Who is the manufacturer of Healgen Rapid Check COVID-19/Flu A&B Antigen Test?

Healgen filed the De Novo classification request for Healgen Rapid Check COVID-19/Flu A&B Antigen Test (DEN240029).

What is the FDA product code for Healgen Rapid Check COVID-19/Flu A&B Antigen Test?

SCA. It is classified under 21 CFR 866.3987 as a Class 2 device in the Microbiology panel.

What is the regulatory pathway for Healgen Rapid Check COVID-19/Flu A&B Antigen Test?

De Novo classification (submission type: Direct).

Who can help prepare an FDA De Novo submission like Healgen Rapid Check COVID-19/Flu A&B Antigen Test?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA De Novo submissions. See https://innolitics.com/services/regulatory/ or contact https://innolitics.com/contact.

Healgen Rapid Check COVID-19/Flu A&B Antigen Test

DEN240029 · Healgen · SCA · Oct 7, 2024 · Microbiology

Device Facts

Record ID	DEN240029
Device Name	Healgen Rapid Check COVID-19/Flu A&B Antigen Test
Applicant	Healgen
Product Code	SCA · Microbiology
Decision Date	Oct 7, 2024
Decision	DENG
Submission Type	Direct
Regulation	21 CFR 866.3987
Device Class	Class 2
Attributes	Pediatric

Intended Use

The Healgen Rapid Check COVID-19/Flu A&B Antigen Test is a lateral flow immunochromatographic assay intended for the qualitative detection and differentiation of influenza A, and influenza B nucleoprotein antigens and SARS-CoV-2 nucleocapsid antigen directly in anterior nasal swab samples from individuals with signs and symptoms of respiratory tract infection. Symptoms of respiratory infections due to SARS-CoV-2 and influenza can be similar. This test is for non-prescription home use by individuals aged 14 years or older testing themselves, or adults testing individuals aged 2 years or older. All negative results are presumptive and should be confirmed with an FDA-cleared molecular assay when determined to be appropriate by a healthcare provider. Negative results do not rule out infection with influenza, SARS-CoV-2 or other pathogens. Individuals who test negative and experience continued or worsening respiratory symptoms, such as fever, cough and/or shortness of breath, should therefore seek follow-up care from their healthcare provider. Positive results do not rule out co-infection with other respiratory pathogens and therefore do not substitute for a visit to a healthcare provider or appropriate follow-up.

Device Story

Lateral flow immunochromatographic assay; detects influenza A, influenza B, and SARS-CoV-2 nucleoprotein/nucleocapsid antigens. Input: anterior nasal swab samples. Operation: user-performed at home; sample applied to test device; visual interpretation of results via lateral flow. Output: qualitative test result (positive/negative). Intended for lay users; no healthcare provider intervention required for testing. Results are preliminary; negative results require molecular confirmation if symptoms persist. Positive results do not rule out co-infection. Benefits: rapid, accessible screening for respiratory pathogens in home settings.

Clinical Evidence

Prospective multi-center clinical study (N=1,122) of symptomatic lay users. Compared to RT-PCR. SARS-CoV-2: PPA 92.0% (95% CI: 83.6-96.3%), NPA 99.0% (95% CI: 98.2-99.5%). Flu A: PPA 92.5% (95% CI: 82.1-97.0%), NPA 99.9% (95% CI: 99.5-100.0%). Flu B: PPA 90.5% (95% CI: 77.9-96.2%), NPA 99.9% (95% CI: 99.5-100.0%). Usability study (N=51) showed 96.8% of critical tasks performed correctly and 97.5% accuracy in result interpretation.

Technological Characteristics

Lateral flow immunochromatographic assay; utilizes antibodies for viral target identification. Components include test device and reagents for antigen detection. Designed for non-prescription home use. Requires design verification/validation including LoD, inclusivity, cross-reactivity, microbial interference, and stability testing. No specific materials or software algorithms described.

Indications for Use

Indicated for symptomatic individuals aged 2 years or older with signs of respiratory tract infection. For non-prescription home use by individuals aged 14+ testing themselves, or adults testing individuals aged 2-13.

Regulatory Classification

Identification

A multi-analyte respiratory virus antigen detection test is an in vitro diagnostic device intended for the detection and/or differentiation of respiratory viruses directly from respiratory clinical specimens. The device is intended to be performed at the site of sample collection, does not involve sample storage and/or transport.

Related Devices

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Submission Summary (Full Text)

{0}------------------------------------------------ Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health & Human Services logo on the left and the FDA logo on the right. The FDA logo features the letters "FDA" in blue, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in a smaller font size. # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Healgen Rapid Check COVID-19/Flu A&B Antigen Test DECISION SUMMARY #### I. Background Information: ### A De Novo Number DEN240029 # B Applicant Healgen # C Proprietary and Established Names Healgen Rapid Check COVID-19/Flu A&B Antigen Test # D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | |--------------------|----------------|---------------------------------------------------------------------------------|--------------| | SCA | Class II | 21 CFR 866.3987 – Multi- analyte respiratory virus antigen detection test | Microbiology | #### II. Submission/Device Overview: ### A Purpose of Submission De Novo request for evaluation of automatic class II designation for the Healgen Rapid Check COVID-19/Flu A&B Antigen Test ### B Measurand Influenza type A and type B nucleoprotein and SARS-CoV-2 nucleocapsid antigens # C Type of Test Qualitative Lateral flow Immunoassay #### III. Indications for Use: ### A Intended Use(s): See Indications for Use below Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1}------------------------------------------------ ### B Indication(s) for Use: The Healgen Rapid Check COVID-19/Flu A&B Antigen Test is a lateral flow immunochromatographic assay intended for the qualitative detection and differentiation of influenza A, and influenza B nucleoprotein antigens and SARS-CoV-2 nucleocapsid antigen directly in anterior nasal swab samples from individuals with signs and symptoms of respiratory tract infection. Symptoms of respiratory infections due to SARS-CoV-2 and influenza can be similar. This test is for non-prescription home use by individuals aged 14 years or older testing themselves, or adults testing individuals aged 2 years or older. All negative results are presumptive and should be confirmed with an FDA-cleared molecular assay when determined to be appropriate by a healthcare provider. Negative results do not rule out infection with influenza, SARS-CoV-2 or other pathogens. Individuals who test negative and experience continued or worsening respiratory symptoms, such as fever, cough and/or shortness of breath, should therefore seek follow-up care from their healthcare provider. Positive results do not rule out co-infection with other respiratory pathogens and therefore do not substitute for a visit to a healthcare provider or appropriate follow-up. ### C Special conditions for Use Statement(s): OTC - Over The Counter ### D Special Instrument Requirements: Not Applicable #### IV. Device/System Characteristics: ### A Device Description: The Healgen Rapid Check COVID-19/Flu A&B Antigen Test is an immunochromatographic assay that uses highly sensitive monoclonal antibodies to detect nucleoprotein antigens from SARS-CoV-2, influenza virus types A and B in anterior nasal swab (ANS) samples from symptomatic individuals. The test device is composed of a plastic housing, known as a cassette that contains a test strip with the following parts: sample pad, reaction membrane, and absorbing pad. The reagent pad contains colloidal gold conjugated with monoclonal antibodies (mAb) specific to SARS-CoV-2, Influenza A, and Influenza B target proteins. The reaction membrane contains different analyte specific antibodies to capture the target proteingold-mAb complexes at the respective test lines. Excess liquid and reagents are absorbed by the absorbing pad. The Healgen Rapid Check COVID-19/Flu A&B Antigen Test does not use biotin-Streptavidin/avidin chemistry in any of the steps for coupling reagents. ### B Principle of Operation: When the test sample is added into the sample well (S) of the cassette, mAb conjugates dried in the reagent pad are dissolved and migrate along with the sample, across the reaction lines on the membrane. The reaction lines contain different antibodies that are also analyte specific and bind {2}------------------------------------------------ to the target protein-gold-mAb complexes and immobilize them on the membrane, resulting in a visible red test line. Results completely develop after 15 minutes. Reactions for each virus occur independently at their respective locations on the test reaction membrane. If the sample contains influenza type A or B antigens, a pink-to-red test line (A or B) will develop; if SARS-CoV-2 antigens are present, a pink-to-red test line (T) will develop. The procedural control lines (C) must always appear. Healgen Rapid Check COVID-19/Flu A&B Antigen Test is validated for testing direct samples without transport media. The technical principle for influenza A virus and influenza B virus is identical to that of the SARS-CoV-2 antigen test strip as shown in figure 1 below. Image /page/2/Figure/3 description: The image shows a diagram of a COVID-19 test strip. The diagram illustrates how the test works with and without the presence of the COVID-19 antigen. The test strip includes a sample pad, conjugate pad, test line, control line, and absorbent pad. The diagram also shows the different components of the test, such as gold particles, COVID-19 antigens, gold-labeled COVID-19 specific antibodies, COVID-19 specific capture antibodies, and anti-mouse IgG. Figure 1: Schematic of the Healgen Rapid Check COVID-19/Flu A&B Antigen Test Strip Both the test strips enclosed in the test device independently feature an internal control, denoted directly on the test device as "C" for user interface. Test strip specific control lines are needed to indicate that each respective test strip is working adequately in each lay user performed test. The control line contains goat anti-mouse IgG antibodies that capture the excess gold-labeled mouse antibody preloaded in the reagent pad. The controls must be positive for a sample to provide a valid result to demonstrate that the test reagents are functional and correctly performed. If the control line is not detected, the sample result is invalid. Test results are displayed as Positive, Negative, or Invalid. # C Interpretation of Results: The qualitative results of the Healgen Rapid Check COVID-19/Flu A&B Antigen Test are visually interpreted by the user. Examples of the positive, and invalid results interpretations are provided within the "Interpreting the Result" section of the QRI. Individuals can scan a QR code within the QRI. This code directs the test user to a complete list of test result interpretations prepared and provided online at: https://www.healgen.com/covid19-influenza-a-b. Results interpretation is described in the below figure. {3}------------------------------------------------ | Visual for Positions of Result Lines | | |-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Image: test cassette | C = Control Line Flu A, Flu B, COVID = Test Line indicating presence/absence of Flu B = Influenza B Flu A = Influenza A COVID = SARS-CoV-2 | | Invalid (No Result) | Invalid | | A valid result must display a pinkish-red Control Line in the control region 'C' of both the result windows. If no or only one window of the test cassette shows a pinkish-red Control Line in the control region 'C', the assay is invalid and cannot be interpreted no matter the presence of any positive Test lines in the cassette windows. Invalid tests should be repeated with a new test. | Missing 'C' line on ONE or BOTH strips Image: three invalid test cassettes Note: The 3 images displayed are examples only; additional invalid outcomes are possible and can include test strips with or without test lines for Flu A, Flu B and/or COVID. | | Negative Result | Valid Negative Result | | A negative sample will produce a single pinkish-red Control Line in the control region 'C' of the window, indicating a negative result. This Control Line means that the detection part of the test was done correctly, sample was added, but no COVID-19 antigen was detected. | BOTH "C" lines must be PRESENT Image: valid negative test cassette | | Positive Result | Valid Positive Result | | A positive specimen will produce a single pinkish-red Test Line and a single pinkish-red Control Line. This means that COVID-19 antigen was detected and that the detection part of the test performed correctly. Specimens with low levels of antigen may give a faint Test Line | BOTH "C" lines must be PRESENT Image: text | {4}------------------------------------------------ Image /page/4/Figure/0 description: The image shows a diagram of COVID-19 and Flu test results. There are six different test results shown, including COVID-19 Positive, Flu A Positive, Flu B Positive, COVID-19 & Flu A Positive, COVID-19 & Flu B Positive, COVID-19 & Flu A & Flu B Positive, and Flu A & Flu B Positive. Each test result shows the control line (C) and the test lines for Flu A, Flu B, and COVID-19. The positive results are indicated by the presence of a red line in the test line area. Figure 2: Results Interpretation #### Standards/Guidance Documents Referenced: V. | Document Number | Title | Publishing Organization | |-------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------| | EP25-A | Evaluation of Stability of In Vitro Diagnostics Reagents | CLSI | | EP05-A3 | Evaluation of Precision of Quantitative Measurement Procedures | CLSI | | EP12-A2 | User Protocol for Evaluation of Qualitative Test Performance | CLSI | | ISO 15223-1 | Medical Devices - Symbols to be used with information to be supplied by the manufacturer | ANSI AAMI ISO | | ISO 10993-5:2009 | Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity | ANSI AAMI ISO | | ISO 10993-10:2010 | Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization | ANSI AAMI ISO | | ISO 11135:2014 | Sterilization of health-care products – Ethylene oxide – Requirements for the development, validation and routine control of a sterilization process for medical devices | ANSI AAMI ISO | | ISO 10993-7 | Biological evaluation of medical devices -Part 7: Ethylene oxide sterilization residuals. | ANSI AAMI ISO | {5}------------------------------------------------ #### VI. Performance Characteristics: # A Analytical Performance: # 1. Precision/Reproducibility: The Precision study for the Healgen Rapid Check COVID-19/Flu A&B Antigen Test was evaluated in two different in-house studies using the same 3 lots of test kits and the same operators. The strains used for testing were PROtrol inactivated SARS-CoV-2 lineage BA.5; omicron variant, PROtrol inactivated influenza A/Guangdong-Maonan/SWL1536/19, and PROtrol inactivated influenza B/Washington/02/19. Study 1 was conducted by 2 trained operators. Three sample levels (2X LoD co-spiked, 5X LoD co-spiked and Negative Pooled Nasal Wash) were tested on each day, one replicate per run, per operator, and per lot of devices. Two (2) runs (morning and afternoon) were conducted each day per operator, per lot, per day. This exact testing scheme was carried out over 10 days (same 3 sample levels tested, on the same 3 lots, by the same 2 operators, in 2 runs per day). This resulted in 120 total tests per sample level. All samples were randomized and blinded for each day. For all three lots and operators, the results for this study shown in the Table 1 below were identical and concordant with the expected results. Study 2 was specifically conducted to further evaluate potential differences between lots. The study used negative samples (without virus analytes) and very low positive samples at 0.75x LoD, commonly referred to as high negative sample. Samples were prepared near the C95 concentration for all three analytes and were randomized and blinded. This supplemental precision testing was carried out over 3 days only, but otherwise followed the same study design as above. This resulted in 72 total tests per analyte and sample level (24 replicates for each analyte with each lot). Data from this testing are integrated into Table 1 below. The random errors of the testing procedure across different days and runs, paired with an operator's ability to read the intensity for samples with very low analyte concentration (commonly referred to as 'high negative samples') is expected to confound lot-specific variability and to have a significant impact on the precision estimates for high negative samples such as the 0.75 x LoD sample tested in this second part of the precision assessment. This is supported by the stratified data for study 2 that demonstrated imprecision also between runs and days (data not shown). Taken together, the results of both precision assessments demonstrate a test precision and a lotto-lot precision that are consistent with the expectations for the analyte concentration in the samples, the test's technology, and the test's LoD. The between-lot variability does not impact low concentrated samples equal to or above 2 x LoD of the test. {6}------------------------------------------------ | | | Lot 1 | | Lot 2 | | Lot 3 | | Total | | |---------------|----------------|--------|----------------|--------|----------------|--------|----------------|------------------------------------|------------| | Sample | Analyte | Count* | % Agreement | Count* | % Agreement | Count* | % Agreement | Percent Lot-to-Lot Agreement | 95% CI | | Negative | SARS- CoV-2 | 0/64 | 100% | 0/64 | 100% | 0/64 | 100% | 100% | 98.0-100% | | | Flu A | 0/64 | 100% | 0/64 | 100% | 0/64 | 100% | 100% | 98.0-100% | | | Flu B | 0/64 | 100% | 0/64 | 100% | 0/64 | 100% | 100% | 98.0-100% | | 0.75 x LoD | SARS- CoV-2 | 20/24 | 83.3% | 22/24 | 91.7% | 17/24 | 70.8% | 81.9% | 71.5-89.1% | | | Flu A | 15/24 | 62.5% | 15/24 | 62.5% | 15/24 | 62.5% | 62.5% | 50.9-72.8% | | | Flu B | 18/24 | 75.0% | 17/24 | 70.8% | 14/24 | 58.3% | 68.0% | 56.6-76.7% | | 2 x LoD | SARS- CoV-2 | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | | | Flu A | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | | | Flu B | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | | 5 x LoD | SARS- CoV-2 | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | | | Flu A | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | | | Flu B | 40/40 | 100% | 40/40 | 100% | 40/40 | 100% | 100% | 93.9-100% | Table 1: Summary Results for Lot-Lot Precision study (Operators 1 and 2 Combined) * The total number of replicates included in this table is different sample concentrations due to the study being performed in two parts. Please refer to the study description above for more information. # 2. Linearity: This is a qualitative test without numerical data output and linearity is not applicable. # 3. Analytical Specificity/Interference: # Cross-reactivity and Microbial Interference: Cross Reactivity and Microbial Interference studies were conducted to determine if other respiratory pathogens/flora that could be present in a direct nasal swab samples could cause a false-positive test result or interfere with a true positive result. A panel of viruses, bacteria, fungi, and pooled nasal wash was used for these studies. Final target organism concentrations were 1.0 x 105 PFU/mL/1 x 105 TCID50/mL for viruses, and 1.0 x 106 cfu/mL for bacteria and fungi. When the target concentration was not achievable due to the stock culture, the highest concentration possible was tested without dilution. Dilutions for cross-reactivity testing were made in pooled negative swab matrix in saline (NCM Saline). Each organism was tested in replicates of three (3) without SARS-CoV-2/ FluA/FluB present in the sample. All testing was randomized and blinded. Organisms that did not cause a false-positive result were further evaluated for microbial interference by testing PNW spiked with low-level UV inactivated SARS-CoV-2, live Flu A virus, and live Flu B virus isolate (3X single analyte LoD) in the presence of potentially interfering organism at a high titer in triplicate. If interference was observed at the level tested, an additional titration study was performed to determine the highest microorganism concentration that does not produce interference with the Healgen Rapid Check COVID-19/Flu A&B Antigen test device. {7}------------------------------------------------ Neither cross-reactivity nor interference was observed for any of the organisms at the concentrations tested with the Healgen Rapid Check COVID-19/Flu A&B Antigen test device. The summary of cross-reactivity and microbial interference results are shown in the table below. | | Concentrations | | Cross- | Microbial | |---------------------------------------------------------------------------------------------------------|--------------------------|-----------|----------------------|---------------------------| | Organism | Tested | Units | Reactivity | Interference | | SARS-CoV-1 | 1.25E+05 | PFU/ml | ND* | ND | | MERS-coronavirus | 1.47E+05 | TCID50/mL | ND | ND | | Human coronavirus OC43 | 7.00E+05 | TCID50/mL | ND | ND | | Human coronavirus 229E | 1.58E+05 | TCID50/mL | ND | ND | | Human coronavirus NL63 | 8.00E+04 | TCID50/mL | ND | ND | | Human coronavirus HKU1 a | 1:20 dilution | NA | NA | ND | | Adenovirus, Type 1 (Adenoid 71) | 2.23E+05 | TCID50/mL | ND | ND | | Adenovirus Type 7, Type 7A (Species B) | 1.58E+05 | TCID50/mL | ND | ND | | Cytomegalovirus, Strain AD-169 | 7.05E+04 | TCID50/mL | ND | ND | | Epstein Barr Virus, Strain B95-8 | 1.83E+06 | CP/mL | ND | ND | | Human Metapneumovirus (hMPV), Strain TN/91-316 | 3.50E+05 | TCID50/mL | ND | ND | | Parainfluenza virus 1, Strain FRA/29221106/2009 | 2.00E+05 | TCID50/mL | ND | ND | | Parainfluenza virus 2, Strain Greer | 1.75E+05 | TCID50/mL | ND | ND | | Parainfluenza virus 3, Strain C243 | 7.00E+05 | TCID50/mL | ND | ND | | Parainfluenza virus 4, Strain N/A | 2.39E+05 | TCID50/mL | ND | ND | | Enterovirus Type (e.g. 68), Species D Type 68 | 2.23E+05 | TCID50/mL | ND | ND | | Respiratory syncytial virus A, Strain A-2 | 3.50E+05 | TCID50/mL | ND | ND | | Respiratory syncytial virus B, Strain CH93(18)-18 | 2.29E+05 | TCID50/mL | ND | ND | | Rhinovirus 1A, Strain N/A | 7.05E+04 | TCID50/mL | ND | ND | | Bordetella pertussis, Strain A639 | 2.50E+08 | CFU/mL | ND | ND | | Candida albicans, Strain Z006 | 6.03E+06 | CFU/mL | ND | ND | | Chlamydia pneumoniae, Strain Z500 | 4.33E+06 | IFU/mL | ND | ND | | Corynebacterium xerosis | 2.30E+07 | CFU/mL | ND | ND | | Escherichia coli, Strain mcr-1 | 1.79E+08 | CFU/mL | ND | ND | | Hemophilus influenzae, type b; Eagan | 9.68E+06 | CFU/mL | ND | ND | | Lactobacillus sp., Lactobacillus Acidophilus, Strain Z048 | 1.21E+07 | CFU/mL | ND | ND | | Legionella spp pneumophila, Strain Philadelphia-1 | 6.50E+06 | CFU/mL | ND | ND | | Moraxella catarrhalis, Strain 59632 | 2.50E+08 | CFU/mL | ND | ND | | Mycoplasma pneumoniae, Strain PI 1428 | 2.50E+07 | CFU/mL | ND | ND | | Mycobacterium tuberculosis avirulent, Strain H37Ra-1 | 4.15E+06 | CFU/mL | ND | ND | | Neisseria meningitidis, serogroup A | 3.43E+06 | CFU/mL | ND | ND | | Neisseria sp. Elongata Z071 | 2.68E+08 | CFU/mL | ND | ND | | Pneumocystis jirovecii, | 1.30E+07 | CFU/mL | ND | ND | | Organism | Concentrations Tested | Units | Cross- Reactivity | Microbial Interference | | Strain W303-Pji | | | | | | Pseudomonas aeruginosa, Strain N/A | 3.45E+08 | CFU/mL | ND | ND | | Staphylococcus aureus Protein A producer, e.g., Cowan strain, NCTC 8530 [S11]; Cowan's serotype 1 | 2.60E+08 | CFU/mL | ND | ND | | Staphylococcus epidermidis (PCI 1200) | 9.00E+07 | CFU/mL | ND | ND | | Streptococcus salivarius, Strain C699 [S30D] | 1.01E+06 | CFU/mL | ND | ND | | Streptococcus pneumoniae, Strain Z022 | 1.81E+07 | CFU/mL | ND | ND | | Streptococcus pyogenes, Strain MGAS 8232 | 7.50E+07 | CFU/mL | ND | ND | | Measles, Strain Edmonston | 8.48E+05 | TCID50/mL | ND | ND | | Mumps (Isolate 1) | 8.48E+05 | TCID50/mL | ND | ND | Table 2:Summary of Cross-reactivity and Microbial Interference Results {8}------------------------------------------------ *ND - Not Detected a1:10 dilution of cultured stock HKU1 sample from Emory #### Competitive Interference: Competitive interference of the test's analytes was tested with different combinations of low (3x LoD) and high concentrations of Flu A, Flu B and SARS-CoV-2 spiked together onto a swab and then tested with one lot of Healgen Rapid Check COVID-19/Flu A&B Antigen test device. The study used inactivated SARS-CoV-2 but live influenza A and B virus strains. The table below summarizes the results of the competitive interference study. For each condition tested all three replicates tested at the low target analyte condition tested positive in the presence of a second target analyte at high concentrations. No false positive results were observed for analytes not present in the sample. | | Analyte Concentration Added to Sample* | | | |----------------------------------------|-----------------------------------------------------|-------------|-------------| | | (No. positive replicates / No. of total replicates) | | | | | Flu A | Flu B | SARS-CoV-2 | | Analyte Concentration Added Results | High 3/3 | Low 3/3 | - 0/3 | | Analyte Concentration Added Results | High 3/3 | - 0/3 | Low 3/3 | | Analyte Concentration Added Results | Low 3/3 | High 3/3 | - 0/3 | | Analyte Concentration Added Results | - 0/3 | High 3/3 | Low 3/3 | | Analyte Concentration Added Results | Low 3/3 | - 0/3 | High 3/3 | | Analyte Concentration Added Results | - 0/3 | Low 3/3 | High 3/3 | Table 3: Competitive Interference Results Summary * SARS-CoV-2 strain - 1X LoD - 3.95E+02 TCID50/mL Flu A - H3N2:A/Darwin/6/2021 - 1X LoD - 2.09E+02 TCIDso/mL Flu B - Yamagata: B/Florida/4/2006 - 1X LoD-1.46E+01 TCID50/mL {9}------------------------------------------------ # Exogenous and Endogenous Interference Study The Healgen Rapid Check COVID-19/Flu A&B Antigen test was evaluated for performance in the presence and absence of potentially interfering substances that might be present in a respiratory specimen. Interfering substances testing was performed using a panel of endogenous and exogenous substances tested at concentrations listed in the below table. Negative specimens were evaluated in triplicates to confirm that the potentially interfering substances would not cause false positive results with the test. Negative clinical matrix (pooled nasal wash) was co-spiked with SARS-CoV-2 USA WA1/2020, Flu A H1N1pdm09/A Victoria/4897/2022, and Flu B Yamagata/B/Florida/4/2006, and then mixed 1:1 with interfering substance. Final concentration for each analyte was 3x LoD (based on the established single analyte LoD). Negative nasal wash has been demonstrated to be equivalent to the anterior nasal swab matrix in a matrix equivalency study. Testing was performed in triplicate to confirm that SARS-CoV-2, Flu A and Flu B could still be detected if the test substances were present in the sample. All testing was randomized and blinded. Test results are summarized in the table below. With the exception of Flu Mist Quadrivalent live influenza vaccine, none of the substances caused a false-positive test result in unspiked samples. While the presence of Flu Mist Quadrivalent live influenza vaccine at 15% v/v concentration did not interfere with the detection of true positive results of the 3x LoD co-spiked samples, the vaccine also resulted in positive results for Flu A and Flu B (as expected based on the composition of the vaccine). Hand sanitizer cream lotion and hand sanitizer 80% ethanol fast drying at 15% v/v showed false negative results for Flu B, but detected all analytes at 7.5% v/v. | Interfering Substance | Concentration | Cross-Reactivity (no analyte) (# pos/ # total) | | | Interference (3x co-spiked analyte LoD) (# pos/ # total) | | | |--------------------------------------------------|-------------------------|------------------------------------------------------|-------|-------|----------------------------------------------------------------|-------|-------| | | | SARS- CoV-2 | Flu A | Flu B | SARS- CoV-2 | Flu A | Flu B | | Human Whole Blood (EDTA tube) | 4% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Leukocytes | 1.67 x 10^6 cells/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Throat Lozenges (Menthol/Benzocaine) | 3 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Mucin, bovine submaxillary gland | 2.5 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Zinc (Therazinc throat Spray) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Naso GEL (NeilMed) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal Drops (Phenylephrine) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal Spray (Oxymetazoline) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal Spray (Cromolyn) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal Corticosteroid (Dexamethasone) | 1 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal Corticosteroid (Fluticasone Propionate) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | ### Table 4: Interfering Substances Study Results {10}------------------------------------------------ | Interfering Substance | Concentration | Cross-Reactivity (no analyte) (# pos/ # total) | | | Interference (3x co-spiked analyte LoD) (# pos/ # total) | | | |------------------------------------------------------------------------------------------|---------------|------------------------------------------------------|-------|-------|----------------------------------------------------------------|-------|-------| | | | SARS- CoV-2 | Flu A | Flu B | SARS- CoV-2 | Flu A | Flu B | | Nasal gel (Galphimia glauca, Histanium hydrocloricum, Luffa operculate, Sulfur) | 1.25% | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Homeopathic allergy relief (Histaminum hydrochloricum) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Zicam nasal spray (Galphimia glauca, Luffa operculata) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Nasal spray (Alkalol) | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Sore Throat Phenol Spray | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Tobramycin | 4 $μ$ g/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Mupirocin | 10 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Anti-viral drug (Remdesvir) | 10 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Tamiflu (Oseltamivir) | 5 mg/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | FluMist | 15% v/v | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | (Quadrivalent/Live) | 0.15% v/v | 0/3 | 0/3 | 0/3 | NA | NA | NA | | Zanamivir | 282 ng/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Biotin | 3500 ng/mL | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Body & Hand Lotion | 0.5% w/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Body Lotion, with 1.2% dimethicone | 0.5% w/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Hand Lotion | 5% w/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Hand Sanitizer with Aloe, 62% ethyl alcohol | 5% w/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Hand Sanitizer cream | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 0/3 | | lotion | 7.5% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Hand Sanitizer, 80% | 15% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 0/3 | | ethanol | 7.5% v/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | | Hand soap liquid gel | 10% w/v | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | # 4. Assay Reportable Range: This section is not applicable as this device is a qualitative assay. # 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): ### Internal Controls: Both the test strips enclosed in the test device independently feature an internal control, denoted directly on the test device as "C" for user interface. Test strip specific control lines are needed to indicate that each respective test strip is working adequately in each lay user performed test. The control line contains IgG antibodies that capture the excess gold-labeled mouse antibody preloaded in the reagent pad. These controls have to be positive for all valid test results to {11}------------------------------------------------ demonstrate that the test reagents are functional, and the tests correctly performed. If the control lines are not detected, the sample result is invalid. # External Controls: External Control testing is not performed by lay users and is therefore not applicable to OTC tests. External controls are therefore not included in the test kit. # Stability # Real Time Stability: Three lots of the Healgen Rapid Check COVID-19/Flu A&B Antigen Test kits were subjected to a summer profile (40°C for 8 hours and then 30°C for 4 hours) and then a winter profile (- 10°C for 8 hours and then 18°C for 4 hours) to simulate the anticipated shipping/handling times and temperatures expected for unopened kits, after which they were stored at 2-8℃ or 30+3℃, respectively. The test panel comprised of negative clinical matrix, 2x LoD and 5x LoD of inactivated SARS-CoV-2, and live Flu A and Flu B viruses. Testing was performed at time 0 (baseline) and month 1, 3, 6, 9, 12, 15, and 18. Testing will continue for months 21, 24 and 27. All study data are 100% concordant with expected results and support a shelf-life of up to 15 months. The shelf life will be updated as additional passing time points will become available. # Open Kit Stability Study: In this study, the amount of time a test device can be left outside of its packaging was assessed using a test panel comprised of five (5) negative samples (clinical matrix: PNW) and five (5) cospiked low positive samples (2X single analyte LoD of SARS-CoV-2, Flu A, and Flu B cospiked together into PNW). PNW was demonstrated to be equivalent to negative nasal swab matrix in a matrix equivalency study. Device packaging was opened and testing was performed at zero (0) hours to establish baseline. Thereafter, devices were stored for one-hour and twohour, respectively at 30±1℃ (the worst-case condition for a room temperature claim). All study data before and after storage of the open kits were 100% concordant with the expected results. ### Transport Stability: Simulated winter and summer transport temperature conditions were used to evaluate the worstcase shipping and handling of unopened components of the Healgen COVID-19/Flu A&B Ag Combo Rapid test over an extended period. The functional performance of Healgen's test device is assessed by comparing the pre- (T0) and post-distribution (Td) results of a test panel comprised of pooled negative nasal wash (PNW) samples and co-spiked low positive samples (3X single analyte LoD with SARS-CoV-2, Flu A, and Flu B, together contrived in PNW). PNW was demonstrated to be an equivalent negative clinical matrix to negative nasal swab matrix in a matrix equivalency study. All results were as expected for all time points. ### 6. Detection Limit: ### Single Analyte LoD: The LoD of the device was performed to determine the lowest detectable concentration of SARS-CoV-2, influenza A and influenza B at which at least 95% of all true positive replicates are consistently detected as positive. The LoD was assessed for each analyte in two parts, a preliminary range finding study, followed by a confirmatory LoD study. A preliminary LoD was {12}------------------------------------------------ determined by first testing serial ten-fold dilutions of live influenza A and B, and inactivated SARS-CoV-2 virus stocks diluted into either pooled negative swab matrix (PNSM) or pooled nasal wash (PNW) in 3 replicates per dilution. Single analyte virus dilutions (50 µL/swab) were each spiked onto dry sterile swabs and tested per the IFU. The preliminary LoD results for each individual virus strain is shown in below tables. Table 5: Preliminary LoD - SARS-CoV-2 | Isolate/Lineage | SARS-CoV-2 (TCID50/mL) | SARS-CoV-2 (TCID50/Swab) | #Positive/# Total | # of device lots tested | |---------------------------------------------------|---------------------------|-----------------------------|----------------------|----------------------------| | | 3.16E+05 | 1.58E+04 | 3/3 | | | | 3.16E+04 | 1.58E+03 | 3/3 | | | | 3.16E+03 | 1.58E+02 | 3/3 | | | | 1.58E+03 | 7.90E+01 | 3/3 | | | USA-WA1-2020 (UV inactivated) | 7.90E+02 | 3.95E+01 | 3/3 | 1 | | | 3.95E+02 | 1.98E+01 | 3/3 | | | | 3.16E+02 | 1.58E+01 | 2/3 | | | | 1.58E+02 | 7.90E+00 | 0/3 | | | | 3.09E+06 | 1.5E+05 | 3/3 | | | | 3.09E+05 | 1.5E+04 | 3/3 | | | | 3.09E+04 | 1.5E+03 | 3/3 | 1 | | USA-WA1-2020 (Heat inactivated) | 3.09E+03 | 1.5E+02 | 3/3 | | | | 1.5E+03 | 7.5E+01 | 1/3 | | | | 3.09E+02 | 1.5E+01 | 0/3 | | | | 2.19E+03 | 1.09E+02 | 9/9 | | | USA/COR-22-063113/2022 (BA.5, Omicron variant) | 1.09E+03 | 5.45E+01 | 9/9 | 3 | | | 5.47E+02 | 2.73E+01 | 4/9 | | | | 2.19E+02 | 1.09E+01 | 2/3 | | ### Table 6: Preliminary LoD - Influenza A | Isolate/Lineage | Strain | SARS-CoV-2 (TCID50/mL) | SARS-CoV-2 (TCID50/Swab) | #Positive/ # Total | # of device lots tested | |-----------------|--------------------------------|---------------------------|-----------------------------|-----------------------|----------------------------| | | | 4.17E+04 | 2.09E+03 | 3/3 | | | | | 4.17E+03 | 2.09E+02 | 3/3 | | | | | 4.17E+02 | 2.09E+01 | 3/3 | | | H3N2 | A/Darwin/6/2021 | 2.09E+02 | 1.05E+01 | 3/3 | 1 | | | | 1.04E+02 | 5.20E+00 | 3/3 | | | | | 5.21E+01 | 2.61E+00 | 3/3 | | | | | 4.17E+01 | 2.09E+00 | 2/3 | | | | | 4.17E+04 | 2.09E+03 | 0/3 | | | | | 2.02E+04 | 1.01E+03 | 3/3 | | | | | 2.02E+03 | 1.01E+02 | 3/3 | | | | | 2.02E+02 | 1.01E+01 | 3/3 | | | H1N1 | pdm09:A/Victoria/48 97/2022 | 1.01E+02 | 5.05E+00 | 1/3 | 1 | | | | 5.05E+01 | 2.53E+00 | 0/3 | | | | | 2.53E+01 | 1.27E+00 | 0/3 | | | | | 2.02E+01 | 1.01E+00 | 0/3 | | {13}------------------------------------------------ | Isolate/Lineage | Strain | SARS-CoV-2 (TCID50/mL) | SARS-CoV-2 (TCID50/Swab) | #Positive/ # Total | # of device lots tested | |-----------------|-------------------------------------|---------------------------|-----------------------------|-----------------------|----------------------------| | | A/California/07/2009 pdm09 | 2.1E+05 | 5.85E+02 | 3/3 | 1 | | | | 2.1E+04 | 5.85E+01 | 3/3 | | | | | 2.1E+03 | 5.85E | 3/3 | | | | | 1.05E+03 | 5.25E+01 | 3/3 | | | | | 5.25E+02 | 2.6E+01 | 1/3 | | | | | 2.1E+02 | 1.05E+01 | 0/3 | | | | Guangdong- Maonan/SWL 1536/19 | 5.62E+01 | 2.81 | 9/9 | 3 | | | | 5.62 | 2.81E-01 | 0/3 | | # Table 7: Preliminary LoD - Influenza B | Isolate/Lineage | Strain | SARS-CoV-2 (TCID50/ mL) | SARS-CoV-2 (TCID50/Swab) | #Positive/# Total | # of device lots tested | |-----------------|-----------------------------------------------|-------------------------------|-----------------------------|----------------------|----------------------------| | Yamagata | B/Florida/4/2006 | 1.17E+04 | 5.85E+02 | 3/3 | | | | | 1.17E+03 | 5.85E+01 | 3/3 | | | | | 1.17E+02 | 5.85E+00 | 3/3 | | | | | 5.85E+01 | 2.93E+00 | 3/3 | 1 | | | | 2.93E+01 | 1.47E+00 | 3/3 | | | | | 1.46E+01 | 7.30E-01 | 3/3 | | | | | 1.17E+01 | 5.85E-01 | 1/3 | | | | B/Washington/02/2019 | 3.16E+05 | 1.58E+04 | 3/3 | | | | | 3.16E+04 | 1.58E+03 | 3/3 | | | | | 3.16E+03 | 1.58E+02 | 3/3 | | | | | 1.58E+03 | 7.90E+01 | 1/3 | 1 | | | | 7.90E+02 | 3.95E+01 | 0/3 | | | | | 3.95E+02 | 1.98E+01 | 0/3 | | | | | 3.16E+02 | 1.58E+01 | 0/3 | | | Victoria | | 1.75E+04 | 8.75E+02 | 9/9 | | | | B/Washington/02/2019 (PROtrol inactivated) | 8.75E+03 | 4.37E+02 | 0/3 | 3 | | | | 1.75E+03 | 8.75E+01 | 0/3 | | | | | | | | | | | B/Florida/78/2015 | 1.7E+06 | 8.5E+04 | 3/3 | | | | | 1.7E+05 | 8.5E+03 | 3/3 | | | | | 1.7E+04 | 8.5E+02 | 3/3 | 1 | | | | 8.5E+03 | 4.25E+02 | 1/3 | | | | | 1.7E+03 | 8.5E+01 | 0/3 | | LoD confirmatory testing was then performed individually for each virus by testing 20 replicates at the virus' preliminary (1X) LoD concentration, as determined above. For the LoD to be confirmed, at least 95% of the replicates (≥19/20) needed to test positive. Results of the LoD confirmation testing for each virus are summarized in the table below. {14}------------------------------------------------ | Analyte | Isolate/ Lineage | Strain | LoD Concentration (TCID50/mL) | LoD Concentration (TCID50/swab) | #Positive /# Total | # device lots tested | |----------------|-------------------------------------------------------|--------------------------------------------------------------|-------------------------------------|---------------------------------------|-----------------------|-------------------------------| | | USA-WA1/2020 (UV inactivated) | NA | 3.95E+02 | 1.98E+01 | 20/20 | 1 | | SARS- CoV-2 | USA-WA1/2020 (Heat inactivated) | NA | 3.09E+03 | 1.5E+02 | 60/60 | 3 | | | USA/COR-22- 063113/2022 (BA.5, Omicron variant) | NA | 1.09E+03 | 5.45E+01 | 58/60 | 3 | | Flu A | H3N2 | Darwin/6/21 | 2.09E+02 | 1.05E+01 | 20/20 | 1 | | | H1N1 | Victoria/4897/22 | 2.02E+02 | 1.01E+01 | 20/20 | 1 | | | | A/California/07/200 9 pdm09 | 1.05E+03 | 5.25 | 60/60 | 3 | | | | Guangdong- Maonan/SWL 1536/19 (PROtrol inactivated) | 5.62E+01 | 2.81 | 60/60 | 3 | | | Yamagata | Florida/04/06 | 1.46E+01 | 7.30E-01 | 20/20 | 1 | | Flu B | Victoria | Washington/02/19 | 1.58E+03 | 7.90E+01 | 20/20 | 1 | | | Victoria | Washington/02/19 (PROtrol inactivated) | 1.75E+04 | 8.75E+02 | 58/60 | 3 | | | Victoria | B/Florida/78/2015 | 1.7E+04 | 8.5E+02 | 60/60 | 3 | Table 8: Confirmatory LoD # Co-spiked LoD: After the single analyte LoDs were established for the candidate device, co-spiked LoD equivalency testing with all three test analytes present in the sample, was conducted to characterize performance with samples that contain more than one analyte at low concentrations. All analytes that are successfully detected by the candidate device when co-spiked at their single analyte LoD, may be co-spiked into positive sample/s used in the analytical studies. Based on the individual analyte specific 1x LoDs, co-spiked samples were prepared by mixing all three viruses (one strain each of SARS-CoV-2, Flu A and Flu B). The 1x co-spiked LoD concentration was tested with the candidate device in twenty (20) replicates and was considered confirmed (i.e., equivalent to the established single analyte LoD) if ≥19/20 replicates were positive for concentrations within 2x LoD of the established single analyte LoD. The Healgen Rapid check COVID-19/Flu A&B Antigen Test demonstrated co-spike equivalency for all analytes, SARS-CoV-2, Flu A and Flu B, to their respective established single analyte 1X LoD. The summary of the co-spike LoD is shown in the below table. {15}------------------------------------------------ | Virus | Fold LoD | LoD Concentration (TCID50/mL) | LoD Concentration per Swab (TCID50/swab) | # Positive Replicates | |--------------------------------------------|----------|----------------------------------|------------------------------------------------|--------------------------| | SARS-CoV-2 (USA-WA1/2020) | 1X | 3.95 x 102 | 2.0 x 101 | 20/20 | | Flu A H1N1 (pdm09:A/Victoria/4897/2022) | 1X | 2.02 x 102 | 1.0 x 101 | 20/20 | | Flu B Yamagata (B/Florida/4/2006) | 1X | 1.46 x 101 | 7.3 | 20/20 | Table 9: Summary of Co-Spike Equivalency LoD Results # NIBSC 21/368 -WHO International Standard: The sponsor tested the sensitivity of the test with the 1st WHO International Standard for SARS-CoV-2 antigen (NIBSC code: 21/368) spiked into pooled negative swab matrix (PNSM). The unitage of this material has an assigned value of 5,000 International Units of SARS-CoV-2 antigen per ampoule when reconstituted per instructions. A 2-fold dilution series was made to determine the preliminary LoD, which was measured using one device lot and triplicate measurements (n=3). The measurements were done by adding 50μl of each dilution directly to the test swab and processing the sample per the test's ORI. The preliminary LoD was determined to be 250 IU/ml (or 12.5 IU/swab). The LoD confirmatory study was performed using 20 replicates (n=20) per dilution. The lowest concentration at which a minimum of 95% of results were positive was confirmed to be 250 IU/ml or 12.5 IU/Swab as shown below. | Table 10: LOD with the 1st WHO International Standard for SARS-CoV-2 Antigen (NIBSC | | | |-------------------------------------------------------------------------------------|--|--| | code:21/368) | | | | Dilution (IU/ml) | Preliminary LoD | | Confirmatory LoD | | | |------------------|-----------------------|---------|---------------------|-----------------------|---------| | | Dilution (IU/swab) | Results | Dilution (IU/ml) | Dilution (IU/swab) | Results | | $4x10^3$ | 200 | 3/3 | | | | | $2x10^3$ | 100 | 3/3 | | | | | $1x10^3$ | 50 | 3/3 | | | | | $5x10^2$ | 25 | 3/3 | $5x10^2$ | 25 | 20/20 | | $2.5x10^2$ | 12.5 | 3/3 | $2.5x10^2$ | 12.5 | 19/20 | | $1.25x10^2$ | 6.25 | 2/3 | $1.25x10^2$ | 6.25 | 10/20 | | $6.25x10^1$ | 3.125 | 0/3 | | | | # High-dose Hook Effect Study: The hook effect study was conducted to evaluate if high levels of antigen present in the sample could result in a false negative test result. In this study, 50uL of the highest concentration possible for UV inactivated SARS-CoV-2 virus stock and for live influenza A and influenza B virus stocks were spiked onto sterile swabs for triplicate measurements, and swabs were tested on the device per IFU of the candidate device. Testing showed no hook effect for SARS-CoV-2, Flu A, Flu B at the concentrations listed in the table below. {16}------------------------------------------------ | Virus | Strain | Subtype / Lineage | Virus Concentration [TCID50/mL*] | Virus Concentration [TCID50/swab] | # Positive/ # Tested | |-------------|----------------------|----------------------|----------------------------------------|-----------------------------------------|-------------------------| | SARS-CoV-2 | USA-WA1/2020 | N/A | 3.16E+06 | 1.58E+05 | 3/3 | | Influenza A | A/Victoria/4897/2022 | H1N1 | 2.02E+05 | 1.01E+04 | 3/3 | | Influenza A | A/Darwin/6/2021 | H3N2 | 4.17E+05 | 2.09E+04 | 3/3 | | Influenza B | B/Washington/02/2019 | Victoria | 3.16E+06 | 1.58E+05 | 3/3 | | Influenza B | B/Florida/4/2006 | Yamagata | 1.17E+05 | 5.85E+03 | 3/3 | Table 11: Summary of High Dose Hook Effect Results *Concentration in the sample solution applied to dry swab #### Inclusivity Study: Inclusivity testing was conducted to determine if the candidate device can detect different strains of SARS-CoV-2, Flu A and Flu B. The Healgen Rapid Check COVID-19/Flu A&B Antigen Test employs the identical test strip as included in the previously cleared COVID-19-only Healgen Rapid COVID-19 Antigen Test (K232377). Inclusivity data for SARS-CoV-2 as obtained for K232377 therefore apply equally to the Healgen Rapid Check COVID-19/Flu A&B Antigen Test. Data derived from testing of commercially obtained Alpha (B.1.1.7), delta (B.1.617.2), omicron (B.1.1.529), beta (B.1.351), gamma (P1) and kappa (B.1.617.1) SARS-CoV-2 virus strains demonstrated the test strip's inclusivity of all tested variants at low concentrations (see FDA's Decision Summary for K232377 (fda.gov)). A selection of temporal, geographic and genetically diverse Influenza A and B strains were tested on the Healgen Rapid Check COVID-19/Flu A&B Antigen Test for inclusivity. A series of ten-fold dilutions of each virus strain was made in pooled negative nasal matrix (PNSM). For each replicate tested in the study, 50uL of the dilution was pipetted on a fresh sterile swab. Once the ten-fold breakpoint was established for each of the strains, an additional series of three twofold dilutions was made from the lowest positive ten-fold dilution of each virus and triplicates were tested to demonstrate inclusivity. Contemporary strains (within the past 5 years) were prioritized over older strains. The lowest concentrations that tested positive for relevant influenza virus strains by the candidate device are shown in the table below. | Virus | Virus Strains | Concentration | Units | |--------------|--------------------------------------|---------------|-----------| | Flu A - H1N1 | A/ California/04/2009 | 2.80E+03 | TCID50/mL | | | A/ Brisbane/02/2018 | 1.51E+02 | TCID50/mL | | | A/ Michigan/45/2015 | 9.30E+00 | TCID50/mL | | | A/ Guangdong-Maonan/SWL 1536/2019 | 1.04E+03 | TCID50/mL | | | A/ NY/03/2009 | 2.29E+04 | TCID50/mL | | | A/ Indiana/02/2020 | 9.70E+06 | CEID50/mL | | | A/Wisconsin/588/2019 | 1.4E+04 | FFU/mL | | | A/ Sydney/5/2021 | 4.80E+03 | TCID50/mL | | | A/ Hawaii/66/2019 | 3.70E+07 | CEID50/mL | | | A/ Wisconsin/67/2022 | 1.05E+03 | TCID50/mL | | | A/New York/21/2020 | 2.6E+05 | FFU/mL | Table 12: Inclusivity Results - Minimal Detectable Concentrations of Flu Variants {17}------------------------------------------------ | Virus | Virus Strains | Concentration | Units | |---------------------------------------|----------------------------------------------|---------------|-----------| | Flu A – H3N2 | A/Tasmania/503/2020 | 6.5E+04 | FFU/mL | | | A/Hong Kong/2671/2019 | 3.1E+06 | CEID50/mL | | | A/Hong Kong/45/2019 | 1.5E+04 | FFU/mL | | | A Alaska/01/2021 | 1.50E+04 | FFU/mL | | Flu A– H1N1 | A/Indiana/08/2011 | 8.10E+02 | TCID50/mL | | Flu A– H1N2 | A/Ohio/09/2015 | 7.0E+05 | CEID50/mL | | Flu A- H5N1 | A/Minnesota/19/2011 | 8.00E+06 | CEID50/mL | | | A/mallard /Wisconsin/2576/2009 | 2.10E+05 | GE/mL | | | A/mallard /Wisconsin/2576/2009 | 800,000 | CEID50/mL | | | A/Bovine/Ohio/B24OSU- 439/2024 | 1,550 | TCID50/mL | | Flu A- H5N6 | A/duck/Guangxi/S11002/2024 | 3.38E+05 | EID50/mL | | Flu A- H5N8 | A/duck/Guangxi/S10888/2024 | 7.90E+05 | EID50/mL | | Flu A- H7N3 | A/goose/Liaoning/S1266/2021 | 1.69E+05 | EID50/mL | | | A/northern pintail/Illinois/10OS3959/2010 | 7.0E+05 | CEID50/mL | | Flu B -Victoria Lineage | B/ Brisbane/60/2008 | 6.45E-01 | TCID50/mL | | | B/Colorado/6/2017 | 5.85E+00 | TCID50/mL | | | B/Texas/02/2013 | 6.13E+00 | TCID50/mL | | | B/ Michigan/01/2021 | 2.85E+03 | TCID50/mL | | Flu B - Yamagata Lineage | B/Texas/06/2011 | 8.00E+05 | CEID50/mL | | | B/Utah/09/2014 | 1.26E+02 | TCID50/mL | | | B/Wisconsin/1/10 | 1.78E+01 | TCID50/mL | | Flu B - non-Victoria, non-Yamagata | B/Maryland/1/1959 | 1.69E+03 | CEID50/mL | ### Assay Cut-Off: Not applicable as this is a qualitative visually read assay without numeric raw data. ### B Comparison Studies: #### Method Comparison: Please refer to section VI.…