Predicate Devices

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Reference Devices

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AI/ML (Artificial Intelligence / Machine Learning)

No
The device description explicitly states that "No software or instrumentation is required for use of the test" and describes it as a "visually-read test," indicating it is a simple lateral-flow immunoassay without computational components.

Therapeutic

No
This device is a diagnostic test kit for detecting syphilis antibodies, not a therapeutic device used for treatment.

Diagnostic

Yes

Explanation: The device is intended for the detection of Treponema pallidum (syphilis) antibodies in human whole blood, which is a diagnostic purpose. Although the information states that positive results alone are not sufficient for diagnosis and require further laboratory testing, the device's function is to identify individuals suspected of syphilis, which directly contributes to the diagnostic process.

SaMD (Software as a Medical Device)

No

The device description explicitly states, "No software or instrumentation is required for use of the test." and describes a physical, visually-read lateral-flow assay.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's for the "detection of Treponema pallidum (syphilis) antibodies in human whole blood (capillary)". This involves testing a biological sample (blood) outside of the body to gain information about a medical condition (syphilis).
Device Description: The description details a "qualitative rapid membrane immunochromatographic assay" that uses a "single-use, lateral-flow, visually-read test" with "human capillary whole blood". This is a typical description of an in vitro diagnostic test.
Performance Studies: The document describes clinical performance studies where the device's results are compared to other laboratory tests using biological samples (venous blood). This is a standard process for evaluating the performance of an IVD.

The definition of an In Vitro Diagnostic (IVD) is a medical device that is used to perform tests on samples such as blood, urine, or tissues to detect diseases or other conditions. The First To Know Syphilis Test clearly fits this definition.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The First To Know Syphilis Test is a qualitative rapid membrane immunochromatographic assay for the detection of Treponema pallidum (syphilis) antibodies in human whole blood (capillary). This test is intended for over the counter (OTC) consumer use in individuals suspected of syphilis. Positive test results with the First To Know Syphilis Test alone are not sufficient to diagnose syphilis infection and must be followed by additional laboratory testing through a health care provider to confirm a diagnosis of syphilis.

This test is not a substitute for visits to a healthcare provider. The information provided by this product should not be used to start, stop or change any treatments without a healthcare provider.

Results of testing with the First To Know Syphilis Test will likely be positive for individuals previously diagnosed with syphilis, even if they were successfully treated. The First To Know Syphilis Test cannot determine whether there has been re-infection with syphilis.

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

SBZ

Device Description

The First To Know Syphilis Test is a single-use, lateral-flow, visually-read test that is intended for over-the-counter use by lay persons. All components of the test are contained in the cassette; no additional reagents are required to run the test. Procedure controls are intrinsic to the cassette. A single [~40 uL] drop of human capillary whole blood is added to the cassette. No software or instrumentation is required for use of the test. There are no accessories required to use the test. The test is a chromatographic immunoassay that delivers results in 15 minutes.

Mentions image processing

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Mentions AI, DNN, or ML

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Input Imaging Modality

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Anatomical Site

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Indicated Patient Age Range

The study enrolled sexually active lay users between the ages of 18 and 64.

Intended User / Care Setting

OTC consumer use

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

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

Clinical performance for the First To Know Syphilis Test was established through a prospective study enrolling individuals of various races, ethnicities, pregnancy status, HIV status, education, and annual income at six geographically diverse clinical sites. The study enrolled sexually active lay users between the ages of 18 and 64. Participants were provided with the First To Know Syphilis Test kit and labeling who completed testing, including set up and sample collection, in a simulated home environment. Site observers were present to assess the ease of use of the test but did not aid the lay users in performing the test. Following testing, venous blood samples were collected from each participant for comparator testing using two separate treponemal tests and one non-treponemal test. The positive comparator result was determined by at least two reactive results. The study consisted of 1424 prospectively enrolled lay users from September 2021 through October 2023. Of the 1424 enrolled subjects, 45 were excluded due to issues such as incomplete paperwork, subject withdrawal, screening failures, and blood draw failures, leaving 1379 evaluable specimens. Of these, 27 were excluded due to invalid comparator results, seven (7) were excluded due to laboratory errors, and 75 were excluded due to subject device invalid results, leaving 1270 First To Know Syphilis Test results with paired valid comparator results.

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

Clinical Studies:

Study Type: Prospective study
Sample Size: 1270 evaluable specimens (from an initial enrollment of 1424 participants)
Key Results:
- Positive Percent Agreement (PPA) = 93.4% (99/106) (95% CI: 87.0-96.8%)
- Negative Percent Agreement (NPA) = 99.5% (1158/1164) (95% CI: 98.9-99.8%)
- The invalid rate for the device was 5.6% (75/1345).

Staged Syphilis Testing:

Study Type: Evaluation using characterized serum samples.
Sample Size: 125 serum samples (25 Primary Syphilis, 56 Secondary Syphilis, 16 Early Latent Syphilis, 28 Late Latent Syphilis).
Key Results: All 125 serum samples tested demonstrated positive results with the First To Know Syphilis Test.

Precision/Reproducibility Study 1:

Study Type: Precision study
Sample Size: Panels of non-reactive, high non-reactive, low reactive, and moderate reactive samples tested in duplicate over three days with three lots by operators from three sites. (e.g., for Site 1, Lot 1-3, 3 operators, 6 samples each = 54 tests per reactivity level per site per lot)
Key Results: The percent positive results for the moderate reactive and low reactive samples were 100% across all lots, operators, and sites for Site 1 and Site 3. For Site 2, Moderate Reactive was 100% and Low Reactive was 100% for Lot 1 and 2, and 94.4% for Lot 3. The percent positive results for the high non-reactive and non-reactive samples were 0% across all lots, operators, and sites. No significant differences observed within run, between run, between sites, or between operators.

Precision/Reproducibility Study 2:

Study Type: Reproducibility study
Sample Size: Panels of non-reactive, high non-reactive, low reactive, and moderate reactive samples tested for five days by operators from three sites. (e.g., for each site, 45 samples per reactivity level = 135 total for each reactivity level across all sites).
Key Results: The percent positive results for the moderate reactive and low reactive samples were 100% (135/135) and 98.5% (133/135), respectively. The percent positive results for the high non-reactive and non-reactive samples were 0% (0/135) and 0% (0/135), respectively. No significant differences observed within run, between run, between sites, or between operators.

Cross-reactivity Study:

Study Type: Analytical specificity study.
Sample Size: Serum samples (10 replicates each) containing antibodies to various bacteria, viruses, and other medically relevant conditions.
Key Results: One out of ten samples containing Epstein-Barr Virus (EBV) antibodies and one out of ten samples containing Rheumatoid Factor showed a false positive result. All other tested cross-reactants showed 0% positive.

Interfering Substances Study:

Study Type: Analytical interference study.
Sample Size: 23 potential interferents spiked into pools of negative whole blood, and low positive test samples prepared. Three replicates tested for positive and negative samples for each interferent.
Key Results: Three out of three positive samples containing Gamma-globulin interferent at a concentration of 60 mg/mL resulted in invalid results. All other samples provided expected results.

Hook Effect Study:

Study Type: High Dose Hook Effect study.
Sample Size: Series of 10 high titer positive concentrations prepared neat and serially diluted. Tested in duplicate.
Key Results: All spiked samples were 100% positive at dilutions tested at 1:160 or lesser, demonstrating no observed Hook Effect.

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

Positive Percent Agreement (PPA) = 93.4% (99/106) (95% CI: 87.0-96.8%)
Negative Percent Agreement (NPA) = 99.5% (1158/1164) (95% CI: 98.9-99.8%)
Clinical Sensitivity: 93.4% (95% CI: 87.0-96.8%)
Clinical Specificity: 99.5% (95% CI: 98.9-99.8%)

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

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

Not Found

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

Not Found

Regulation Number and Section

N/A

Summary

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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: a symbol on the left and the text "FDA U.S. FOOD & DRUG ADMINISTRATION" on the right. The symbol is a stylized representation of a caduceus, a traditional symbol of medicine, while the text clearly identifies the organization as the U.S. Food and Drug Administration.

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR First To Know Syphilis Test DECISION SUMMARY

I Background Information:

A De Novo Number

DEN230090

B Applicant

NOWDiagnostics

C Proprietary and Established Names

First To Know Syphilis Test

D Regulatory Information

| Product
Code(s) | Classification | Regulation
Section | Panel |
|--------------------|----------------|--------------------------------------------------------------------------------------------------------------|-------------------|
| SBZ | Class II | 21 CFR 866.3986 - Test
for detection of antibodies
associated with syphilis
performed by lay users. | MI - Microbiology |

Submission/Device Overview: II

A Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the First To Know Syphilis Test.

B Measurand:

Antibodies to Treponema pallidum

C Type of Test:

Chromatographic Lateral Flow Immunoassay

Indications for Use: III

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

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A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

The First To Know Syphilis Test is a qualitative rapid membrane immunochromatographic assay for the detection of Treponema pallidum (syphilis) antibodies in human whole blood (capillary). This test is intended for over the counter (OTC) consumer use in individuals suspected of syphilis. Positive test results with the First To Know Syphilis Test alone are not sufficient to diagnose syphilis infection and must be followed by additional laboratory testing through a health care provider to confirm a diagnosis of syphilis.

This test is not a substitute for visits to a healthcare provider. The information provided by this product should not be used to start, stop or change any treatments without a healthcare provider.

Results of testing with the First To Know Syphilis Test will likely be positive for individuals previously diagnosed with syphilis, even if they were successfully treated. The First To Know Syphilis Test cannot determine whether there has been re-infection with syphilis.

C Special Conditions for Use Statement(s):

OTC - Over The Counter

D Special Instrument Requirements:

Not applicable.

IV Device/Svstem Characteristics:

A Device Description:

The First To Know Syphilis Test is a single-use, lateral-flow, visually-read test that is intended for over-the-counter use by lay persons. All components of the test are contained in the cassette; no additional reagents are required to run the test. Procedure controls are intrinsic to the cassette. A single [~40 uL] drop of human capillary whole blood is added to the cassette. No software or instrumentation is required for use of the test. There are no accessories required to use the test. The test is a chromatographic immunoassay that delivers results in 15 minutes.

B Principle of Operation:

The First To Know Syphilis Test is a chromatographic lateral-flow immunoassay. It consists of a small plastic test cassette with a nitrocellulose membrane, attached to a conjugate pad next to a sample Fill Zone. The test begins when a person adds a blood drop to the edge of the cassette where the blood is drawn into the Fill Zone. When enough sample is in the Fill Zone, the sample flows into a dry porous test strip composed of a plasma-separating membrane and a series of analytical membranes. As the sample flows through the membranes, syphilis-specific antibody in the sample binds to the test strip at the test band location. The appearance of a visible test band indicates the sample contains a detectable level of syphilis antibody. An internal control line is

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present to verify the sample has flowed through the membrane and the test components are working properly.

V Standards/Guidance Documents Referenced:

ISO 14971 Third Edition 2019-12 "Medical Devices - Application of Risk Management to Medical Devices

ISO 15223-1 Fourth Edition 2021-07 "Medical Devices - Symbols to be used with information to be supplied by the manufacturer - Part 1: General requirements"

CLSI EP07 3rd Edition "Interference Testing in Clinical Chemistry"

CLSI EP25-A "Evaluation of Stability of In Vitro Diagnostic Reagents: Approved Guideline"

VI Performance Characteristics:

A Analytical Performance:

1. Precision/Reproducibility:

Study 1

A precision study was conducted to assess the variability of the First To Know Syphilis Test across lots, operators, sites and days. This study was conducted by operators from three sites using panels of blind coded specimens containing non-reactive, high non-reactive, low reactive, and moderate reactive samples. Operators tested panels of samples with three different lots in duplicate over three days of testing. The percent positive results for the moderate reactive and low reactive samples were 100% across all lots, operators, and sites. The percent positive results for the high non-reactive and non-reactive samples were 0% across all lots, operators, and sites. There were no significant differences observed within run (replicates tested by one operator), between run (three different days), between sites (three sites), or between operators (nine operators). The lot-to-lot precision study qualitative results (percent positive results and counts) are presented in the tables below:

| Site 1 | | Moderate
Reactive | Low
Reactive | High Non-
Reactive | Non-
Reactive |
|--------|------------|----------------------|-----------------|-----------------------|------------------|
| | | | | | |
| Lot 1 | Operator 1 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 2 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 3 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) |
| Lot 2 | Operator 1 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 2 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 3 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) |
| Lot 3 | Operator 1 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 2 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Operator 3 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) |

Table 1. Lot-to-Lot Precision (Site 1)

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Operator 3	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
Overall	18/18 (100%)	18/18 (100%)	0/18 (0%)	0/18 (0%)

	Site 2
		Moderate
Reactive	Low Reactive	High Non-
Reactive	Non-
Reactive
Lot 1	Operator 4	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 5	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 6	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Overall	18/18 (100%)	18/18 (100%)	0/18 (0%)	0/18 (0%)
Lot 2	Operator 4	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 5	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 6	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Overall	18/18 (100%)	18/18 (100%)	0/18 (0%)	0/18 (0%)
Lot 3	Operator 4	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 5	6/6 (100%)	6/6 (100%)	0/6 (0%)	0/6 (0%)
	Operator 6	6/6 (100%)	5/6 (83.3%)	0/6 (0%)	0/6 (0%)
	Overall	18/18 (100%)	17/18 (94.4%)	0/18 (0%)	0/18 (0%)

Table 2 Lot-to-Lot Precision (Site 2)

Table 3. Lot-to-Lot Precision (Site 3)

| | | Site 3 | Moderate
Reactive | Low
Reactive | High Non-
Reactive | Non-
Reactive |
|-------|------------|--------------|----------------------|-----------------|-----------------------|------------------|
| Lot 1 | Operator 7 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 8 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 9 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) | |
| Lot 2 | Operator 7 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 8 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 9 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) | |
| Lot 3 | Operator 7 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 8 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Operator 9 | 6/6 (100%) | 6/6 (100%) | 0/6 (0%) | 0/6 (0%) | |
| | Overall | 18/18 (100%) | 18/18 (100%) | 0/18 (0%) | 0/18 (0%) | |

Study 2

A reproducibility study of the First To Know Syphilis Test was conducted by operators from three sites using panels of blind coded specimens containing non-reactive, high non-reactive, low reactive, and moderate reactive samples. Operators tested multiple samples of each panel member for five days. The percent positive results for the moderate reactive and low reactive samples were 100% (135/135) and 98.5% (133/135), respectively. The percent positive results for the high non-reactive and non-reactive samples were 0% (0/135) and 0% (0/135), respectively. There were no significant differences observed within run (replicates tested by one operator), between run (five different days), between sites (three sites), or between

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operators (nine operators). The Reproducibility Study site-to-site qualitative results (percent positive results and counts) are presented in the table below.

Sample	Site 1		Site 2		Site 3		Overall Results
	% Positive	Count	% Positive	Count	% Positive	Count	% Positive	Count
Moderate Reactive	100%	45/45	100%	45/45	100%	45/45	100%	135/135
Low Reactive	100%	45/45	95.6%	43/45	100%	45/45	98.5%	133/135
High Non-Reactive	0%	0/45	0%	0/45	0%	0/45	0%	0/135
Non-Reactive	0%	0/45	0%	0/45	0%	0/45	0%	0/135

Table 4. Site-to-Site Reproducibility

2. Linearity:

Not applicable. This test is a qualitative test.

3. Analytical Specificity/Interference:

Cross-reactivity

Cross-reactivity was evaluated by testing serum samples containing antibodies to various bacteria, viruses, and other medically relevant conditions with the First To Know Syphilis Test. Each serum was tested in ten (10) replicates to determine whether it will provide a false positive result when tested with the First To Know Syphilis Test. One out of ten samples containing Epstein-Barr Virus (EBV) antibodies and one out of ten samples containing Rheumatoid Factor showed a false positive result. A limitation is included in the labeling, warning of the possibility of false positive results with sera containing EBV antibodies or Rheumatoid Factor. Results of the Cross-reactivity study are shown in the table below.

Table 5. Cross-reactivity

| Potential Cross-reactant | # of
Positives / #
Tested | % Positive |
|----------------------------|---------------------------------|------------|
| Anti-Lyme IgG and IgM | 0/10 | 0% |
| Anti-Gonorrhea | 0/10 | 0% |
| Anti-Chlamydia trachomatis | 0/10 | 0% |
| Anti-Leptospirosis | 0/10 | 0% |
| Anti-Trichomonas | 0/10 | 0% |
| Anti-Toxoplasma gondii | 0/10 | 0% |
| Anti-Cytomegalovirus | 0/10 | 0% |
| Anti-Epstein-Barr Virus | 1/10 | 10% |
| Anti-Hepatitis A Virus | 0/10 | 0% |
| Anti-Hepatitis B Virus | 0/10 | 0% |

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Anti-Hepatitis C Virus	0/10	0%
Anti-nuclear Antibody	0/10	0%
Anti-HIV Type 1 & Type 2	0/10	0%
Hemodialysis Patient serum	0/10	0%
Anti-Herpes Simplex Virus	0/10	0%
Rheumatoid Factor positive serum	1/10	10%
Human Anti-Mouse Antibody	0/10	0%
Heterophile Antibodies	0/10	0%
Anti-Human Papillomavirus	0/10	0%
Anti-Human T-cell Lymphotropic
Virus	0/10	0%

Interfering Substances

An analytical study was performed to assess the potential interference effects of 23 substances naturally present in blood specimens or that may be artificially introduced into the blood. To prepare the samples, various potential interferents were first spiked into pools of negative whole blood. Then, low positive test samples were prepared by spiking treponemal antibody positive plasma into the pool of whole blood containing the specific interferent. Negative samples were prepared from the pool of negative whole blood containing the specific interferent but without treponemal antibodies. Three replicates were tested each for the positive and negative samples. The ratio of spiked serum to whole blood did not exceed 10% for samples tested.

Table 6. Interfering Substances

| Potential
Interferent | Concentration
Tested | # Invalid Results
/ Negative +
Positive Samples
Tested | # of Negative
Results / # of
Negative
Samples Tested | # of Positive
Results / # of
Positive
Samples
Tested |
|--------------------------|---------- | Acetaminophen | 0.2 mg/mL | Acetylcysteine | 1.66 mg/mL | Aspirin | 0.65 mg/mL | Albumin | 50 mg/mL | Ampicillin | 0.2 mg/mL | Ascorbic acid | 0.2 mg/mL | Bilirubin | 2 mg/mL | Cefoxitin | 0.66 mg/mL | Cholesterol | 2.5 mg/mL | Cyclosporine | 1.4 mg/mL | Doxycycline | 0.03 mg/mL | Gamma-globulin | 60 mg/mL | Hemoglobin | 20 mg/mL | Heparin | 3000 U/L | Ibuprofen | 0.5 mg/mL | K2EDTA | 0.8 mg/mL | Levodopa | 6.5 mg/mL | Methyldopa | 0.015 mg/mL | Metronidazole | 0.12 mg/mL ---------------|-----------------------------------------------------------------|---------------------------------------------------------------|------------------------------------------------------------------|
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 3/6 | 3/3 | 0/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |
| 0/6 | 3/3 | 3/3 |

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Phenylbutazone	5 mg/mL	0/6	3/3	3/3
Rifampin	0.064 mg/mL	0/6	3/3	3/3
Theophylline	0.04 mg/mL	0/6	3/3	3/3
Triglycerides	2.5 mg/mL	0/6	3/3	3/3

Three out of three positive samples containing Gamma-globulin interferent at a concentration of 60 mg/mL resulted in invalid results after testing with the First To Know Syphilis Test. All other samples provided the expected result when tested in the presence of the above interferents at the stated concentrations. A limitation is added to the labeling stating that presence of gammaglobulin may cause invalid test results.

Hook Effect

The Hook/Prozone effect is interference due to excess analyte in the sample resulting in a signal intensity lower than expected, and in extreme cases it can cause false negative/non-reactive results. A High Dose Hook Effect study was conducted to determine if a hook effect would be observed at high concentrations of the analyte (i.e., a false negative at high concentrations of anti-syphilis antibody). A series of 10 high titer positive concentrations were prepared neat and also serially diluted in negative plasma and tested in duplicate on the First To Know Rapid Syphilis Test. All spiked samples were 100% positive at dilutions tested at 1:160 or lesser. This study demonstrates there is no observed Hook Effect for high concentrations of anti-Syphilis antibody tested herein.

1. Assay Reportable Range:
  Not applicable. This test is a qualitative test.
1. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

Stability

Unopened kit stability

A multi-lot kit stability study was conducted to establish the shelf-life of the First To Know Syphilis Test. Test kits were stored at temperatures of 12℃ and 33℃ for a period of 24 months. Three different lots were tested at month 0, 1, and 3, and every three months afterward until month 24 (10 total timepoints) with 10 replicates per lot, for a total of 300 tests (10 tests x 3 lots x 10 timepoints). Samples for testing included negative whole blood, normal human serum, . low reactive serum, and moderate reactive serum. All tests produced the expected results for the first 18 months of the study. The first failure was observed in one lot at the 21 months timepoint. The study results demonstrated the stability of the First To Know Syphilis Test for up to 18 months when stored within the stated temperature range of 15°C to 30°C.

Shipping stability

A kit stability study was conducted for a period of 10 days to evaluate the stability of the First To Know Syphilis Test under conditions representing the extreme cold and hot temperatures and durations anticipated during shipping. Samples for testing included negative whole blood, low reactive serum, and moderate reactive serum. Twenty-five replicates were tested per time point per condition for each positive panel member. Five replicates were tested per time point per condition for the negative panel member. One false negative result from the low reactive sample was observed at the four-hour time point for the 43℃ condition. This is acceptable because

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95% positive results were generated with low reactive sample. All other results were as expected. The study results demonstrate the stability of the First To Know Syphilis Test under anticipated shipping conditions.

6. Detection Limit:

Not Applicable.

1. Assay Cut-Off:
  The First To Know Syphilis Test was optimized by testing known positive samples prepared from serum specimens obtained from clinically-positive individuals. The samples were prepared by diluting the serum specimens to levels that corresponded to a negative, high-non reactive (C5), low reactive (C95), and moderate reactive (2x C95) concentration as determined by an FDA-cleared total Syphilis IgG/IgM assay. Twenty individual samples were prepared for the C5 and C95 levels and tested using three lots of the First To Know Syphilis Test providing 60 individual data points for each concentration. The results demonstrated the First To Know Syphilis Test can accurately and consistently detect low reactive (C95) samples (60/60 positive).

B Comparison Studies:

1. Method Comparison:
  See Clinical Studies section below.
1. Matrix Comparison:
  Not applicable.

C Clinical Studies:

Clinical performance for the First To Know Syphilis Test was established through a prospective study enrolling individuals of various races, ethnicities, pregnancy status, HIV status, education, and annual income at six geographically diverse clinical sites. The study enrolled sexually active lay users between the ages of 18 and 64. Participants were provided with the First To Know Syphilis Test kit and labeling who completed testing, including set up and sample collection, in a simulated home environment. Site observers were present to assess the ease of use of the test but did not aid the lay users in performing the test. Following testing, venous blood samples were collected from each participant for comparator testing using two separate treponemal tests and one non-treponemal test. The positive comparator result was determined by at least two reactive results. The study consisted of 1424 prospectively enrolled lay users from September 2021 through October 2023. Of the 1424 enrolled subjects, 45 were excluded due to issues such as incomplete paperwork, subject withdrawal, screening failures, and blood draw failures, leaving 1379 evaluable specimens. Of these, 27 were excluded due to invalid comparator results, seven (7) were excluded due to laboratory errors, and 75 were excluded due to subject device invalid results, leaving 1270 First To Know Syphilis Test results with paired valid comparator results. Clinical performance for the First To Know Syphilis Test is summarized in the table below. The invalid rate for the device was 5.6% (75/1345).

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Table 7. Clinical Performance

| | Comparator Algorithm
Positive | Comparator Algorithm
Negative | |
|--------------------------------------------------|----------------------------------|----------------------------------|------|
| First To
Know
Syphilis
Test Positive | 99 | 6 | 105 |
| First To
Know
Syphilis
Test
Negative | 7 | 1158 | 1165 |
| | 106 | 1164 | 1270 |

Positive Percent Agreement (PPA) = 93.4% (99/106) (95% CI: 87.0-96.8%)

Negative Percent Agreement (NPA) = 99.5% (1158/1164) (95% CI: 98.9-99.8%)

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

The PPA with the comparator method for the test is 93.4% (95% CI: 87.0-96.8%)

Clinical Specificity:

Please refer to Section VI.C (Clinical Studies) above for the clinical validation. The NPA with the comparator method is 99.5% (95% CI: 98.9-99.8%)

Staged Syphilis Testing

Characterized serum samples from known positive Syphilis patients clinically diagnosed at various stages of the disease we obtained for testing using the First To Know Syphilis Test. A total of 125 serum samples were tested with the First To Know Syphilis Test. The panel included serum samples from patients with Primary Syphilis (n=25), Secondary Syphilis (n=56), Early Latent Syphilis (n=16), and Late Latent Syphilis (n=28). All 125 serum samples tested demonstrated positive results with the First To Know Syphilis Test.

D Clinical Cut-Off:

Not applicable

E Expected Values/Reference Range:

In the First To Know Syphilis Test clinical study, 1270 specimens were tested during September 2021 - October 2023 and determined to be evaluable. There were 105 positive results by the device corresponding to a positivity of 8.3%. Since the First To Know Test does not constitute a full Syphilis testing algorithm, the actual prevalence is undetermined.

F Other Supportive Performance Characteristics Data:

Usability and User Comprehension

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Test usability was assessed in two studies. For the first. all 1345 participants enrolled in clinical study were observed while performing testing and difficulties were noted. A second study enrolled 20 participants to assess lay users' execution of the First To Know Syphilis test workflow using the written instructions alone. Following both studies, a questionnaire was issued to participants to assess ease of use. The results of both studies demonstrated that the First To Know Syphilis test is easy to use by lay users.

User label comprehension was assessed using two sets of questionnaires. One questionnaire was issued to all 1345 participants following the original clinical study. A second questionnaire was issued to 40 participants in a supplemental label comprehension study (20 participants in the supplemental usability study also participated in the supplemental label comprehension study). Both questionnaires were aimed at evaluating users' understanding of key communication messages (e.g., FAO messages and warnings) found in the labeling. The results of user label comprehension demonstrated that the First To Know Syphilis Test labeling is easy to understand by lay users.

2. Frequently Asked Ouestions

To improve user label comprehension, the labeling includes a Frequently Asked Questions (FAQ) section. The FAQ section was created to provide users information to adequately understand the purpose, limitations, and meaning of the test results as well as where users can access additional information regarding Syphilis pathology and epidemiology. The concepts covered in the FAQ section include:

. The purpose of the test and description of the test and the analyte.
. Who should and who should not use the test (self-selection).
. Meaning of the test results.
When to re-test (e.g., following an invalid result). .
Follow-up for appropriate health management. .
1. FMEA, Residual risks and Mitigations

A comprehensive hazard analysis of the First To Know Syphilis Test was conducted in accordance with ISO 14971:2019. Three separate FMEA analyses were performed, one each for Process (Manufacturing), Design (First To Know Test design), and Use (Human Factors) of the device. The hazard analyses included identification of the potential hazard, likelihood of occurrence, severity of potential harm, hazard control measure(s), and assignment of pre- and post-control risk levels. The elements considered included device design, operator errors (i.e., human factors), and environmental factors.

For risks which reached level 2 or 3, mitigations were put in place to reduce the Risk to acceptable levels. These mitigations were implemented via the following means:

. Instructions for Use including warnings and precautions
Clinical studies were performed to demonstrate safety and effectiveness .
Flex studies based on Use FMEA risks .
1. Flex Studies

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The operational limits of the device were evaluated in a series of studies simulating conditions of use outside of the intended use environment or in instances of user errors. Studies were run by trained operators using a testing panel of contrived samples, unless otherwise noted. Each study also contained a control condition in which tests were run within the specifications indicated by the instructions for use. All test conditions were run with five replicates per condition with the following exceptions: Hand Contaminants - 25 replicates; Atmospheric Pressure - 10 replicates.

The results demonstrate that the test is robust to stresses of environmental conditions and potential user error.

Study Title	Objective	Outcome
Hand
Contaminants	To assess test performance when the
following common hand contaminants were
present at the test site: hand sanitizer, hand
lotion, alcohol, hand soap, and water.	The testing panel included a negative,
low reactive, and moderate reactive.

All results were as expected. |
| Household
contaminants | To assess test performance when the
following common hand contaminant were
present in the sample: (b)(4) hand sanitizer,
(b)(4) (b)(4) (b)(4) Air Freshener,
nail polish, and Alcohol wipes. | The testing panel included a negative,
low reactive, and moderate reactive.

False results were observed in the
presence of (b)(4) and (b)(4) . All
other results were as expected.

Mitigations include instructions to wipe
finger with alcohol wipe before starting
test. |
| Device angle and
orientation
during loading | To assess test performance when sample was
loaded at different angles and orientations. | The testing panel included a high non-
reactive and a low reactive.

All results were as expected. |
| Device angle and
orientation
during run | To assess test performance when the test was
run upside down and at a 90° angle. | The testing panel included a negative
and a low reactive.

All results were as expected. |
| Sample loading
location | To assess test performance when sample was
loaded at the following locations: on the fill
line, at the opposite end, and in the viewing
window. | The testing panel included a high non-
reactive and a low reactive.

Sample loaded in the viewing window
produced two false negative results. All
other results other than the control
condition were invalid.

False results are mitigated by
instructions and graphics showing how
to correctly load sample. |
| Unsealed
Packaging | To assess test performance when the device
has been left out of the packaging under the
following conditions: 15°C and 30% RH or
30°C and 90% RH for up to three days. | The testing panel included a negative,
low reactive, and moderate reactive. |
| | | |
| Sample loading
volume | To assess test performance with sample
loading volumes outside of 40 $ \mu $ L. The
following sample loading volumes were
tested: 15, 20, 25, 30, 35, 50, 60, 70, 80, 90,
and 100 $ \mu $ L. | Devices left at 30°C and 90% RH for 1
day produced 4 false negative results
each for low reactive and moderate
reactive and 1 invalid result for low
reactive. One of the five moderate
reactive was positive at this condition.
All other results were as expected.

False results are mitigated by device
labeling not to use open or damaged
tests. |
| Temperature and
humidity during
testing | To assess test performance while running the
test outside the recommended conditions. The
following conditions were tested: -20°C and
ambient humidity, 4°C and