Flowflex Plus RSV + Flu A/B + COVID Rapid Test

K261212 · ACON Laboratories, Inc. · SCA · Jul 10, 2026 · Microbiology

Device Facts

• Record ID: K261212
• Device Name: Flowflex Plus RSV + Flu A/B + COVID Rapid Test
• Applicant: ACON Laboratories, Inc.
• Product Code: SCA · Microbiology
• Decision Date: Jul 10, 2026
• Decision: SESE
• Submission Type: Dual Track
• Regulation: 21 CFR 866.3987
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

The Flowflex Plus RSV + Flu A/B + COVID Rapid Test is a lateral flow immunoassay intended for the qualitative detection and differentiation of respiratory syncytial virus (RSV), influenza A, influenza B, and SARS-CoV-2 protein antigens directly in anterior nasal swab samples from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to RSV, influenza, and SARS-CoV-2 can be similar. All negative results are presumptive and should be confirmed with a molecular assay, if necessary, for patient management. Negative results do not rule out infection with RSV, influenza, or SARS-CoV-2 and should not be used as the sole basis for treatment or patient management decisions. Positive results do not rule out bacterial infection or co-infection with other viruses.

Device Story

Lateral flow immunoassay; detects RSV, influenza A, influenza B, and SARS-CoV-2 protein antigens; input is anterior nasal swab sample; visual qualitative readout; intended for symptomatic individuals; aids in clinical differentiation of respiratory infections; negative results require molecular confirmation; positive results do not exclude co-infections.

Clinical Evidence

Prospective clinical study (Oct 2024–Apr 2025) at 10 US sites with 1,263 symptomatic subjects (≥6 months). Compared to FDA-cleared RT-PCR. Results: SARS-CoV-2 PPA 91.6%, NPA 99.9%; Flu A PPA 92.9%, NPA 99.8%; Flu B PPA 92.9%, NPA 99.9%; RSV PPA 94.1%, NPA 99.8%.

Technological Characteristics

Lateral flow immunochromatographic assay in sandwich format. Two-strip cassette: Strip 1 (SARS-CoV-2, Flu A, Flu B), Strip 2 (RSV). Components: backing card, sample/reagent pads, nitrocellulose membrane, absorbent pad. Visual readout. Storage: 2-30°C. Shelf life: 15 months. No electronic components or software.

Indications for Use

Indicated for symptomatic individuals aged 6 months or older with signs of respiratory tract infection. For prescription use by healthcare professionals in CLIA-waived settings.

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.

Submission Summary (Full Text)

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**FDA** U.S. FOOD & DRUG
ADMINISTRATION

# **510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY**

# **I Background Information:**

# **A 510(k) Number**

K261212

# **B Applicant**

ACON Laboratories, Inc.

# **C Proprietary and Established Names**

Flow*flex* Plus RSV + Flu A/B + COVID Rapid 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 for Submission:**

To obtain substantial equivalence determination for the Flow*flex* Plus RSV + Flu A/B + COVID Rapid Test.

# **B Measurand:**

Protein antigens from RSV, SARS-CoV-2, Influenza A and Influenza B

# **C Type of Test:**

Qualitative Lateral flow immunoassay

# **III Intended Use/Indications for Use:**

# **A Intended Use(s):**

See Indications for Use below.

# **B Indication(s) for Use:**

The Flow*flex* Plus RSV + Flu A/B + COVID Rapid Test is a lateral flow immunoassay intended for the qualitative detection and differentiation of respiratory syncytial virus (RSV), influenza A, influenza B, and SARS-CoV-2 protein antigens directly in anterior nasal swab samples from

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to RSV, influenza, and SARS-CoV-2 can be similar.

All negative results are presumptive and should be confirmed with a molecular assay, if necessary, for patient management. Negative results do not rule out infection with RSV, influenza, or SARS-CoV-2 and should not be used as the sole basis for treatment or patient management decisions.

Positive results do not rule out bacterial infection or co-infection with other viruses.

**C Special Conditions for Use Statement(s):**

Rx - For Prescription Use Only

IVD - For In Vitro Diagnostic Use

**D Special Instrument Requirements:**

Not applicable

**IV Device/System Characteristics:**

**A Device Description:**

The Flowflex Plus RSV + Flu A/B + COVID Rapid Test is a lateral flow immunoassay in sandwich format intended for the qualitative detection and differentiation of RSV, influenza A, influenza B, and SARS-CoV-2 protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of respiratory tract infection. This test is for healthcare professional use.

The test package is composed of:

- 27 Test Cassettes
- 27 Extraction Buffer Tubes
- 25 Sterile Nasal Swabs
- 25 Pediatric Swab Guards
- 1 Tube Holder
- 1 Positive Control Swab (composed of RSV, influenza A, influenza B, and SARS-CoV-2 non-infectious recombinant antigens dried onto a swab with red stick, containing 0.05% Proclin 300 as a preservative).
- 1 Negative Control Swab (composed of negative control buffer dried onto a swab with blue stick, containing 0.05% Proclin 300 as a preservative)
- 1 English Package Insert
- 1 English Quick Reference Instructions

The Test Cassette is assembled with two test strips in a plastic housing.

***Test Strip Structure:***

The test strips in the plastic housing include backing card (provides structural support during use), sample receiving and particles pads, nitrocellulose (NC) membrane, test lines for each analyte and absorption pad. The test lines and the control lines are pre-coated with analyte specific antibody on the membrane.

The following two strips are included in the cassette housing:

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### Strip #1 (SARS-CoV-2 + Flu A + Flu B):

Includes: 1) particle pad containing monoclonal antibody conjugates with colored particles (SARS-CoV-2, Flu A, and Flu B antibody conjugates) and colored rabbit IgG for the control line; 2) NC membrane containing three test lines that are pre-coated with SARS-CoV-2, Flu A, and Flu B antibodies separately labeled as “CoV”, “A”, and “B” respectively on the cartridge and one control line, pre-coated with goat anti-rabbit IgG antibody (Fig 1) labeled as “Ctl” on the cartridge.

Fig 1: Strip 1


### Strip #2 (RSV):

Includes: 1) particle pad containing monoclonal antibody conjugates with colored particles (RSV antibody conjugates) and colored rabbit IgG for the control line; 2) NC membrane containing one test line pre-coated with RSV antibody labeled as “R” on the test cartridge and one control line, pre-coated with goat anti-rabbit IgG antibody (Fig 2) labeled as “Ctl” on the cartridge.

Fig 2: Strip 2


In both strips, attached to the NC membrane are the sample pad (which absorbs and distributes the sample), the reagent pad (containing reagents for antigen binding and detection), and the absorbent pad (which collects the excess liquid sample and reagents).

### Swab Guard:

For collecting nasal swabs from children less than 2 years, lay users employ a swab guard which serves as an added safety measure, preventing the nasal swab from going deeper than intended, limiting the risk of injury in children. In order to use, lay users slide the guard onto the swab from the stick end through the guard opening, and push it up until it clicks into place at the base of the foam head (Fig 3).

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Fig 3: Infant Swab Guard


### B Principle of Operation:

Nasal swabs are collected from individuals with signs and symptoms of respiratory infection. The swab is then processed in extraction buffer and then added to the sample well of the test cassette. When an adequate volume of the processed specimen is assed to the sample well of the test cassette, the specimen migrates by capillary action across each of the two test strips. SARS-CoV-2, Flu A, Flu B, or RSV antigen, if present in the specimen, will react with the specific antibody-coated colored particles. The mixture then migrates towards the membrane by capillary action to bind to the specific antibody on the NC membrane, producing a visible colored test line in the related test line region (CoV/A/B/R).

To serve as a procedural control, the colored rabbit IgG labeled particle will bind to goat anti-rabbit IgG on each NC membrane on each test strip to produce a colored line on the control line (Ctl) in the control line region. Formation of the control lines serves as an internal control indicating that proper volume of specimen has been added, and membrane wicking has occurred.

#### Interpretation of Results:

After dispensing the test specimen into the sample well, the result should be read at 15 minutes. Results should not be read before 15 minutes and after 30 minutes.

Table 1: Result Interpretation

| Result | Interpretation | Example Image |
| --- | --- | --- |
| Positive | If the control (Ctl) line is visible on both strips and any red or pink line appear at “CoV”, “A”, “B”, or “R”, no matter how faint, the test is positive for that virus. |  |

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| Result | Interpretation | Example Image |
| --- | --- | --- |
| **Negative** | If both control (Ctl) lines are visible, but the test line(s) (CoV, A, B, R) is/are not visible, the test is negative. | |
| **Invalid** | If the control (Ctl) line is not visible on either or both test strips, even if any test line is visible in the result window, the test is invalid. A new sample should be collected and re-tested with a new cartridge. | |

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

Flow/lex Plus RSV + Flu A/B + COVID Home Test

### B Predicate 510(k) Number(s):

K251749

### C Comparison with Predicate(s):

| Device & Predicate Device(s): | K261212 | Predicate: K251749 |
| --- | --- | --- |
| Device Trade Name | Flow/lex Plus RSV + Flu A/B + COVID Rapid Test | Flow/lex Plus RSV + Flu A/B + COVID Home Test |
| Intended Use/Indications For Use | The Flow/lex Plus RSV + Flu A/B + COVID Rapid Test is a lateral flow immunoassay intended for the qualitative detection and differentiation of respiratory syncytial virus (RSV), influenza A, influenza B, and SARS-CoV-2 protein antigens directly in anterior nasal swab samples from individuals with signs and symptoms of | The Flow/lex Plus RSV + Flu A/B + COVID Home Test is a lateral flow immunoassay intended for the qualitative detection and differentiation of respiratory syncytial virus (RSV), influenza A, influenza B, and SARS-CoV-2 protein antigens directly in anterior nasal swab samples from |

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| | respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to RSV, influenza, and SARS-CoV-2 can be similar. All negative results are presumptive and should be confirmed with a molecular assay, if necessary, for patient management. Negative results do not rule out infection with RSV, influenza, or SARS-CoV-2 and should not be used as the sole basis for treatment or patient management decisions. Positive results do not rule out bacterial infection or co-infection with other viruses. | individuals with signs and symptoms of respiratory tract infection. Symptoms of respiratory infections due to RSV, influenza, and SARS-CoV-2 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 six (6) months 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 RSV, influenza, SARS-CoV-2 or other pathogens. Individuals who test negative and/or experience continued or worsening 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. |
| --- | --- | --- |
| **General Device Characteristic Similarities** | | |
| Regulation Number | 21 CFR 866.3987 | Same |

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| Assay Technique | Lateral flow immunochromatographic assay, visually read | Same |
| --- | --- | --- |
| Analytes | SARS-CoV-2, influenza A, influenza B and RSV protein antigens | Same |
| Intended Specimen | Direct anterior nasal swabs | Same |
| Time to result | 15 minutes | Same |
| Detection Period | Within 5 days of symptom onset | Same |
| Sample Collection Method | Nasal swab supplied in kit; includes pediatric swab guard for ages less than 2 years. | Nasal swab supplied in kit; includes pediatric swab guard for ages 6-23 months. |
| Usage | Single use test | Same |
| Storage Temperature | 36-86 °F (2- 30°C) | Same |
| General Device Characteristic Differences | | |
| Patient Use Setting | For prescription use only by healthcare providers in CLIA-waived settings | Over the counter use |

### VI Standards/Guidance Documents Referenced:

| Document | Title | Publisher | Applicable Study |
| --- | --- | --- | --- |
| Special Controls under 21 CFR 866.3987 (multi-analyte respiratory virus antigen detection test) | Reclassification order for DEN240029 and special controls under 21 CFR 866.3987.pdf | FDA/CDRH | All Studies |

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

#### A Analytical Performance:

1. Precision/Reproducibility:

a. Lot-to-lot Precision:

The multi-lot precision for the Flowflex Plus RSV + Flu A/B + COVID Rapid Test was evaluated in two different in-house studies to assess variability between reagent lots, days, runs and operators.

Study 1 was conducted by 2 trained operators each of whom tested seven samples with various analyte concentrations and combinations as described below:

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a. Negative
b. 2X LoD SARS-CoV-2
c. 2X LoD Flu A
d. 2X LoD Flu B
e. 2X LoD RSV
f. 2X LoD Flu B & RSV
g. 2X LoD SARS-CoV-2 & Flu B & RSV

Each operator tested two sample replicates per run across two runs using three lots of devices. Runs were performed in the morning and afternoon over 10 days. This design (2 replicates/run/lot x 2 runs/operator x 2 operators x 3 lots x 10 days) resulted in 240 replicates per sample. All samples were prepared in pooled nasal swab matrix (PNSM). The study was performed in randomized and blinded manner.

Results for this study are shown in table below and were concordant with the expected results. Since the study obtained 100% agreement for all samples, both with and without analytes, the data were not stratified by the individual sources of variation.

Table 2: Summary Results of Multi-lot Precision Study (Study 1)

| Sample level | Analyte | Obtained results per analyte / Expected results per analyte | Total percent lot-to-lot agreement | 95% CI |
| --- | --- | --- | --- | --- |
| Negative | SARS-CoV-2 | (240/240) | 100.0% | 98.5-100.0% |
| | Flu A | (240/240) | 100.0% | 98.5-100.0% |
| | Flu B | (240/240) | 100.0% | 98.5-100.0% |
| | RSV | (240/240) | 100.0% | 98.5-100.0% |
| 2X LoD | SARS-CoV-2 | (240/240) | 100.0% | 98.5-100.0% |
| | Flu A | (240/240) | 100.0% | 98.5-100.0% |
| | Flu B | (240/240) | 100.0% | 98.5-100.0% |
| | RSV | (240/240) | 100.0% | 98.5-100.0% |
| | Flu B + RSV | (480/480) | 100.0% | 98.5-100.0% |
| | Flu B + RSV+ SARS-CoV-2 | (720/720) | 100.0% | 98.5-100.0% |

Study 2 was specifically conducted to assess difference between lots, because study 1 resulted in 100% agreement across all sources of variation and did not allow to conclusively assess lot-to-lot variability of the test. Study 2 was performed using one negative sample without any analyte and two low positive samples with the following analyte combinations, each at 0.75X LoD (i.e., below the concentration tested in study 1 and near the analytes' C95 concentration):

i. Negative
ii. 0.75X LoD Flu A & SARS-CoV-2
iii. 0.75X LoD Flu B & RSV

All samples were randomized and tested in a blinded manner. The testing was carried out over 3 days only but otherwise followed the same study design as Study 1. Seventy two data points per sample were collected (2 replicates/run/lot x 2 runs/day x 2 operators x 3 days x 3 lots = 72 data points).

Procedural random errors across various days, combined with the operator's ability to

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visually read test line intensities of samples with low analyte concentrations are expected to confound variability between lots and to have an impact on the precision estimates of a device for very low positive samples below the LoD. However, the results with concentrations at 0.75X LoD that yielded results with less than 100% showed minor variability between lots and were not deemed significant. The results are summarized below.

Table 3: Summary of Supplemental Precision Study (Study 2)

| Analyte Concentration | Analyte | Lot 1 | | Lot 2 | | Lot 3 | | Lot-to-Lot Agreement | | 95% CI | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | n/N^{1} | % Agmt^{2} | n/N^{1} | % Agmt^{2} | n/N^{1} | % Agmt^{2} | n/N^{1} | | % Agmt^{2} |
| Negative | SARS-CoV-2 | | 24/24 | 100.0% | 24/24 | 100.0% | 24/24 | 100.0% | 72/72 | 100.0% | 95.0-100.0% |
| | Flu A | | 24/24 | 100.0% | 24/24 | 100.0% | 24/24 | 100.0% | 72/72 | 100.0% | 95.0-100.0% |
| | Flu B | | 24/24 | 100.0% | 24/24 | 100.0% | 24/24 | 100.0% | 72/72 | 100.0% | 95.0-100.0% |
| | RSV | | 24/24 | 100.0% | 24/24 | 100.0% | 24/24 | 100.0% | 72/72 | 100.0% | 95.0-100.0% |
| Analyte Concentration | Analyte | | n/N^{3} | % Pos | n/N^{3} | % Pos | n/N^{3} | % Pos | n/N^{3} | % Pos | 95% CI |
| 0.75X LoD | Flu A+ SARS-CoV-2 | Flu A | 16/24 | 66.7% | 15/24 | 62.5% | 13/24 | 54.2% | 44/72 | 61.1% | 48.9-72.4% |
| | | SARS-CoV-2 | 23/24 | 95.8% | 18/24 | 75.0% | 21/24 | 87.5% | 62/72 | 86.1% | 75.9-93.1% |
| | Flu B+ RSV | Flu B | 19/24 | 79.2% | 22/24 | 91.7% | 15/24 | 62.5% | 56/72 | 77.8% | 66.4-86.7% |
| | | RSV | 17/24 | 70.8% | 18/24 | 75.0% | 14/24 | 58.3% | 49/72 | 68.1% | 56.0-78.6% |

$^{1}$ number of agreement with expected results per analyte/total number tested.

$^{2}$ Agreement.

$^{3}$ number of positive samples/total number tested.

### b. Multi-Site Reproducibility/Near-Cutoff study:

A multi-site reproducibility study was conducted to assess the performance of the candidate device using a contrived sample panel comprised of a true negative, a high negative pooled (0.3X LoD for FluA/FluB/RSV and 0.1X LoD for SARS-CoV-2), a low positive (1X LoD for each virus), and a moderate positive (3X LoD for each virus) sample for each analyte. The study was conducted by untrained operators in CLIA waived sites for over 5 days.

Contrived samples were prepared by spiking the diluted RSV, influenza A, influenza B or SARS-CoV-2 virus into the negative matrix to the required concentrations. Each diluted sample (50 μL) was directly applied onto the sample collection swab head. True negative swab samples were prepared by applying fifty (50 μL) of negative matrix directly onto the swab head.

Prepared contrived samples were randomized and blinded to each operator at three CLIA-waived sites. Nine (9) operators conducted the testing with each operator testing ten (10) panels with each panel consisting of eight (8) panel members. For each panel member, 90

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data points were collected (1 replicate/run x 2 runs/day x 3 operators/site x 3 sites x 5 days = 90 data points).

The results are shown in Table 4 below. All data met the predefined acceptance criteria, and no significant differences were observed among sites, operators, days and runs.

**Table 4: Summary of Multi-Site Reproducibility Study**

| Sample | # Positives / # Total (% Positive Rate) | | | Total Sample count (% Positive Rate) | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | | Site 3 |
| True Negative | RSV | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| Pooled High Negative | RSV | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 0/30 (0.0%) | 1/30 (3.3%) | 0/30 (0.0%) | 1/90 (1.1%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| 1X LoD RSV | RSV | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| 1X LoD Flu A | RSV | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |

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| Sample | # Positives / # Total (% Positive Rate) | | | Total Sample count (% Positive Rate) | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | | Site 3 |
| | Flu A | 30/30 (100.0%) | 30/30 (100.0%) | 29/30 (96.6%) | 89/90 (98.8%) |
| | Flu B | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| 1X LoD Flu B | RSV | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| 1X LoD SARS-CoV-2 | RSV | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | SARS-CoV-2 | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| 3X LoD RSV& Flu B | RSV | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | Flu A | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | Flu B | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | SARS-CoV-2 | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |

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| Sample | # Positives / # Total (% Positive Rate) | | | Total Sample count (% Positive Rate) | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | | Site 3 |
| 3X LoD RSV & Flu A & SARS-CoV-2 | RSV | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | Flu A | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |
| | Flu B | 0/30 (0.0%) | 0/30 (0.0%) | 0/30 (0.0%) | 0/90 (0.0%) |
| | SARS-CoV-2 | 30/30 (100.0%) | 30/30 (100.0%) | 30/30 (100.0%) | 90/90 (100.0%) |

## 2. Linearity:

This is a qualitative assay and linearity is not applicable.

## 3. Analytical Specificity/Interference:

### a. Cross-Reactivity and Microbial Interference:

Cross reactivity and microbial interference studies were conducted to determine if other respiratory pathogens/microbial flora that may be present in nasal swab samples could cause a false positive test result or interfere with the detection of a true positive result and cause a false negative result. A panel of related viruses, high prevalence disease agents, and normal or pathogenic flora were used for these studies.

For the cross-reactivity study, dilutions of the panel organisms were made in pooled anterior nasal swab matrix (PNSM) and tested in triplicates in the absence of SARS-CoV-2, influenza A, influenza B, RSV A and RSV B. No cross-reactivity was observed with the organisms tested for any of the 4 analytes (Table 4).

For the microbial interference study, dilutions of the panel organisms were made in PNSM, in the presence of low levels (3x LoD) of SARS-CoV-2 (Omicron XBB; USA/CA-Stanford109-S21/2022), influenza A (H1N1; A/Guangdong-Maonan/SWL1536/19), influenza B (Victoria; B/Hong Kong/574/19), RSV A (2006 isolate), and RSV B [CH93(18)-18] and tested in triplicate. No microbial interference was observed for any of the 4 analytes tested (Table 5).

**Table 5: Cross-Reactivity and Microbial Interference Study Results**

| Virus/Microorganism | Concentration | Units | # Positive / # Total | |
| --- | --- | --- | --- | --- |
| | | | Cross-Reactivity^{1} | Interference^{1} |
| Adenovirus Type 7A | 3.16 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Adenovirus 1 | 1.05 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |

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| Virus/Microorganism | Concentration | Units | # Positive / # Total | |
| --- | --- | --- | --- | --- |
| | | | Cross-Reactivity^{1} | Interference^{1} |
| *Bordetella pertussis* | 5.04 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Candida albicans* | 3.32 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Escherichia coli* | 2.37 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Haemophilus influenzae* | 9.85 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Neisseria mucosa* | 6.52 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Corynebacterium jeikeium* | 3.68 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Legionella pneumophila* | 5.98 x 10^{9} | CFU/mL | 0/3 | 3/3 |
| *Moraxella catarrhalis* | 1.37 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| Mumps Virus | 4.92 x 10^{6} | TCID_{50}/mL | 0/3 | 3/3 |
| *Mycobacterium tuberculosis* | 4.60 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Mycoplasma pneumoniae* | 1.37 x 10^{7} | CCU/mL | 0/3 | 3/3 |
| *Neisseria meningitidis* | 2.04 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Neisseria sicca* | 3.23 x 10^{9} | CFU/mL | 0/3 | 3/3 |
| Parainfluenza Virus Type 1 | 3.80 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Parainfluenza Virus Type 2 | 3.39 x 10^{6} | TCID_{50}/mL | 0/3 | 3/3 |
| Parainfluenza Virus Type 3 | 1.15 x 10^{6} | TCID_{50}/mL | 0/3 | 3/3 |
| Parainfluenza Virus Type 4 | 9.55 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| *Pseudomonas aeruginosa* | 5.50 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Staphylococcus aureus* | 5.41 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Staphylococcus epidermidis* | 3.52 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Streptococcus pyogenes* | 2.36 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Streptococcus pneumoniae* | 1.55 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Streptococcus salivarius* | 4.07 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| Corona Virus Strain: 229E | 2.09 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Corona Virus Strain: OC43 | 3.80 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Corona Virus, Strain: NL63 | 1.78 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Cytomegalovirus (CMV) | 2.09 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Human Metapneumovirus | 1.02 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Rhinovirus | 2.09 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| Measles Virus | 2.53 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| MERS-coronavirus | 1.12 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| *Lactobacillus plantarum* | 3.13 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| Enterovirus | 1.05 x 10^{5} | TCID_{50}/mL | 0/3 | 3/3 |
| *Pneumocystis jirovecii* | 5.18 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| *Chlamydophila (Chlamydia) pneumoniae* | 1.40 x 10^{7} | IFU/mL | 0/3 | 3/3 |
| *Chlamydia trachomatis* | 1.78 x 10^{7} | IFU/mL | 0/3 | 3/3 |
| Epstein Barr Virus | 4.02 x 10^{7} | CP/mL | 0/3 | 3/3 |
| *Corynebacterium diphtheriae* | 3.45 x 10^{8} | CFU/mL | 0/3 | 3/3 |
| *Saccharomyces cerevisiae* | 3.28 x 10^{7} | CFU/mL | 0/3 | 3/3 |
| Human Coronavirus HKU1: MINF-1596^{2} | 1:10 dilution | N/A | 0/3 | 3/3 |
| Human Coronavirus HKU1: MINF-1597 | 1:10 dilution | N/A | 0/3 | 3/3 |
| Human Coronavirus HKU1: MINF-1598^{2} | 1:10 dilution | N/A | 0/3 | 3/3 |
| Human Coronavirus HKU1: MINF-1601^{2} | 1:10 dilution | N/A | 0/3 | 3/3 |

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| Virus/Microorganism | Concentration | Units | # Positive / # Total | |
| --- | --- | --- | --- | --- |
| | | | Cross-Reactivity^{1} | Interference^{1} |
| Human Coronavirus HKU1: MINF-1602^{2} | 1:10 dilution | N/A | 0/3 | 3/3 |

$^{1}$ All analytes in the sample replicates were in agreement with the expected result.

$^{2}$ Human Coronavirus HKU1 (clinical specimens) were confirmed using a 510(k)-cleared PCR assay.

### b. Competitive Inhibition:

A competitive inhibition study was conducted to evaluate the potential for a high concentration of one target analyte to interfere with the detection of another target analyte at low concentration. Testing was performed in triplicate with different combinations of low (2.5-3X LoD for single analyte) and high (highest achievable) concentrations of SARS-CoV-2, influenza A, influenza B, and RSV. The results showed no competitive interference between SARS-CoV-2, influenza A, influenza B, and RSV as listed in Table 6 below.

**Table 6: Competitive Inhibition Study Results**

| Combination # | Viral Targets in Sample | | | | # of Positive Results / # of Replicates Tested | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Flu A | Flu B | SARS-CoV-2 | RSV | Flu A | Flu B | SARS-CoV-2 | RSV |
| 1 | Low | Negative | Negative | High | 3/3 | 0/3 | 0/3 | 3/3 |
| 2 | Negative | Low | Negative | High | 0/3 | 3/3 | 0/3 | 3/3 |
| 3 | Negative | Negative | Low | High | 0/3 | 0/3 | 3/3 | 3/3 |
| 4 | Low | Negative | High | Negative | 3/3 | 0/3 | 3/3 | 0/3 |
| 5 | Negative | Negative | High | Low | 0/3 | 0/3 | 3/3 | 3/3 |
| 6 | Negative | Low | High | Negative | 0/3 | 3/3 | 3/3 | 0/3 |
| 7 | Low | High | Negative | Negative | 3/3 | 3/3 | 0/3 | 0/3 |
| 8 | Negative | High | Low | Negative | 0/3 | 3/3 | 3/3 | 0/3 |
| 9 | Negative | High | Negative | Low | 0/3 | 3/3 | 0/3 | 3/3 |
| 10 | High | Low | Negative | Negative | 3/3 | 3/3 | 0/3 | 0/3 |
| 11 | High | Negative | Low | Negative | 3/3 | 0/3 | 3/3 | 0/3 |
| 12 | High | Negative | Negative | Low | 3/3 | 0/3 | 0/3 | 3/3 |

### c. Endogenous/Exogenous Substances Interference:

Flowflex Plus RSV + Flu A/B + COVID Rapid Test was evaluated for performance in the presence of potentially interfering substances that might be present in upper respiratory tract specimens. Substances that are commonly found on the hands were also tested. Potentially interfering substances were prepared and diluted in anterior nasal swab matrix (PNSM) to the recommended concentration. Virus negative specimens were evaluated in triplicate to confirm that the potentially interfering substances were not cross-reactive with the test.

To assess interference, positive samples were prepared in PNSM at 3X LoD using each analyte individually to assess interference. Positive and negative samples were evaluated in the presence of interfering substances in triplicate to confirm that these substances do not interfere with detection of SARS-CoV-2, influenza A, influenza B and RSV.

False positive results were detected for Flumist (live attenuated influenza vaccine,

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intranasal) at 15% v/v and 3% v/v for both Flu A and Flu B, and at 1.5% v/v for Flu A only; no cross reactivity was observed at 0.2% v/v.

No interference was observed amongst the rest of the substances tested as shown in Table 7.

**Table 7: Interfering Substances Study Results**

| Interfering Substance | Concentration | Cross-Reactivity (without analyte) (# Pos/ # Total) | | | | Interference (with analyte) (# Pos/ # Total) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV | SARS-CoV-2 | Flu A | Flu B | RSV |
| Equate Sore Throat Oral Anesthetic Spray (Phenol) | 5% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| ZICAM Cold Remedy | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Equate Nasal Four Nasal Spray (Phenylephrine HCl) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Nasal spray (Cromolyn sodium nasal solution) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Nasal spray (Oxymetazoline HCl) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Equate Nasal Spray Premium | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Equate Nasal Allergy Spray (Triamcinolone Acetonide) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Nasonex 24 HR Allergy (Mometasone furoate) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Nasal gel | 1.25% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Homeopathic nasal wash | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Homeopathic allergy relief (histaminum hydrochloricum) | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Ribavirin (RSV antiviral drug) | 10 mg/ml | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Oseltamivir Phosphate (Tamiflu) | 5 mg/ml | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Whole Blood | 2.5% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Hand sanitizer | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Hand Soap | 10% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Equate Budesonide Nasal Spray | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Flonase sensimist allergy relief spray | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Fluticasone Propionate Nasal Spray | 15% v/v | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |

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| Interfering Substance | Concentration | Cross-Reactivity (without analyte) (# Pos/ # Total) | | | | Interference (with analyte) (# Pos/ # Total) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV | SARS-CoV-2 | Flu A | Flu B | RSV |
| Tobramycin | 50 μg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Dyclonine Hydrochloride | 2 mg/mL | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mucin (from bovine submaxillary glands) | 2.5 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| HALLS (Menthol) | 3 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Sore Throat & Cough Lozenges (Benzocaine, Dextromethorphan HBr) | 3 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Beclomethasone | 5 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mupirocin | 10 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Flunisolide | 5 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Dexamethasone | 5 mg/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Biotin | 3500 ng/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Leukocytes | 2.5x10^{6} cells/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Flumist (Influenza Vaccine Intranasal) | 15% v/v | 0/3 | 3/3 | 3/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| | 3% v/v | 0/3 | 3/3 | 3/3 | 0/3 | NT* | NT* | NT* | NT* |
| | 1.5% v/v | 0/3 | 3/3 | 0/3 | 0/3 | NT* | NT* | NT* | NT* |
| | 0.2% v/v | 0/3 | 0/3 | 0/3 | 0/3 | NT* | NT* | NT* | NT* |
| Molnupiravir | 10 mg/ml | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Remdesivir (covid-19 antiviral drug) | 10 mg/ml | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Zanamivir | 5 mg/ml | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |

*NT: Not Tested

#### 4. Detection Limit and Assay Reportable Range:

##### a. Limit of Detection (LoD):

###### i. Single Analyte Limit of Detection (LoD):

A limit of detection (LoD) study was conducted to determine the lowest detectable concentration of heat-inactivated SARS-CoV-2, live influenza A (H1N1 and H3N2), live influenza B (Victoria and Yamagata) and live RSV (A and B) at which atleast 95% of all true

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positive replicates show a positive result. The LoD study was determined using a two-step method: a preliminary range finding study, followed by a confirmatory LoD study.

A preliminary LoD was determined by first testing serial ten-fold dilutions of virus stocks diluted into PNSM in 5 replicates for three device lots for a total of 15 replicates per dilution. A 50 µL sample of each virus diluted in PNSM was pipetted onto the dry swab and tested per the IFU. The lowest concentration at which all tested replicates were positive was treated as the preliminary LoD. The preliminary LoD of each virus was confirmed by testing an additional twenty samples for each viral stock at the preliminary LoD concentration as well as three-folds above and below the preliminary LoD. Acceptance criteria for confirmation of the LoD were that at least 95% of the replicates test positive for each device lot. The results of LoD confirmation testing for each virus are summarized in Table 8.

**Table 8: Limit of Detection of Flowflex Plus RSV + Flu A/B + COVID Rapid Test**

| Virus | Subtype/Lineage | Strains | Virus concentration | | # Positive/# Total | % Detected |
| --- | --- | --- | --- | --- | --- | --- |
| | | | TCID_{50}/mL | TCID_{50}/Swab | | |
| SARS-CoV-2 | Omicron XBB | USA/CA-Stanford-109 S21/2022 | 5.95 x 10^{4} | 2.98 x 10^{3} | 60/60 | 100.0% |
| | Wild Type | USA-WA1/2020 | 1.27 x 10^{3} | 6.35 x 10^{1} | 60/60 | 100.0% |
| Flu A | H1N1 | A/Guangdong-Maonan/SWL1536/19 | 3.90 x 10^{3} | 1.95 x 10^{2} | 60/60 | 100.0% |
| | | A/Victoria/4897/22 | 3.89 x 10^{1} | 1.95 x 10^{0} | 60/60 | 100.0% |
| | H3N2 | A/Darwin/6/2021 | 1.56 x 10^{2} | 7.80 x 10^{0} | 60/60 | 100.0% |
| Flu B | Victoria | B/Hong Kong/574/19 | 5.01 x 10^{2} | 2.51 x 10^{1} | 60/60 | 100.0% |
| | | B/Alabama/02/17 | 3.90 x 10^{2} | 1.95 x 10^{1} | 60/60 | 100.0% |
| | Yamagata | B/Phuket/3073/13 | 1.86 x 10^{1} | 9.30 x 10^{-1} | 60/60 | 100.0% |
| RSV | A | 2006 Isolate | 1.05 x 10^{3} | 5.25 x 10^{1} | 60/60 | 100.0% |
| | B | CH93 (18)-18 | 4.17 x 10^{2} | 2.09 x 10^{1} | 59/60 | 98.3% |

# *ii. Co-spiked Multi-Analyte LoD:*

After single analyte LoDs were determined, a co-spike equivalency study was conducted to assess whether the LoD of the Flowflex Plus RSV + Flu A/B + COVID Rapid Test for the four combined viruses (SARS-CoV-2, Flu A, Flu B, and RSV) was the same as for each virus tested separately.

The equivalency LoD study was validated by testing 20 replicates with the four pooled viruses at the final concentrations of 1X LoD, 10 replicates at 3X LoD, 10 replicates at 0.3X LoD, and five replicates with negative samples. In parallel, the same conditions and number of replicates were tested with individual viruses. 50 µL of each sample was applied to each dry swab and were tested with the device according to the IFU.

All replicates produced 100% positive results for each analyte at 3X and 1X LoD. At 0.3X LoD, the positive rate dropped as expected. Similar results were obtained when each analyte was tested individually. There was no impact on the sensitivity when all analytes were co-spiked. This study supports the use of co-spiked samples in subsequent analytical studies.

# *iii. International Standard Material NIBSC code: 21/368 – Limit of Detection:*

The LoD of the Flowflex Plus RSV + Flu A/B + COVID Rapid Test was also determined by evaluating different dilutions of the International Standard for SARS-CoV-2 antigen (NIBSC code: 32/368) in negative pooled nasal swab matrix. The International Standard for SARS-CoV-2 containing lyophilized SARS-CoV-2 antigen was reconstituted in 0.25 mL of ultra-

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pure water (for a final concentration of 20,000 IU/mL). The LoD was determined as the lowest virus concentration that was detected $\geq 95\%$ of the time (i.e., concentration at which at least 19/20 replicates tested positive).

The preliminary LoD concentration was determined by testing a series of 2-fold dilutions with one device lot, starting with a 5-fold pre-dilution from the stock concentration. Each concentration in the preliminary LoD study was tested in 5 replicates. The lowest concentration with 5/5 positive results was considered the preliminary LoD. For each replicate, 50 $\mu$L of virus dilution was applied to a swab and the swab was processed according to the IFU.

The preliminary LoD concentration was tested with an additional 20 replicates to confirm the LoD. Concentrations 2-fold higher, and 2-fold lower the preliminary LoD were also tested with 20 replicates to further define the LoD. Samples were prepared as for the preliminary LoD study above. To confirm the LoD, at least 19 of 20 replicates should be positive per lot. The results are summarized in Table 9.

**Table 9: LoD study Results for the International Standard**

| Testing | Concentration (IU/mL) | Concentration on dry swab (IU/swab) | # Positive / # Total |
| --- | --- | --- | --- |
| Preliminary LoD (10-fold dilutions) | 4.0 x 10^{3} | 2 x 10^{2} | 5/5 |
| | 2.0 x 10^{3} | 1 x 10^{2} | 5/5 |
| | 1.0 x 10^{3} | 0.5 x 10^{2} | 5/5 |
| | **5.0 x 10^{2}** | **2.5 x 10^{1}** | **5/5** |
| | 2.5 x 10^{2} | 1.2 x 10^{1} | 0/5 |
| | 1.2 x 10^{2} | 0.6 x 10^{1} | 0/5 |
| Confirmatory LoD | 1.0 x 10^{3} | 0.5 x 10^{2} | 20/20 |
| | **5.0 x 10^{2}** | **2.5 x 10^{1}** | **20/20** |
| | 2.5 x 10^{2} | 1.2 x 10^{1} | 0/20 |

# **b. High Dose Hook effect:**

A high-dose hook effect study was performed to assess whether very high concentrations of the target analyte could lead to false negative results. One lot of test cassettes was evaluated using three sample concentrations: negative, 2X LoD, and the original stock concentration of the analyte. The analytes were tested individually, and all samples were tested in triplicates. For each test, a dry swab was used to absorb 50 $\mu$L of either negative matrix or analyte sample (2X LoD or stock concentration) and were processed according to the IFU. No evidence of a high-dose hook effect was observed for any of the virus stocks or concentrations tested as shown in Table 10.

**Table 10: High-Dose Hook Effect Study Results**

| Sample | Concentration (TCID_{50}/mL) | # Pos/ # Total | | | |
| --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV |
| Negative | N/A | 0/3 | 0/3 | 0/3 | 0/3 |
| SARS-CoV-2 (High dose) | 5.95 x 10^{6} | 3/3 | 0/3 | 0/3 | 0/3 |
| SARS-CoV-2 (2X LoD) | 1.19 x 10^{5} | 3/3 | 0/3 | 0/3 | 0/3 |
| Flu A (H1N1) (High dose) | 1.17 x 10^{5} | 0/3 | 3/3 | 0/3 | 0/3 |
| Flu A (H1N1) (2X LoD) | 7.80 x 10^{3} | 0/3 | 3/3 | 0/3 | 0/3 |

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| Sample | Concentration (TCID_{50}/mL) | # Pos/ # Total | | | |
| --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV |
| Flu B (Victoria) (High dose) | 5.01 x 10^{5} | 0/3 | 0/3 | 3/3 | 0/3 |
| Flu B (Victoria) (2X LoD) | 1.00 x 10^{3} | 0/3 | 0/3 | 3/3 | 0/3 |
| RSV A (High dose) | 1.05 x 10^{6} | 0/3 | 0/3 | 0/3 | 3/3 |
| RSV A (2X LoD) | 2.10 x 10^{3} | 0/3 | 0/3 | 0/3 | 3/3 |

# **c. Inclusivity:**

Analytical reactivity testing for the Flowflex Plus RSV + Flu A/B + COVID Rapid Test was performed to ensure that the device can adequately detect a variety of strains for the SARS-CoV-2, influenza A, influenza B, and RSV viruses. A selection of temporally, geographically, and genetically diverse SARS-CoV-2, influenza and RSV strains were tested for inclusivity. A series of ten-fold dilutions of each virus strain was spiked into PNSM and tested to determine an approximate LoD of the test for each virus. The lowest concentration with 100% positive replicates was identified and additional 3-fold dilutions above and below that approximate LoD were tested to demonstrate inclusivity.

Based on the dilution series, the minimum detectable concentration was defined as the lowest concentration for which all five replicates were detected. Results summarized in Table 11, Table 12, Table 13 and Table 14 demonstrate that the test tests can detect analytes across a range of viral strains.

**Table 11: Inclusivity Results for SARS-CoV-2 Strains**

| SARS-CoV-2 Subtypes | Pathogens | Concentration (TCID_{50}/mL) | #Pos / # Total |
| --- | --- | --- | --- |
| Alpha | B.1.1.7; (USA/CA_CDC_5574/2020) | 3.41 x 10^{5} | 5/5 |
| Beta | B.1.351; (South Africa/KRISP-K005325/2020) | 1.51 x 10^{4} | 5/5 |
| Delta | B.1.617.2; (USA/PHC658/2021) | 4.17 x 10^{3} | 5/5 |
| Gamma | P.1; Gamma (Japan/TY7-503/2021) | 8.47 x 10^{4} | 5/5 |
| Omicron | B.1.1.529; (USA/MDHP20874/2021) | 1.29 x 10^{2} | 5/5 |
| | BA.2.3; (USA/MDHP24556/2022) | 3.90 x 10^{3} | 5/5 |
| | BA.4.6; (USA/MDHP35538/2022) | 1.15 x 10^{4} | 5/5 |
| | BA.5;(USA/COR-22-063113/2022) | 2.53 x 10^{4} | 5/5 |
| | BF.5; (USA/MD-HP34985/2022) | 8.80 x 10^{3} | 5/5 |
| | BF.7; (USA/NY-Wadsworth-22042128-01/2022) | 1.26 x 10^{4} | 5/5 |
| | BQ.1; (USA/NY-Wadsworth-22050462-01/2022) | 1.43 x 10^{4} | 5/5 |
| | BQ.1.1; (USA/MD-HP38861/2022) | 2.93 x 10^{3} | 5/5 |
| | JN.1.4; (USA/NY-Wadsworth-23068107-01/2023) | 5.43 x 10^{3} | 5/5 |
| | EG.5.1; (USA/MD-HP47946/2023) | 1.22 x 10^{4} | 5/5 |
| | JG.3; (USA/NY-Wadsworth-23067147-01/2023) | 7.88 x 10^{4} | 5/5 |
| | HV.1 USA/MD-HP49152/2023 | 8.73 x 10^{3} | 5/5 |

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**Table 12: Inclusivity Results for Influenza A Strains**

| Flu A Subtypes | Pathogens | Concentration (TCID_{50}/mL) | # Pos / # Total |
| --- | --- | --- | --- |
| H1N1 | A/California/07/09 | 1.70 x 10^{2} | 5/5 |
| | A/Mexico/4108/09 | 8.10 x 10^{3} | 5/5 |
| | A/New York/18/09 | 1.26 x 10^{4} | 5/5 |
| | A/Michigan/45/15 | 2.70 x 10^{2} | 5/5 |
| | A/New Caledonia/20/99 | 4.20 x 10^{3} | 5/5 |
| | A/Solomon Islands/03/06 | 5.62 x 10^{1} | 5/5 |
| | A/PR/8/34 | 5.01 x 10^{2} | 5/5 |
| | A/Wisconsin/67/22 | 3.87 x 10^{1} | 5/5 |
| | A/Connecticut/11/2023 | 9.33 x 10^{3} | 5/5 |
| | A/Baltimore/JH-22400/2022 | 8.90 x 10^{3} | 5/5 |
| H3N2 | A/Brisbane/10/07 | 1.05 x 10^{3} | 5/5 |
| | A/Perth/16/09 | 1.87 x 10^{3} | 5/5 |
| | A/Kansas/14/17 | 1.51 x 10^{5} | 5/5 |
| | A/Norway/466/14 | 4.17 x 10^{4} | 5/5 |
| | A/Texas/50/12 | 1.26 x 10^{3} | 5/5 |
| | A/Victoria/361/11 | 6.20 x 10^{2} | 5/5 |
| | A/Wisconsin/67/05 | 4.70 x 10^{2} | 5/5 |
| | A/Michigan/173/20 | 5.20 x 10^{3} | 5/5 |
| | A/Kumamoto/102/02 | 3.87 x 10^{2} | 5/5 |
| | A/Tasmania/503/20 | 1.41 x 10^{4} | 5/5 |
| H5N1 | A/Chicken/Liaoning/SD007/2017 | 1.30 x 10^{4} | 5/5 |
| | A/Vietnam/1194/2004 | 1.10 x 10^{4} | 5/5 |
| H5N8 | A/H5N8 | 3.67 x 10^{2} | 5/5 |
| H7N3 | A/Waterfowl/Chenhu/367-1/2021 | 5.00 x 10^{3} | 5/5 |

**Table 13: Inclusivity Results for Influenza B Strains**

| Flu B Subtypes | Pathogens | Concentration (TCID_{50}/mL) | #Pos / # Total |
| --- | --- | --- | --- |
| Victoria | B/Malaysia/2506/04 | 3.16 x 10^{3} | 5/5 |
| | B/Michigan/01/21 | 1.17 x 10^{4} | 5/5 |
| | B/Singapore/WUH4618/21 | 3.90 x 10^{4} | 6/6 |
| | B/Washington/02/19 | 6.27 x 10^{3} | 5/5 |
| Yamagata | B/Florida/04/06 | 1.17 x 10^{2} | 5/5 |
| | B/Texas/06/11 | 3.80 x 10^{3} | 5/5 |
| | B/Utah/09/14 | 4.17 x 10^{2} | 5/5 |
| | B/Wisconsin/01/10 | 1.41 x 10^{2} | 5/5 |

**Table 14: Inclusivity Results for RSV Strains**

| RSV Subtypes | Pathogens | Concentration (TCID_{50}/mL) | # Pos / # Total |
| --- | --- | --- | --- |
| RSV A | RSV A NY-Wadsworth-22055420-01/2022 | 4.20 x 10^{3} | 5/5 |
| | RSV A Long | 1.60 x 10^{6} | 5/5 |
| | RSV A2 | 1.60 x 10^{6} | 5/5 |
| | RSV B NY-Wadsworth-22055413-01/2022 | 3.53 x 10^{3} | 5/5 |
| | RSV B 18537 | 1.60 x 10^{3} | 5/5 |

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| RSV Subtypes | Pathogens | Concentration (TCID_{50}/mL) | # Pos / # Total |
| --- | --- | --- | --- |
| RSV B | RSV B, Isolate: 3/2015 | 1.69 x 10^{3} | 5/5 |

# **d. Assay Reportable Range:**

Not applicable; the device is a binary qualitative assay that is visually read.

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

# **a. Controls:**

# **i. Internal Controls:**

A built-in internal procedural control is incorporated into the test device to ensure that the test is functioning properly and correct use of the device. The internal control is part of the test strip membrane and is therefore automatically run within the development time of each test. The internal procedural control consists of IgG antibodies that are immobilized at the “Ctl” line of the test membrane in the device and captures leftover, unbound IgG complexes to generate a color signal.

# **ii. External Controls:**

Flowflex Plus RSV + Flu A/B + COVID Rapid Test contains positive and negative control swabs. Positive Control Swab with a red stick is composed of a RSV, influenza A, influenza B, and SARS-CoV-2 non-infectious recombinant antigen dried onto a swab, containing 0.05% Proclin 300 as a preservative. The negative Control Swab with a blue stick is composed of negative control buffer dried onto a swab, containing 0.05% Proclin 300 as a preservative.

Reproducibility of the external controls were assessed using three lots of the external control swabs (one lot per site) with the following study design: 1 replicate/operator/day x 3 operators/site x 3 sites x 5 days = 45 replicates. All controls produced expected results for each operator on each day of testing as summarized in Table 15.

**Table 15: External Control Swabs Reproducibility Study Results**

| External Control | Lot No. | # Positive / # Total (% Agreement) | | | |
| --- | --- | --- | --- | --- | --- |
| | | RSV | Flu A | Flu B | SARS-CoV-2 |
| Positive Control | Lot 1 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) |
| | Lot 2 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) |
| | Lot 3 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) |
| Negative Control | Lot 1 | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) |

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| External Control | Lot No. | # Positive / # Total (% Agreement) | | | |
| --- | --- | --- | --- | --- | --- |
| | | RSV | Flu A | Flu B | SARS-CoV-2 |
| | Lot 2 | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) |
| | Lot 3 | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) | 0/15 (100%) |

The real time stability of Flowflex Plus RSV + Flu A/B + COVID Positive and negative control swabs were evaluated using three different lots at two different temperature conditions of 2-8°C and 30°C. At each designated timepoint, the control swabs were removed from 2-8°C and 30°C and equilibrated to room temperature prior to testing. Each control swab was processed and tested with the Flowflex Plus RSV + Flu A/B + COVID Rapid Test kits per QRI. 5 replicates of the positive control swabs and 5 replicates of the negative control swabs were tested at each timepoint for each lot. At the time of clearance, all study data met the predefined acceptance criteria and showed that the external control swabs are stable for up to 3 months.

# b. Stability:

# i. Real-time Stability:

A real-time stability study was conducted to evaluate stability and determine the shelf-life of the unopened kit. To validate shelf-life, three unopened Flowflex Plus RSV + Flu A/B + COVID Rapid Test kit lots were stored at 2°C and 30°C. At defined intervals, an assessment of each lot was conducted with the following panel of test samples: negative clinical matrix, and co-spiked positive samples with inactivated SARS-CoV-2, live Flu A, Flu B, or RSV viruses, each at 3x LoD. Fifty (50) μL of each sample was applied directly to the swab and tested according to the instructions for use (IFU). Five replicates of each sample were tested for each time point and baseline testing was performed within one month of each manufactured lot. At the time of clearance, all study data met the protocol defined acceptance criteria and support storage of the test kits from 2-30°C for up to 15 months.

# ii. Shipping Stability:

The purpose of this study was to evaluate the stability of the unopened kits under the worst-case scenario for anticipated handling and shipping times and temperatures. Three sets of freeze/thaw cycles were conducted to mimic cold shipping conditions. After the final thaw, the kits were stored at 55°C for 35 days. Three lots were tested, and two concentrations of the samples (negative and 3x LoD co-spiked positive samples) were tested in 5 replicates at multiple timepoints (0, 7, 14, 21, 28 and 35 days). Testing was performed after the test kits return to room temperature. Results showed 100% agreement with the expected result across all analytes, lots, and timepoints, with no performance changes observed compared to baseline. These findings demonstrate that the assay remains stable under worst-case temperature conditions.

# 6. Assay Cut-Off:

Not applicable as this is a qualitative visually read assay without numeric data.

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## B Comparison Studies:

### 1. Method Comparison with Predicate Device:

See section C (clinical studies) below.

### 2. Matrix Comparison:

Not Applicable, the device only uses one matrix (i.e., anterior nasal swab).

## C Clinical Studies:

### 1. Clinical Performance Study:

A prospective clinical study was conducted from October 2024 to April 2025 to assess the performance of the Flowflex Plus RSV + Flu A/B + COVID Rapid Test when compared to an FDA cleared RT-PCR assay. The study prospectively enrolled symptomatic subjects at 10 geographically distinct study sites located in the United States. Enrolled subjects were aged 6 months or older who exhibited symptoms of infection consistent with COVID-19, influenza, or RSV at the time of collection and who were within 5 days post symptom onset (DPSO).

Testing was performed in a simulated home environment by lay users. A nasopharyngeal swab was collected first by the study operator, placed into transport tube containing viral transport medium, and shipped on dry ice to a central lab for testing with a highly sensitive RT-PCR comparator assay. Thereafter, an anterior nasal (AN) swab was collected per the candidate test's quick reference instructions (QRI). The swabs were either self-collected by a lay user aged ≥14 years or collected by an adult (parent/ guardian) from individuals aged ≥6 months old. Samples were then immediately tested with the Flowflex Plus RSV + Flu A/B + COVID Rapid Test according to the test QRI.

A total of 1263 anterior nasal swab samples were collected from symptomatic individuals within 5 days of respiratory symptom onset and met the inclusion criteria for the analysis, of which 1257 samples were evaluable for SARS-CoV-2 and 1261 samples evaluable for Flu A/B and RSV respectively. The subject demographics are shown in Table 16.

**Table 16: Subject Demographics**

| Specific Demographic | Lay user collecting and testing | Self-collecting and testing | Overall |
| --- | --- | --- | --- |
| | (N=634) | (N=629) | (N=1263) |
| **Age** | | | |
| Mean (SD) | 5.9 (4.0) | 48.6 (19.6) | 27.2 (25.6) |
| Median [Min, Max] | 5 [0.5, 17] | 49 (14, 91) | 14 (0.5, 91) |
| **Age Group** | | | |
| ≥ 6 months - < 2 years of age | 114 (18.0%) | 0 (0.0%) | 114 (9.0%) |
| 2-5 years of age | 212 (33.4%) | 0 (0.0%) | 212 (16.8%) |
| 6-13 years of age | 296 (46.7%) | 0 (0.0%) | 296 (23.4%) |
| 14-21 years of age | 12 (1.9%) | 75 (11.9%) | 87 (6.9%) |
| 22-59 years of age | 0 (0.0%) | 314 (49.9%) | 314 (24.9%) |
| ≥ 60 years of age | 0 (0.0%) | 240 (38.2%) | 240 (19.0%) |
| **Sex at Birth** | | | |

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| Specific Demographic | Lay user collecting and testing | Self-collecting and testing | Overall |
| --- | --- | --- | --- |
| | (N=634) | (N=629) | (N=1263) |
| Female | 300 (47.3%) | 355 (56.4%) | 655 (51.9%) |
| Male | 334 (52.7%) | 274 (43.6%) | 608 (48.1%) |
| **Ethnicity** | | | |
| Hispanic/Latino | 140 (22.1%) | 75 (11.9%) | 215 (17.0%) |
| Not Hispanic/Latino | 494 (77.9%) | 554 (88.1%) | 1048 (83.0%) |

The performance of the Flowflex Plus RSV + Flu A/B + COVID Rapid Test when compared to FDA-cleared highly sensitive RT-PCR molecular assays are presented in the tables below.

**Table 17: SARS-CoV-2 Clinical Performance**

| SARS-CoV-2 | Comparator Positive | Comparator Negative | Total |
| --- | --- | --- | --- |
| **Candidate Positive** | 131 | 1 | 132 |
| **Candidate Negative** | 12 | 1113 | 1125 |
| **Total** | 143 | 1114 | 1257* |
| Positive Percent Agreement (PPA) | 91.6% (131/143) (95% C.I.: 85.9% - 95.1%) | | |
| Negative Percent Agreement (NPA) | 99.9% (1113/1114) (95% C.I.: 99.5% - 100.0%) | | |

*6 samples excluded due to invalid results with comparator methods

**Table 18: Influenza A Performance**

| Influenza A | Comparator Positive | Comparator Negative | Total |
| --- | --- | --- | --- |
| **Candidate Positive** | 236 | 2 | 238 |
| **Candidate Negative** | 18 | 1005 | 1023 |
| **Total** | 254 | 1007 | 1261* |
| Positive Percent Agreement (PPA) | 92.9% (236/254) (95% C.I.: 89.1% - 95.5%) | | |
| Negative Percent Agreement (NPA) | 99.8% (1005/1007) (95% C.I.: 99.3% - 100.0%) | | |

*2 samples excluded due to invalid results with comparator methods

**Table 19: Influenza B Performance**

| Influenza B | Comparator Positive | Comparator Negative | Total |
| --- | --- | --- | --- |
| **Candidate Positive** | 91 | 1 | 92 |
| **Candidate Negative** | 7 | 1162 | 1169 |
| **Total** | 98 | 1163 | 1261* |
| Positive Percent Agreement (PPA) | 92.9% (91/98) (95% C.I.: 86.0% - 96.5%) | | |
| Negative Percent Agreement (NPA) | 99.9% (1162/1163) (95% C.I.: 99.5% - 100.0%) | | |

*2 samples excluded due to invalid results with comparator methods

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**Table 20: RSV Performance**

| RSV | Comparator Positive | Comparator Negative | Total |
| --- | --- | --- | --- |
| Candidate Positive | 159 | 2 | 161 |
| Candidate Negative | 10 | 1090 | 1100 |
| **Total** | **169** | **1092** | **1261*** |
| Positive Percent Agreement (PPA) | 94.1% (159/169) (95% C.I.: 89.5% - 96.8%) | | |
| Negative Percent Agreement (NPA) | 99.8% (1090/1092) (95% C.I.: 99.3% - 100.0%) | | |

\*2 samples excluded due to invalid results with comparator methods

**Table 21: RSV Performance Stratified by Age Group**

| Age Group | Number of Subjects Tested | Candidate Positives | Comparator Positives | PPA (95% CI) |
| --- | --- | --- | --- | --- |
| 6-11 months | 51 | 21 | 21 | 100.0% (84.5%, 100.0%) |
| 12-17 months | 47 | 17 | 17 | 100.0% (81.6%, 100.0%) |
| 18-23 months | 16 | 5 | 5 | 100.0% (56.6%, 100.0%) |
| **< 2 years** | **114** | **43** | **43** | **100.0% (91.8%, 100.0%)** |
| 2-5 years | 212 | 75 | 83 | 90.4% (82.1%, 95.0%) |
| 6-13 years | 296 | 20 | 21 | 95.2% (77.3%, 99.2%) |
| 14-21 years | 87 | 6 | 6 | 100% (60.9%, 100.0%) |
| 22-59 years | 314 | 8 | 9 | 88.9% (56.5%, 98.0%) |
| >=60 years | 238 | 7 | 7 | 100.0% (64.6%, 100.0%) |
| **Total** | **1261** | **159** | **169** | **94.1% (89.5%, 96.8%)** |

# 2. Clinical Sensitivity:

Please refer to Section VI.C (Clinical Studies) above for clinical validation.

# 3. Clinical Specificity:

Please refer to Section VI.C (Clinical Studies) above for clinical validation.

# 4. Clinical Cut-Off:

The test is a qualitative test with a binary positive/negative signal and there is no clinical cut-off for the test.

# **D Expected Values/Reference Range:**

A patient sample is expected to be negative for RSV, Influenza A, Influenza B and SARS-CoV-2.

# **VIII Proposed Labeling:**

The labeling supports the finding of substantial equivalence for this device.

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# **IX Conclusion:**

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

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