |
| SARS-CoV-2 | Negative | 0% (0/10) | 0% (0/10) |
| Flu A | Negative | 0% (0/10) | 0% (0/10) |
| Flu B | Negative | 0% (0/10) | 0% (0/10) |

# C Clinical Studies:

# 1. Prospective Study:

The clinical performance of the VELO Respiratory Test to detect influenza A, influenza B, and SARS-CoV-2 was evaluated in a prospective clinical study using paired anterior nasal swab (ANS) specimens collected from individuals with signs and symptoms of upper respiratory viral infection. Testing of clinical samples was performed with the VELO Respiratory Test in nine (9) CLIA waived healthcare facilities (e.g., physician offices, primary care / outpatient clinics, and urgent care centers) in various geographical locations with 15 untrained test operators. The results of all three viral targets were compared to results from an FDA-cleared, CLIA waived RT-PCR assay (comparative reference method).

Prospective clinical specimens were collected and tested between December 2024–March 2025. Initial enrollment in the prospective clinical study included 1,815 anterior nasal swab specimens. Of these, 97 specimens were excluded from the performance analysis for major protocol deviations. Table 12 provides a summary of the demographic information for the remaining 1,718 subjects enrolled in the clinical study.

Table 12. Subject Demographics- Prospective Symptomatic Population

| Characteristics | Symptomatic Subjects |
| --- | --- |
| Total, N | 1718 |
| Age (years) | |
| Mean | 41.0 |
| Standard Deviation | 17.97 |
| Median | 41 |
| Range (minimum – maximum) | 0 – 92 |
| Age Group (Years), n (%) | |
| ≤ 12 | 78 (4.5%) |
| 13 to ≤18 | 96 (5.6%) |

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In addition, of the 1,718 evaluable specimens collected in the prospective study a further 6 specimens were excluded either due to invalid comparator tests results for all three target analytes or the patient subsequently withdrew from the study. Of the remaining 1,712 evaluable specimens from the prospective clinical study, additional exclusions due to invalid VELO Respiratory Test results upon retest resulted in the following final sample sizes for the performance evaluation of each analyte, influenza A 1,677 (35 excluded), influenza B 1,665 (47 excluded), and SARS-CoV-2 1,670 (42 excluded). All remaining specimens had valid VELO Respiratory Test and comparator test results for their respective targets and the performance is summarized in Table 13. Three (3) influenza A / SARS-CoV-2 coinfections were detected by both the VELO Respiratory Test and the primary reference method.

Table 13. Prospective, Paired ANS Specimen Results For All Targets

| Analyte | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
| --- | --- | --- | --- | --- | --- | --- |
| | TP/(TP+FN) | % | 95% CI | TN/(TN+FP) | % | 95% CI |
| SARS-CoV-2 | 150/156^{a, b} | 96.2 | 91.9 – 98.2 | 1507/1514 | 99.5 | 99.1 – 99.8 |
| Influenza A | 342/370^{a, c} | 92.4 | 89.3 – 94.7 | 1295/1307^{d} | 99.1 | 98.4 – 99.5 |
| Influenza B | 14/15^{e} | 93.3 | 70.2 – 98.8 | 1646/1650 | 99.8 | 99.4 – 99.9 |

a Includes three (3) Influenza A / SARS-CoV-2 coinfections.
b One (1) discrepant specimen tested negative on secondary reference testing.
c Two (2) discrepant specimens tested negative on secondary reference testing.
d Three (3) discrepant specimens tested positive on secondary reference testing.
e One (1) discrepant specimen tested negative on secondary reference testing.

2. Retrospective/Archived Study:

To supplement the prospective data for influenza B, retrospective frozen clinical ANS specimens collected from individuals with signs and symptoms of influenza infection during the 2023-2024 North American respiratory season were evaluated. Frozen paired positive and negative ANS (n=110) specimens prospectively obtained during the 2023-2024 influenza

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season were distributed to a single CLIA-waived site and tested during their daily workflow over a period of 5 days. Of the 110 retrospective specimens with valid comparator results, 12 were excluded due to invalid VELO Respiratory Test results. Retesting was not applicable in this case. All remaining specimens had valid test results for influenza B for the VELO Respiratory Test and a comparator RT-PCR assay and the performance is summarized in Table 14.

Table 14. Retrospective, Paired ANS Specimen Results For Influenza B Results

| Analyte | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
| --- | --- | --- | --- | --- | --- | --- |
| | TP/(TP+FN) | % | 95% CI | TN/(TN+FP) | % | 95% CI |
| Influenza B | 15/16 | 93.8 | 71.7 – 98.9 | 82/82 | 100 | 95.5 – 100 |

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

Not Applicable.

D Clinical Cut-Off:

Not Applicable.

E Expected Values/Reference Range:

The positivity for SARS-CoV-2, Flu A and Flu B, as determined by the VELO Respiratory Test, are shown below (Table 15), stratified by the study site.

Table 15: Overall Positivity Rates Observed During the Clinical Study Stratified by Site

| Clinical Site ID | Site Location | SARS-Cov-2 | | | Influenza A | | | Influenza B | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Total No. | No. Pos. | Exp. Value | Total No. | No. Pos. | Exp. Value | Total No. | No. Pos. | Exp. Value |
| Overall | | 1,670 | 157 | 9.4% | 1,677 | 354 | 21.1% | 1,665 | 18 | 1.1% |
| 1 | Riverside CA | 138 | 1 | 0.7% | 138 | 26 | 18.8% | 138 | 6 | 4.3% |
| 2 | Orange City FL | 73 | 3 | 4.1% | 73 | 14 | 19.2% | 73 | 1 | 1.4% |
| 3 | DeLand FL | 20 | 2 | 10.0% | 20 | 1 | 5.0% | 20 | 0 | 0.0% |
| 4 | Brooklyn NY | 5 | 0 | 0.0% | 5 | 0 | 0.0% | 5 | 0 | 0.0% |
| 5 | Gulfport MS | 232 | 5 | 2.2% | 232 | 13 | 5.6% | 232 | 0 | 0.0% |
| 6 | Birmingham AL | 379 | 49 | 12.9% | 380 | 121 | 31.8% | 377 | 5 | 1.3% |
| 7 | Birmingham AL | 595 | 90 | 15.1% | 600 | 157 | 26.2% | 592 | 6 | 1.0% |
| 8 | Tulsa OK | 215 | 6 | 2.8% | 215 | 19 | 8.8% | 215 | 0 | 0.0% |
| 9 | Salt Lake City, UT | 13 | 1 | 7.7% | 14 | 3 | 21.4% | 13 | 0 | 0.0% |

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F Other Supportive Instrument Performance Characteristics Data:
Not Applicable.

VIII Proposed Labeling:
The labeling supports or the finding of substantial equivalence for this device.

IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

The submitted information in this CLIA waiver application supports a CLIA waiver approval decision.

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