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.

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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Device Performance for First To Know Syphilis Test

Please note: The document does not explicitly list "acceptance criteria" in a separate section with pass/fail thresholds. Instead, it describes various performance studies and their outcomes. The table below infers acceptance criteria based on the reported performance and the overall conclusion of the De Novo request.

Acceptance Criteria (Inferred)	Reported Device Performance
I. Precision/Reproducibility:
- Consistent results across lots, operators, sites, and days	Study 1 (Lot-to-Lot Precision):
	- Moderate Reactive samples: 100% positive across all lots, operators, and sites.
	- Low Reactive samples: 100% positive (Site 1 & 3), 94.4% (Site 2 Lot 3 Operator 6 had 5/6, making overall 17/18).
	- High Non-Reactive and Non-Reactive samples: 0% positive across all lots, operators, and sites.
	Study 2 (Reproducibility):
	- Moderate Reactive samples: 100% (135/135) overall.
	- Low Reactive samples: 98.5% (133/135) overall.
	- High Non-Reactive and Non-Reactive samples: 0% (0/135) overall.
II. Analytical Specificity/Interference:
- Minimal cross-reactivity	Cross-reactivity:
	- 10% false positive with Epstein-Barr Virus (EBV) antibodies (1/10 samples).
	- 10% false positive with Rheumatoid Factor (1/10 samples).
	- 0% cross-reactivity for all other tested substances (Anti-Lyme, Anti-Gonorrhea, Anti-Chlamydia, Anti-Leptospirosis, Anti-Trichomonas, Anti-Toxoplasma, Anti-Cytomegalovirus, Anti-Hepatitis A, B, C, Anti-nuclear Antibody, Anti-HIV 1&2, Hemodialysis Patient serum, Anti-Herpes Simplex Virus, Human Anti-Mouse Antibody, Heterophile Antibodies, Anti-Human Papillomavirus, Anti-Human T-cell Lymphotropic Virus).
- No significant interference from common substances	Interfering Substances:
	- 3/3 positive samples containing Gamma-globulin (60 mg/mL) resulted in invalid results.
	- All other 22 tested substances (Acetaminophen, Acetylcysteine, Aspirin, Albumin, Ampicillin, Ascorbic acid, Bilirubin, Cefoxitin, Cholesterol, Cyclosporine, Doxycycline, Hemoglobin, Heparin, Ibuprofen, K2EDTA, Levodopa, Methyldopa, Metronidazole, Phenylbutazone, Rifampin, Theophylline, Triglycerides) showed 0 invalid results and 100% expected results (3/3 negative, 3/3 positive).
- No Hook Effect observed	Hook Effect: No observed Hook Effect for high concentrations of anti-Syphilis antibody tested, with all spiked samples 100% positive at dilutions of 1:160 or lesser.
III. Stability:
- Shelf-life within specified temperature range	Unopened kit stability: Stable for up to 18 months when stored within 15°C to 30°C. First failure observed in one lot at 21 months.
- Stability under shipping conditions	Shipping stability: All results were as expected, except one false negative from a low reactive sample at 4-hour time point for 43℃ condition (acceptable as >95% positive results were generated).
IV. Assay Cut-Off Performance:
- Consistent detection of low reactive samples	Accurately and consistently detected low reactive (C95) samples (60/60 positive).
V. Clinical Performance:
- High Positive Percent Agreement (PPA)	PPA = 93.4% (99/106) (95% CI: 87.0-96.8%)
- High Negative Percent Agreement (NPA)	NPA = 99.5% (1158/1164) (95% CI: 98.9-99.8%)
- Detection across various syphilis stages	Staged Syphilis Testing: 100% positive results (125/125) for samples from Primary (n=25), Secondary (n=56), Early Latent (n=16), and Late Latent (n=28) Syphilis patients.
- Low invalid rate for lay users	Invalid rate for the device was 5.6% (75/1345).
VI. Usability and User Comprehension:
- Easy to use by lay users	Both studies (clinical study observation and supplemental usability study) demonstrated that the First To Know Syphilis test is easy to use by lay users.
- Labeling is easy to understand	Both questionnaires (clinical study participants and supplemental label comprehension study) demonstrated that the First To Know Syphilis Test labeling is easy to understand by lay users.
VII. Robustness to User Error/Environmental Conditions:
- Tolerates various conditions and potential errors	Flex Studies demonstrated robustness, with mitigations (labeling instructions) implemented for identified false results (e.g., household contaminants like (b)(4) and (b)(4), sample loaded in viewing window, unsealed packaging, incorrect sample volume, device run at -20°C, incorrect read times, device reuse). Control conditions generally yielded expected results.

Study Details:

For Clinical Performance (Comparison Studies C.1. Method Comparison and C.3. Clinical Studies):

Sample size used for the test set and the data provenance:
- Total enrolled participants: 1424
- Evaluable specimens: 1379 (after exclusions for incomplete paperwork, withdrawal, screening/blood draw failures)
- Final evaluable First To Know Syphilis Test results with paired valid comparator results: 1270 (after further exclusions for invalid comparator results, laboratory errors, and device invalid results)
- Data provenance: Prospective study enrolling individuals at six geographically diverse clinical sites in the U.S., conducted from September 2021 through October 2023.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The ground truth was established by "comparator testing using two separate treponemal tests and one non-treponemal test."
- Experts: The document does not specify the number or qualifications of the individuals who performed or interpreted these comparator tests (e.g., laboratory technicians, pathologists, infectious disease specialists). It refers to a "Comparator Algorithm Positive" where the positive comparator result was determined by at least two reactive results from these tests.
Adjudication method (e.g., 2+1, 3+1, none) for the test set:
- The adjudication method for establishing the ground truth was based on a "comparator algorithm" defined as "at least two reactive results" from the three comparator tests (two treponemal, one non-treponemal). This suggests an agreement-based method, effectively a "2 out of 3" rule.
If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, an MRMC comparative effectiveness study was not done. This device is a visually-read, lateral-flow immunoassay intended for over-the-counter (OTC) consumer use by lay persons, without AI assistance. The performance reported is for the device as a standalone OTC test.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- Yes, the primary clinical performance (PPA and NPA) reflects standalone performance in the hands of lay users. While site observers were present to assess ease of use, they "did not aid the lay users in performing the test." Therefore, the reported PPA and NPA represent the device's performance as interpreted by the intended end-user without professional human clinical interpretation or assistance beyond the device's labeling.
The type of ground truth used (expert consensus, pathology, outcomes data, etc):
- The ground truth was established through reference laboratory testing (a "comparator algorithm") using a combination of two treponemal tests and one non-treponemal test, with a positive result requiring at least two reactive outcomes. This is a common and accepted method for serological diagnoses like syphilis. For the "Staged Syphilis Testing," characterized serum samples from known positive Syphilis patients clinically diagnosed at various stages were used, implying a clinical diagnosis as part of that specific ground truth.
The sample size for the training set:
- The document describes optimization of the assay cut-off during development but does not specify a distinct "training set" size in the context of machine learning or AI. For assay optimization to establish the cut-off, it states: "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." This refers to specific concentrations (C5, C95) for assay development rather than a broad training set for an algorithm.
How the ground truth for the training set was established:
- For the assay cut-off optimization, "known positive samples prepared from serum specimens obtained from clinically-positive individuals" were used. These samples were diluted 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." This indicates that an existing, FDA-cleared laboratory assay served as the ground truth reference for defining the concentrations used in optimizing the First To Know Syphilis Test's cut-off.

Summary

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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

ProductCode(s)	Classification	RegulationSection	Panel
SBZ	Class II	21 CFR 866.3986 - Testfor detection of antibodiesassociated with syphilisperformed 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:

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.

C Special Conditions for Use Statement(s):

OTC - Over The Counter

D Special Instrument Requirements:

Not applicable.

IV Device/Svstem Characteristics:

A Device Description:

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		ModerateReactive	LowReactive	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
		ModerateReactive	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	ModerateReactive	LowReactive	High 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	# ofPositives / #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 LymphotropicVirus	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

PotentialInterferent	ConcentrationTested	# Invalid Results/ Negative +Positive SamplesTested	# of NegativeResults / # ofNegativeSamples Tested	# of PositiveResults / # ofPositiveSamplesTested
Acetaminophen	0.2 mg/mL	0/6	3/3	3/3
Acetylcysteine	1.66 mg/mL	0/6	3/3	3/3
Aspirin	0.65 mg/mL	0/6	3/3	3/3
Albumin	50 mg/mL	0/6	3/3	3/3
Ampicillin	0.2 mg/mL	0/6	3/3	3/3
Ascorbic acid	0.2 mg/mL	0/6	3/3	3/3
Bilirubin	2 mg/mL	0/6	3/3	3/3
Cefoxitin	0.66 mg/mL	0/6	3/3	3/3
Cholesterol	2.5 mg/mL	0/6	3/3	3/3
Cyclosporine	1.4 mg/mL	0/6	3/3	3/3
Doxycycline	0.03 mg/mL	0/6	3/3	3/3
Gamma-globulin	60 mg/mL	3/6	3/3	0/3
Hemoglobin	20 mg/mL	0/6	3/3	3/3
Heparin	3000 U/L	0/6	3/3	3/3
Ibuprofen	0.5 mg/mL	0/6	3/3	3/3
K2EDTA	0.8 mg/mL	0/6	3/3	3/3
Levodopa	6.5 mg/mL	0/6	3/3	3/3
Methyldopa	0.015 mg/mL	0/6	3/3	3/3
Metronidazole	0.12 mg/mL	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 AlgorithmPositive	Comparator AlgorithmNegative
First ToKnowSyphilisTest Positive	99	6	105
First ToKnowSyphilisTestNegative	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
HandContaminants	To assess test performance when thefollowing common hand contaminants werepresent at the test site: hand sanitizer, handlotion, alcohol, hand soap, and water.	The testing panel included a negative,low reactive, and moderate reactive.All results were as expected.
Householdcontaminants	To assess test performance when thefollowing common hand contaminant werepresent 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 thepresence of (b)(4) and (b)(4) . Allother results were as expected.Mitigations include instructions to wipefinger with alcohol wipe before startingtest.
Device angle andorientationduring loading	To assess test performance when sample wasloaded at different angles and orientations.	The testing panel included a high non-reactive and a low reactive.All results were as expected.
Device angle andorientationduring run	To assess test performance when the test wasrun upside down and at a 90° angle.	The testing panel included a negativeand a low reactive.All results were as expected.
Sample loadinglocation	To assess test performance when sample wasloaded at the following locations: on the fillline, at the opposite end, and in the viewingwindow.	The testing panel included a high non-reactive and a low reactive.Sample loaded in the viewing windowproduced two false negative results. Allother results other than the controlcondition were invalid.False results are mitigated byinstructions and graphics showing howto correctly load sample.
UnsealedPackaging	To assess test performance when the devicehas been left out of the packaging under thefollowing conditions: 15°C and 30% RH or30°C and 90% RH for up to three days.	The testing panel included a negative,low reactive, and moderate reactive.

Sample loadingvolume	To assess test performance with sampleloading volumes outside of 40 $ \mu $ L. Thefollowing sample loading volumes weretested: 15, 20, 25, 30, 35, 50, 60, 70, 80, 90,and 100 $ \mu $ L.	Devices left at 30°C and 90% RH for 1day produced 4 false negative resultseach for low reactive and moderatereactive and 1 invalid result for lowreactive. One of the five moderatereactive was positive at this condition.All other results were as expected.False results are mitigated by devicelabeling not to use open or damagedtests.
Temperature andhumidity duringtesting	To assess test performance while running thetest outside the recommended conditions. Thefollowing conditions were tested: -20°C andambient humidity, 4°C and <10% RH, 4°Cand 95% RH, 40°C and <10% RH, 40°C and95% RH	The testing panel included a negative,low reactive, and moderate reactive.Devices loaded with 15 $ \mu $ L and 20 $ \mu $ Lproduced invalid results, demonstratingthat the internal control functions asexpected. All other results were asexpected.
		The testing panel included a negative,low reactive, and moderate reactive.Devices run at -20°C produced 5 falsenegative results for low reactive and 3false negative results for moderatereactive samples. All other results wereas expected.False results are mitigated byinstructions to run at room temperature.
Run time	To assess test performance when the resultsare read outside the specified window. Thefollowing read times were tested: 5 min, 10min, 1 hour, 2 hour, 4 hours, and 24 hours.	The testing panel included a negative,low reactive, and moderate reactive.Tests read at 5 minutes producedinvalid results. All other results were asexpected.
Sample LoadingTime	Since it is possible for blood to coagulate inthe loading zone over time, sample wascontinually loaded for up to 3 minutes.	Actual finger stick blood was used.All tests showed continuous capillaryaction for up to 3 minutes and producedvalid results.
Tapping times	To assess test performance when the samplehas been loaded and the device is tapped asfollows: No tapping; tapping one, two, andfive times; tapping twice in the long edge;tapping twice on the loading zone	The testing panel included a lowpositive.All results were as expected.
TappingLocations	To assess test performance when the samplehas been loaded and the device is tapped as	The testing panel included a lowreactive.
	follows: wrist tap (fill zone up); wrist tap (fillzone down); Elbow tap (fill zone up); elbowtap (fill zone down).	All results were as expected.
Device Reuse	To assess test performance after a negativesample was run, allowed to dry for at least 24hours, and then used with a second moderatepositive sample.	After the first loading, all 10 testsproduced valid negative results. Afterthe second loading, all tests remainednegative.
		False results are mitigated by devicelabeling not to reuse.
Dropping	To assess test performance when the test hasbeen dropped before and after sample loading.	The testing panel included a negativeand a low reactive.
Atmosphericpressure	To assess test performance at low pressure(0.5 atm).	The testing panel included a negativeand a low reactive.
Bubbleformation	To assess test performance when bubblesform in the loading zone. Both large andsmall bubbles were tested.	The testing panel included a negative.low reactive, and moderate reactive.
LightingConditions	To assess the effect of the following lightingconditions on results interpretation:incandescent (2700K), fluorescent (3000K),LED (4000K), daylight (5000K)	The testing panel included a negativeand a low reactive.

Table 8. Flex Studies

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Proposed Labeling: VII

The labeling supports the decision to grant the De Novo request for this device.

Identified Risks and Mitigations: VIII

Risks to Health	Mitigation Measures
Risk of false results	Certain labeling information includinglimitations, device descriptions, performanceinformation, and explanations of procedures.Certain design verification and validationincluding certain analytical and clinicalstudies, and risk analysis strategies.
Failure to correctly interpret test results	Certain labeling information includinglimitations, device descriptions, performanceinformation, and explanations of procedures.Certain design verification and validationincluding certain analytical and clinicalstudies, and risk analysis strategies.
Failure to correctly operate the device	Certain labeling information includinglimitations, device descriptions, performanceinformation, and explanations of procedures.

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Risks to Health	Mitigation Measures
	Certain design verification and validationincluding certain analytical and clinicalstudies, and risk analysis strategies.

IX Benefit/Risk Assessment:

A Summary of the Assessment of Benefit: (Final Assessment)

As an OTC test, the probable clinical benefit of this test will be to reach individuals who otherwise would not engage with the healthcare system and therefore increase access to syphilis diagnostic testing. This is critical in light of the recent rise in syphilis infection rates. Specifically, this test may be used by individuals unwilling or unable to go to a healthcare provider to ascertain if they are at risk for syphilis infection, and for the subsequent positive results may prompt these individuals to then seek the appropriate follow-up care. One additional benefit of the device may be that persons unsure if they have ever been exposed to syphilis in their lifetime may find that they have a positive T. pallidum antibody and choose to seek care for potential late latent syphilis - which may prevent progression of disease.

B Summary of the Assessment of Risk:

The risks associated with this device, when used as intended, are those related to the risk of false test results, failure to correctly interpret the test results, and failure to correctly operate the device. False-negative results can cause delays in effective treatment and delay entry into medical care, resulting in ongoing syphilis transmission by individuals unaware of their true syphilis status. False positive results may lead to incorrect diagnosis and unnecessary treatment for syphilis, and delayed diagnosis and treatment of other infections. There is also risk that a lay user may not understand the clinical significance of a positive or negative test result.

C Summary of the Assessment of Benefit-Risk:

The risks associated with the device are mitigated by the special controls that address verification and validation requirements and risk analysis strategies, which help to lower the risk of false test results. In addition, labeling that includes step-by-step instructions and limiting statements and clearly describes subpopulations for whom this test may not be appropriate will help to lower the risks of failure to correctly interpret the test results or operate the test correctly.

In conclusion, while general controls are not sufficient to ensure safety and effectiveness, in light of the special controls, the clinical benefits outweigh the probable risks for the First To Know Syphilis Test.

X Conclusion:

The De Novo request is granted and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:

Product Code(s): SBZ

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Device Type: Test for detection of antibodies associated with syphilis performed by lay users Class: Class II Regulation: 21 CFR 866.3986

Regulation Number and Section

N/A