The Zeus Scientific, Inc AtheNA Multi-Lyte Borrelia VISE-1/ pepC10 Plus Test System is a multiplexed sandwich immunoassay for the qualitative detection of IgG class antibody to recombinant VISE-1 and the IgM class of antibody to synthetic pepC10 in human serum. The AtheNA Multi-lyte Borrelia VISE-1/pepC10 Plus Test System is intended for use with the Luminex® 200 IS and the AtheNA Multi-Lyte data management package in testing serum samples from symptomatic patients or those with a history of Lyme Borreliosis. All positive specimens should be tested with a second-tier test such as Western Blot which if positive, is supportive evidence of infection with B.burgdorferi. Diagnosis of Borreliosis should be made based on the presence of 8.burgdorferi antibodies, history, symptoms and other laboratory data. Negative first or second tier results should not be used to exclude Borreliosis.

Device Description

The AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System is a micro particle immunoassay intended for the qualitative detection of distinct IgG class antibody to VlsE-1 and distinct IgM antibody to pepC10. The assay is a multiplexed immunoassay designed to simultaneously detect, distinguish and identify IgG reactivity to recombinant VISE-1 antigen and IgM reactivity to synthetic pepC10 antigen. The test system is comprised of the AtheNA Multi-Lyte test kit and the Luminex Corp instrument model number Luminex™ 200 IS and software version 3.

AI/ML Overview

Here's an analysis of the acceptance criteria and the studies performed for the Zeus Scientific Inc. AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System, based on the provided text:

Important Note: The provided document is a 510(k) summary, which focuses on demonstrating substantial equivalence to a predicate device. It does not explicitly state "acceptance criteria" in the format of a predefined performance target, but rather presents the results of various validation studies, which imply what would be considered acceptable performance for a diagnostic device in its class. For the purpose of this analysis, I will infer relevant performance metrics from the study results.

1. Table of Acceptance Criteria and Reported Device Performance

Performance Metric Category	Implied Acceptance Criteria (Inferred from context and typical IVD requirements)	Reported Device Performance (AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System)	Relevant Section(s)
Linearity (R-squared)	R-squared value ≥ 0.90	Demonstrated dilutions recovered within acceptance criteria (implies R-squared ≥ 0.90)	7A: Linearity
Analytical Specificity (Interfering Substances - Signal Change)	Signal change < 20% in the presence of interfering substances (for most cases)	- VlsE-1: Most samples showed <20% change. Borderline VlsE-1 showed 32% (high bilirubin) and 27% (high cholesterol) increase. Negative VlsE-1 showed 36% reduction (high hemoglobin), 23% (low bilirubin), 27% (high bilirubin), 25% (low cholesterol), 45% (high cholesterol), 45% (low and high triglycerides). - pepC10: Most samples showed <20% change. Borderline pepC10 showed 24% reduction (high hemoglobin) and 28% increase (low triglyceride). Crucially, these changes in negative samples did not change the qualitative outcome (remained negative), suggesting acceptable specificity despite some signal shifts.	7B: Analytical Specificity-Interfering Substances
Cross-Reactivity	0% cross-reactivity with common interfering antibodies/conditions	0/90 samples showed cross-reactivity with EBV VCA IgG, ANA, Syphilis, CMV IgG, CMV IgM, Rubella IgG, Toxo IgG, VZV IgM, RF.	7C: Cross Reactivity
Performance with Characterized Acute Samples (Agreement with Clinical Diagnosis)	High agreement (e.g., >85%)	100% (21/21) (95% CI: 86.7%-100%)	Table 1 (Study 1)
Performance with Characterized Convalescent Samples (Agreement with Clinical Diagnosis)	High agreement (e.g., >85%)	94% (47/50) (95% CI: 83.5%-98.8%)	Table 1 (Study 1)
Performance with Culture Positive Early Acute Samples (Agreement with Clinical Diagnosis)	Moderate agreement given early stage (no explicit target, but a clear improvement over random)	51.9% (41/79) (95% CI: 40.4%-63.3%)	Table 1 (Study 1)
Performance with Early Convalescent Samples (Agreement with Clinical Diagnosis)	High agreement (e.g., >70%)	78.5% (62/79) (95% CI: 67.8%-86.9%)	Table 1 (Study 1)
Overall Performance with Characterized Samples (Agreement with Clinical Diagnosis)	High overall agreement	74.7% (171/229) (95% CI: 68.5%-80.2%)	Table 1 (Study 1)
Prospective Study (Positive Percent Agreement - PPA)	High PPA compared to predicate ELISA (no explicit target, but expectation is substantial equivalence)	81.4% (162/199) (95% CI: 75.3-86.6)	Table 2 (Study 2)
Prospective Study (Negative Percent Agreement - NPA)	High NPA compared to predicate ELISA (no explicit target)	91.4% (509/557) (95% CI: 88.7-93.6)	Table 2 (Study 2)
Retrospective Study (PPA)	High PPA compared to predicate ELISA	80% (180/225) (95% CI: 74.2-85.0)	Table 3 (Study 3)
Reproducibility (Total %CV)	Acceptable coefficient of variation (typically <20%, lower for higher concentrations)	VlsE-1: 13.5% (Near Cut-off) to 5.3% (High Positive). pepC10: 11.1% (Negative) to 6.2% (High Positive).	Table 6 (Section 9A)
Repeatability (Total %CV)	Acceptable coefficient of variation (typically ≤10-15%)	VlsE-1: 11.4% (High Negative) to 6.9% (High Positive). Note: VIsE-1 Negative 1 is 29.7%, which is high, but these values represent iAU performance, and as noted in Analytical Specificity, large iAU shifts for negative samples did not change qualitative outcome. pepC10: 13.1% (Negative 1) to 5.4% (High Positive).	Table 7 (Section 9A)
Analytical Specificity (Non-endemic population positivity)	Low positivity rate in a non-endemic population	9.8% (39/400)	Table 5 (Study 5)

2. Sample Sizes Used for the Test Set and Data Provenance

The document describes multiple studies with different test sets:

Characterized Samples (Study 1): 229 human serum samples.
- Provenance: Northeastern state Department of Health Laboratory. Implies a mix of acute, convalescent, and culture-proven Lyme disease patients, likely collected prospectively or retrospectively for diagnostic purposes.
Prospective Population (Study 2): 756 unselected samples from patients with an order for a Lyme antibody test.
- Provenance: Samples collected from four sites in the US: a hospital laboratory in the Mid-Atlantic (103 samples), a hospital laboratory in upper Connecticut (100 samples), a hospital laboratory in lower Connecticut (107 samples), and a state Department of Health Lab in the northeast (446 samples). All samples were submitted for Lyme antibody testing and were sequentially numbered, de-identified and archived.
Retrospective Samples (Study 3): 242 samples (124 from Connecticut, 118 from Pennsylvania).
- Provenance: Believed to have screened positive for Borrelia burgdorferi antibodies, tested at a hospital facility in Connecticut and a Pennsylvania hospital laboratory.
CDC Characterized Lyme Panel (Study 4): 40 samples (5 normal blood donors, 35 Borreliosis patients).
- Provenance: Acquired from the CDC.
Analytical Specificity / Endemic & Non-Endemic Controls (Study 5): 700 samples (300 endemic from New England, 400 non-endemic from New Mexico).
- Provenance: Blood donors from New England (endemic) and blood donors/individuals undergoing routine non-infectious testing from New Mexico (non-endemic).
Reproducibility: 5 internal samples (levels: negative, near cut-off, low positive, moderate and high positive).*
Repeatability: 6 internal samples (levels: negative, high negative, near cut-off, low positive, moderate and high positive).*

*For reproducibility and repeatability, reference samples are typically prepared internally or sourced commercially with known characteristics, not representative of general patient populations for diagnostic accuracy evaluation.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

The document does not explicitly state the "number of experts" or their specific "qualifications" for establishing ground truth. However, implicitly:

Clinical Diagnosis: For the "Characterized Samples" (Study 1) and "CDC Characterized Lyme Panel" (Study 4): The ground truth is based on "clinical diagnosis," "history of Borreliosis," "culture proven, early acute Lyme disease," or "patients diagnosed with Borreliosis." This implies a consensus among medical professionals (physicians, infectious disease specialists) using clinical criteria, possibly supported by existing laboratory results.
Western Blot: For discrepant results in prospective and retrospective studies, Western Blot methodology was used to confirm reactivity. Western Blot interpretation is a specialized skill typically performed and verified by laboratory professionals experienced in serology.
Predicate Device: For comparative studies, the predicate ELISA test system was used as a reference assay. The "ground truth" here is the result of the predicate device, which itself would have been FDA cleared based on its own validation by medical laboratory experts.

4. Adjudication Method for the Test Set

The document describes adjudication for discrepancies:

Prospective Samples (Study 2): "If available, western blot testing was performed on the discrepant results."
Retrospective Samples (Study 3): "Western blot testing was done on discrepant results."

This indicates a form of discrepancy resolution using a higher-tier test (Western Blot), which acts as a secondary adjudication method. The document does not specify if multiple readers were involved in the Western Blot interpretation or what consensus rule was applied (e.g., 2+1).

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not explicitly conducted or described in the provided text for evaluating human reader performance with and without AI assistance. This device is an in vitro diagnostic test system (an assay), not an AI-powered image analysis tool that assists human interpretation of images. The comparison is between the new assay system and a predicate ELISA assay, not human readers.

6. Standalone Performance

Yes, standalone performance was done. The entire document describes the performance of the "AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System" as a standalone algorithm (a fully automated assay, without human-in-the-loop performance in terms of interpretative decision-making). The results presented are directly from the device's output. Clinical studies compare its performance against clinical diagnoses, recognized reference standards (Western Blot), and a predicate device.

7. Type of Ground Truth Used

The ground truth for the various studies includes:

Clinical Diagnosis: For characterized samples (e.g., acute, convalescent patients with history of Borreliosis, culture-proven early acute Lyme disease, patients diagnosed with Borreliosis).
Predicate ELISA: For comparison studies (Prospective, Retrospective) where samples were categorized as "reference assay positive" or "reference assay negative" based on the predicate ELISA.
Western Blot: Used as a confirmatory test for discrepant results between the investigational device and the predicate device. For the "Characterized Samples" table, Western Blot results are presented as another comparator for agreement with clinical diagnosis.
CDC Characterized Lyme Panel: Samples from the CDC with established reactivity profiles.
Normal Blood Donors/Non-infectious Individuals: Used to assess specificity/low prevalence.

8. Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning or AI algorithm development. This is an immunoassay, not typically developed using machine learning with distinct training and test sets in the same way an AI diagnostic algorithm would be. The studies described are validation studies for the device's performance, not for training stages.

9. How the Ground Truth for the Training Set Was Established

Since no explicit "training set" for an AI algorithm is mentioned (as the device is an immunoassay), the method for establishing ground truth for a training set is not applicable here. The focus is on the validation of the assay's performance using established methods of clinical and analytical evaluation.

Summary

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Zeus Scientific Inc. 510(k) AtheNA Multi-Lyte Borrelia VlsE-1/ pepC10 Plus Test System Summary of Safety and Effectiveness

Section I: Administrative Information

Submission Purpose: Intent to market referenced device for the qualitative detection of VlsE-1 1. and pepC10 antibodies.
Measurand: VIsE-1 IgG and pepC10 IgM antibodies. 2.
Type of Test: Sandwich Immunoassay. 3.
Applicant: Zeus Scientific, Inc. , PO Box 38, Raritan, NJ 08869 (908)526-3744 4.
Establishment Registration Number: 2242426 5.
Contact: Ewa Nadolczak, Manager of Clinical Affairs (347)731-0402 6. enadolczak@zeusscientific.com
Proprietary Name: AtheNA Multi-Lyte® Borrelia VlsE-1/ pepC10 Plus Test System. 7.
Established name: Borrelia serological reagents. 8.

Section 2: Regulatory Information

Device Classification: Borrelia Serological Reagent 1.
1. Class: Class 2
Product Code: LSR 3.
1. Panel Microbiology
1. Form 3454: Appendix A
1. Device Hazard Analysis: Appendix B

Section 3: Intended Use

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Section 3A: Special Conditions for Use

The AtheNA Multi-Lyte Borrelia VlsE-1/ pepC10 Plus Test System is for in vitro diagnostic use only.

The AtheNA Multi-Lyte Borrelia VlsE-1/ pepC10 Plus Test System is for prescription use only.

Section 4: Device Description

The AtheNA Multi-Lyte Borrelia VIsE-1/pepC10 Plus test system provides the following Components:

Reactive Reagents:

1. All reactive reagents contain sodium azide as a preservative at a concentration of 0.1% (w/v).
1. Multiplexed bead suspension 1. Ready to use, 5.5 mL bottle. The suspension contains separate distinguishable 5.6 micron polystyrene beads that are conjugated with recombinant VlsE1 antigen. The bead mix also contains one bead set designed to detect non-specific antibodies in the patient sample (if present) and four separate bead sets used for assay calibration.
1. Multiplexed bead suspension 2. Ready to use, 5.5 mL bottle. The suspension contains separate distinguishable 5.6 micron polystyrene beads that are conjugated with the following antigens: recombinant VlsE-1 and synthetic pepC10.
1. Conjugate 1: Phycoerythrin conjugated goat anti-human IgG (y chain specific). Ready to use, 15 mL amber bottle.
1. Conjugate 2: Phycoervthrin conjugated goat anti-human IgM (μ chain specific). Ready to use, 15 mL amber bottle.
1. Human positive serum controls. Two, 0.2 mL vials.
1. Human negative serum control. One, 0.2 mL vial.
1. SAVe Diluent®. One 50 mL bottle containing phosphate-buffered-saline. Ready to use. NOTE: the sample diluent will change color in the presence of serum.
1. Wash Buffer Concentrate: dilute 1 part concentrate + 9 parts deionized or distilled water. One bottle containing 10 X concentrate of phosphate buffered saline.

Non-Reactive Reagents:

1. One, dilution plate
1. One, 96-well filtration plate for rinsing the microspheres
1. Data Labels: One label is adhered to the inside lid of the kit box and a second label is inside the kit box.
1. Package Insert providing instructions for use
1. Calibration CD: a compact disc that includes all lot-specific kit calibration values required for specimen analysis and assay quality control

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Instrument and Software

This assay is platform dependant and used in conjunction with the Luminex 100 IS and the AtheNA Multi-Lyte® Test System Data Analysis Package Version 3.0.

Materials required but not provided:

1. AtheNA Multi-Lyte® System (Luminex® instrument)
1. Pipettes capable of accurately delivering 10 to 200 µL
1. Multichannel pipette capable of accurately delivering (10 to 200 µL)
1. Reagent reservoirs for multichannel pipettes
1. Disposable pipette tips
1. Laboratory timer to monitor incubation steps
1. Small bath sonicator
1. Plate shaker capable of shaking at 800 RPM (optional for mixing)
1. Vacuum aspirator and vacuum manifold for washing the microspheres

Section 5: Substantial Equivalence

Examination of enclosed data indicates that the Zeus Scientific, Inc AtheNA Multi-Lyte Borrelia VISE-1/pepC1- Plus Test System for the detection of IgG class antibody to VisE-1 and IgM antibody to pepC10 is substantially equivalent to a commercially marketed test system which has been previously cleared by the FDA for in vitro diagnostic use.

1. Name of Predicate Device: Zeus Scientific Borrelia burgdorferi ELISA Test System
1. Manufacturer of Predicate Device: Zeus Scientific, Inc.
Methodology of Predicate: enzyme-linked immunosorbent assay 3.

Section 5A: Interpretation of Results

Investigational Device: AtheNA Score: raw fluorescence is converted into intermediate AtheNA Unit values (iAU/mL). These values correspond to the amount of human IgM antibody bound to the VISE-1 and/or pepC10 antigen coated beads respectively. iAU/mL values are converted to a single outcome (AtheNA Score).

Investigational Device: AtheNA Scores Negative: <1.0 Positive ≥1.0

Predicate Device: Index Value Negative: ≤ 0.90 Equivocal: 0.91 to 1.09 Positive: ≥ 1.10

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The comparison of the AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus to the predicate device follows, including intended use and various aspects of the procedure.

Comparison of AtheNA Multi-Lyte VlsE-1 PepC10 Plus Test System versus Predicate ELISA

Characteristic	AtheNA Multi-Lyte Borrelia VIsE-1/pepC10 Plus	Predicate ELISA
Use	For in vitro diagnostic use onlyThe Zeus Scientific, Inc AtheNA Multi-Lyte Borrelia VIsE-1/ pepC10 Plus Test System is a multiplexed sandwich immunoassay for the qualitative detection of IgG class antibody to recombinant VIsE-1 and the IgM class of antibody to synthetic pepC10 in human serum. The AtheNA Multi-lyte Borrelia VIsE-1/pepC10 Plus Test System is intended for use in testing serum samples from symptomatic patients or those with a history of Borreliosis.	For in vitro diagnostic use onlyqualitative detection of human (IgG and IgM) antibodies to individual proteins of Borrelia burgdorferi in human serum. This test system should only be used with patients with signs and symptoms that are consistent with Lyme disease. Equivocal or positive results must be supplemented by testing with a standardized Western Blot procedure. Positive supplemental results are supportive evidence of exposure to B. burgdorferi and can be used to support a clinical diagnosis of Lyme disease.
Intended Use
Assay	Immunoassay	Immunoassay
Detection Method	Fluorescent	Colorimetric
Solid Phase	Polystyrene micro particle	Polystyrene micro wells
Antigen Used	Recombinant VIsE-1 antigen and synthetic pepC10 antigen	Inactive Borrelia burgdorferi (B31 Strain)
Specimen Tested	Human Serum	Human Serum
Controls	Two PC and one NC	One PC and one NC
Calibration	Includes Intra-Well Calibration® that provides a separate calibration curve for every sampleHuman IgG and IgM	One calibrator
Analyte Measured		Human IgG and IgM
Sample Dilution	1:21 in SAVe Diluent	1:21 in SAVe Diluent
Sample Incubation	30 +/- 10 minutes at room temperature	25 +/- 5 minutes at room temperature
Post Sample Wash	3x wash (vacuum filtration)	5X wash (manual or automated)
Conjugate	Goat anti-human IgG; y chain specific Goat anti-human IgM; u chain specific	Goat anti-human IgM/IgG
Conjugate Label	Phycoerythrin	horseradish peroxidase
Conjugate Incubation	30 +/- 10 minutes at room temperature	25 +/- 5 minutes at room temperature
Post Conjugate Wash	N/A	5X wash (manual or automated)
Substrate	N/A	TMB
Reading	Read the fluorescence on the beads	Read the optical density against the blank
Data Points	Read a minimum of 50 beads (events) for each bead in the bead mix.	Read one OD value for each control and sample
Math	Multi-point curve, regression analysis	Single point regression
Scale	Intra-Well Calibration determines a unit value for each sample from the regression curve. Unit values for each analyte are converted to a single AtheNA Score.Negative is < 1.0 AtheNA Score, Positive is ≥ 1.0AtheNA Score	Calculate the index value of unknown samples by comparing their OD to the cut-off ODNegative is ≤ 0.90, equivocal is 0.91 to 1.09 and positive is ≥1.1
Interpretation Criteria

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Section 6: Test Principle

The Zeus Scientific, Inc. AtheNA Multi-Lyte Borrelia VlsE1/pepC10 Plus Test System is detect IgG class antibodies in human sera to VISE1 antigen and IgM class antibodies to pepC10 antigen. The test procedure involves four incubation steps:

1. Test sera (properly diluted) are incubated in a filter plate well containing a multiplexed mixture of Bead Suspension-1. The multiplexed Bead Suspension-1 contains a mixture of distinguishable sets of polystyrene microspheres; one of these bead sets is conjugated with the VlsE1 antigen. The bead mix also contains one bead set designed to detect non-specific binding and four separate bead sets used for assay calibration. If present in patient sera, specific antibodies will bind to the immobilized antigen on one or more of the bead sets. The microspheres are rinsed to remove non-reactive serum proteins.
1. Conjugate-1 is added to the micro titer well and the plate is incubated. The conjugate will react with IgG antibody immobilized on the solid phase in step 1. The microspheres are rinsed to remove unbound conjugate.
Bead Suspension-2 is added to the wells. The bead set contains beads conjugated with pepC10 3. and VIsE1 antigens. A second aliquot of test sera at the same dilution as in step 1 is added to the well and mixed. The bead and specimen suspension is incubated. Following incubation, the microspheres are rinsed to remove the non-reacting serum proteins.
ধে Conjugate 2 is added to the micro titer well and the plate is incubated. The conjugate will react with IgM antibody immobilized on the solid phase in step 1 and step 3.
1. The entire bead suspension is then analyzed by the AtheNA Multi-Lyte instrument. The bead set(s) are sorted (identified) and the amount of reporter molecule (PE conjugate) is determined for each bead set. Using the Intra-Well Calibration Technology™ (see following page for an explanation of this technology), internal calibration bead sets are used to convert raw fluorescence into intermediate AtheNA Unit values (iAU/mL). These values correspond to the amount of human IgG and/or human IgM antibody bound to the VISE-1 and/or pepC10 antigen coated beads respectively. iAU/mL values are converted to a single outcome (AtheNA Score).

Section 7: Analytical Performance

7 A: Linearity

A strong positive sample was determined using a FDA cleared test system to determine the sample reactivity. A fresh, clean, dead negative sample was used as the diluent to prepare serial dilutions of the positive sample. Each dilution was tested in duplicate, the mean calculated and the result plotted. The linearity is acceptable if the R-squared value obtained through regression analysis is ≥0.90. The results of this study demonstrated that the dilutions recovered were within the acceptance criteria.

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Section 7B: Analytical Specificity-Interfering Substances

The effect of potential interfering substances on sample results generated using the AtheNA Multi-Lyte Borrelia VIsE-1/pepC10 Plus test system was evaluated with the following possible interfering substances at two different concentrations: bilirubin, albumin, cholesterol, triglycerides, hemoglobin and intralipid. The quantity of analyte in each interfering substance is as follows:

1. Bilirubin: 1mg/dL (low), 15 mg/dL (high)
1. Albumin: 3.5 g/dL (low), 5 g/dL (high)
Cholesterol: 150 mg/dL (low), 250 mg/dL (high) ന്
1. Triglycerides: 150 mg/dL (low), 500 mg/dL (high)
Hemoglobin: 20 g/dL (low), 20 g/dL (high) 5.
1. Intralipid: 300 mg/dL (low), 750 mg/dL (high)

Three samples each for VISE-1 and pepC10 were chosen based on their performance on the AtheNA Multi-Lyte Borrelia VISE-1/pepC10 Plus test system: (strongly reactive and negative and negative). The samples were exposed to the possible interfering substance, tested in duplicate and the mean iAU/mL was determined.

All samples showed less than a 20% change in signal in the Vise-1 study with the exception of the borderline VISE-1 sample which exhibited an increase in signal of 32% with the high spike of bilirubin and an increase in signal of 27% with the high spike of cholesterol. The negative VISE-1 sample showed a reduction in signal of 36% with the high spike of hemoglobin, a change in signal of 23% with the low spike of bilirusin and 27% with the high spike of bilirubin, a change in signal of 25% with the low spike of cholesterol and 45% with the high spike of cholesterol and a change in signal of 45% with both the low and high spikes of triglycerides. The change of signal in these negative samples did not change the qualitative outcome, the results remained negative.

All samples showed less than a 20% change in signal in the exception of the exception of the borderline pepC10 sample which exhibited a reduction in signal of 24% with the high spike of hemoglobin and an increase in signal of 28% with the low spike of triglyceride.

Section 7C: Cross Reactivity

A study was conducted at Zeus Scientific to assess cross reactivity with the Athena Multi-Lyte Borrelia VISE-1/pepC10 Plus test system using sera that were sero-positive to EBV VCA IgG, RF, ANA, Syphilis, CMV IgG, CMV IgM, Rubella, VZV IgM and Toxoplasma. ELISA, IFA and micro-particle immunoassay test systems manufactured by various companies for commercial distribution were used to determine the

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sero-positivity of the samples for each possible cross-reactant were tested. None of the ninety samples showed cross-reactivity with any of the nine analytes tested.

AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Cross Reactivity Study
PossibleCross-Reactants	Positive Results/Number Tested
EBV VCA IgG	0 / 10
ANA	0 / 10
Syphilis	0 / 10
CMV IgG	0 / 10
CMV IgM	0 / 10
Rubella IgG	0 / 10
Toxo IgG	0 / 10
VZV IgM	0 / 10
RF	0 / 10

Section 8: Comparative Data

A total of 410 prospective samples were tested at four sites for the presence of VISE-1 and pepC10 using the AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus test system and predicate ELISA test system commercially marketed for the detection of Borrelia burgdorferi IgG and IgM antibodies namely the Zeus Scientific Borrelia burgdorferi ELISA Test System. These samples were submitted for B. burgdorferi antibody testing, sequentially numbered and archived. All positive samples were furthered tested using western blot methodology to confirm reactivity. 342 retrospective samples, acquired from various sources, were tested at three sites. Additional testing consisted of 100 low prevalence population samples, 300 endemic and 300 non-endemic control samples, 246 random samples submitted for Borrelia burgdorferi antibodies, 229 characterized serum samples and a panel of 40 characterized samples acquired from the CDC.

Site ofTesting	NumberofSamples	Sample Status
1	124	retrospective	previously screened positive by laboratory
1	107	prospective	submitted for B.burgdorferi antibody testing
2	118	retrospective	previously screened positive by laboratory
2	103	prospective	submitted for B.burgdorferi antibody testing
3	446	prospective	submitted for B.burgdorferi antibody testing in 2006
4	100	prospective	submitted for B.burgdorferi antibody testing
4	100	non-endemic controls	samples collected in non-endemic area for testing non-infectious in nature
3	150	endemic controls	samples from blood donors collected in endemic area (New England)
3	150	non-endemic controls	samples from blood donors collected in non-endemic area (New Mexico)
4	150	endemic controls	samples from blood donors collected in endemic area (New England)
4	150	non-endemic controls	samples from blood donors collected in non-endemic area (New Mexico)
3	21	characterized samples	samples from patients with clinical history of Borreliosis (acute)
3	50	characterized samples	samples from patients with clinical history of Borreliosis (convalescent)
3	158	characterized samples	78 paired samples from patients with clinical history of Borreliosis (acute & convalescent)

Summary of Specimens Included in Study:

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Section 8A: Expected results

Internal and external investigators assessed the device's performance with 756 masked samples prospectively collected from patients between the ages of 1 and 94 which were submitted for Lyme antibody testing. Site 1, a hospital laboratory located in the northeast tested 107 samples. Site 2, a hospital laboratory in the northeast tested 103 samples. The third clinical site was a state Department of Health located in the northeast. This facility tested 446 samples collected in the northeast. Demographics for 346 of the 756 samples were unavailable. Site 4, the manufacturer's research facility tested 100 samples collected in Connecticut.

Age	Specimen Group	Positive	Negative
1-9	Males	7	14
	Females	1	15
10-19	Males	1	13
	Females	1	10
20-29	Males		13
	Females		19
30-39	Males	1	19
	Females		30
40-49	Males	3	25
	Females	1	37
50-59	Males	3	41
	Females		34
60-69	Males	4	28
	Females	1	22
70+	Males	7	15
	Females	1	27
Age / SexUnknown		1	15
Total:	Prospective Males	26	168
	Prospective Females	5	194
	Female age Unknown		1
	Age / Sex Unknown	144	215
	Total	175	581

AtheNA Multi-Lyte Vlse-1/pepC10 Results From the Prospective Study

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Internal and external investigators assessed the device's performance with varying populations. The available patient demographics, volume of samples tested and the number of samples which tested positive for each population are summarized in the following table.

AtheNA Multi-Lyte Vlse-1/pepC10 Results from Other Populations
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	Number	Gender		Age	Positive/
Populations	Tested	Male	Female	Range	Tested
Characterized	229	*NA	NA	NA	171/229
Retrospective	242	128	113	4-85	191/242
Endemic Controls	300	NA	NA	NA	38/300
Non-Endemic Controls	400	NA	NA	NA	39/400

*Not available

Section 9: Clinical Studies

Clinical Data Generated for Submission: Method Comparison with Predicate Device Clinical studies consisted of 1,967 serum samples evaluated at a total of four US sites. The following populations were tested.

1. Characterized Samples
1. Prospective Population
1. Retrospective Samples
1. CDC Lyme Panel
1. Endemic and Non-Endemic Control Samples
1. Precision and Reproducibility

PERFORMANCE CHARACTERISTICS

The clinical study consisted of 1,967 serum samples evaluated at four sites located in the United States. All serum samples evaluated for concordance were tested with the ELISA reference assay. Samples that were positive by ELISA were reference assay positive. Samples that were negative by ELISA were reference assay negative.

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Study 1. Characterized Samples: 229 characterized serum samples were acquired and tested at a northeastern state Department of Health Laboratory. 21 samples were acute patients with a history of Borreliosis. 50 samples were from convalescent patients with a history of Borreliosis. 14 of these patients present with neurological, 2 with cardiac and 34 with arthritic symptoms, 79 samples were paired acute (culture proven, early acute Lyme disease) and early convalescent sera from these same patients.

				AtheNA Multi-Lyte	Predicate ELISA				Western Blot
ClinicalDiagnosis	Pos	NegorEqv	Total	% agreementwith clinicaldiagnosis & 95%CI	Pos	NegorEqv	Total	% agreementwith clinicaldiagnosis	Pos	NegorEqv	Total	% agreementwith clinicaldiagnosis
Acute	21	0	21	100% (21/21)86.7%-100%	21	0	21	100% (21/21)86.7%-100%	20	1	21	95.2% (20/21)76.2%-99.9%
Convalescent	47	3	50	94% (47/50)83.5%-98.8%	50	0	50	100% (50/50)94.2%-100%	43	7	50	86% (43/50)73.3%-95.2%
Culture (+)early acute	41	38	79	51.9% (41/79)40.4%-63.3%	37	42	78*	47.4 (37/78)36.0%-59.1%	31	22	53*	58.5% (31/53)44.1%-71.9%
EarlyConvalescent	62	17	79	78.5% (62/79)67.8%-86.9%	73	5	78*	93.6% (73/78)85.7%-97.9%	62	13	75*	82.7% (62/75)72.2%-90.4%
Total	171	58	229	74.7 (171/229)68.5%-80.2%	181	47	227	79.7% (181/227)73.9%-84.8%	156	43	199	78.4% (156/199)72.0%-83.9%

Table 1. Characterized Samples. Summary of Comparative Testing Results.

*invalid sample

*blot results unavailable

Study 2. Prospective Population: A total of 756 unselected samples from patients with an order for a Lyme antibody test were included in the study. The samples submitted for Lyme antibody testing were sequentially numbered, de-identified and archived. After the collection, 103 samples were tested at a hospital laboratory located in the Mid-Atlantic, 100 samples were tested at a hospital laboratory in upper Connecticut, 107 samples were tested at a hospital laboratory in lower Connecticut and 446 samples were tested at a state Department of Health Lab also located in the northeast.

Image /page/9/Figure/6 description: The image shows the title of a table. The title is "Table 2. Prospective Samples. Summary of Comparative Testing Results."

		Predicate ELISA (IgG/IgM)
		Positive	Equivocal	Negative	Site Total	PPA	95% CI
AtheNA Multi-LyteVise-1/pepC10 Plus	Positive	162	3	45	210	81.4% (162/199)	75.3-86.6
	Equivocal	0	0	0	0

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Negative	31	e	ਨੇ ਕਿ	546	91.4% (509/557)	88.7-93.6
Site Total	193	ರಿ	રેડવ	756
test system and negative by ELISA (33 samples had no blot data available).					If available, western blot testing was performed on the discrepant results. 12/31 samples were negative by blot and 9/31 samples werepositive by blot for sera which tested negative on the Athell Multi-yte test system and positive by EUSA (10/31) samples had no blot dataprovided). The 3 equivotal samples by the prediced for second step Western blot testing along with positives.7/45samples tested positive and 5/45 samples tested negative by blot for the discrepant samples that were positive on the Athella Multi-Lyte

Study 3. Retrospective Samples believed to have screened positive for Borrelia burgdorferi antibodies were tested at two external sites. 124 samples were tested in a hospital facility in Connecticut and 118 samples were tested in a Pennsylvania hospital laboratory.

Table 3. Retrospective Samples. Summary of Comparative Testing Results.
		Predicate ELISA (IgG/IgM)
		Positive	Equivocal	Negative	Site Total	PPA	95% CI
AtheNA Multi-Lyte Vise-1/pepC10Plus	Positive	180	7	4	191	80% (180/225)	74.2-85.0
	Equivocal	0	0	0	0
	Negative	39	6	6	51	35.3% (6/17*)	17.3-59.0
	Site Total	219	13	10	242
Western blot testing was done on discrepant results. 2/37 samples were negative by blot and 37/39 samples were positive by blot for sera which testednegative on the AtheNA Multi-Lyte test system and positive by ELISA. 4/4 samples tested positive by blot for the discrepant samples that were positive onthe AtheNA Multi-Lyte test system and negative by ELISA.
*Statistical significance evaluation can not be made on limited number of samples.

Study 4. CDC Characterized Lyme Panel: 40 samples of various reactivity were acquired from the CDC and evaluated internally at the manufacturer's site. 5 samples were from normal blood donors. 35 samples were from patients diagnosed with Borreliosis. The results of the testing are presented here as a means of conveying further information on the performance of this assay with a characterized serum panel. This does not imply an endorsement of the assay by the CDC.

	Table 4. CDC Characterized Lyme Panel. Summary of Comparative Testing Results

	AtheNA Multi-Lyte Borrelia Vise-1/pepC10				Predicate ELISA			Western Blot
		% agreement			% agreement			% agreement
TimeFromOnset	Pos	Neg	Total	with clinical diagnosis	Pos or Eqv	Total	with clinical diagnosis	Pos	Neg	Total	with clinical diagnosis
normals	0	5	5	100% (5/5)	0	5	100% (5/5)	0	5	5	100% (5/5)

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0-1 month	3	0	3	100% (3/3)	3	0	3	100% (3/3)	3	0	3	100% (3/3)
1-2 months	5	4	9	55.6% (5/9)	8	1	9	88.9% (8/9)	6	3	9	66.7% (6/9)
3-12 months	11	5	16	68.8% (11/16)	13	3	16	81.3% (13/16)	11	5	16	68.8% (11/16)
> 12 months	6	1	7	85.7% (6/7)	7	0	7	100% (7/7)	6	1	7	85.7 (6/7)
Total	25	15	40	62.5% (25/40)	31	9	40	77.5% (31/40)	26	14	40	65.0% (26/40)

Analytical Specificity

Study 5. Analytical Specificity: Testing of normal population was done on 300 samples acquired from blood donors in the New England endemic area and 400 samples acquired from blood donors and individuals undergoing routine testing not infectious in nature in the New Mexico non-endemic area.

Table 5. Analytical Specificity.

Sample Type	Volume	Negative	Positive	% Positivity
Endemic	300	262	38	12.7%
Non-endemic	400	361	39	9.8%

% positivity with the predicate was found to be: endemic =14.3%; non-endemic-= 6.5%.

Section 9A: Precision Studies

Reproducibility

Assay reproducibility was evaluated at three external clinical sites. The study was conducted as follows: Five samples were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte assay. Selected samples were negative, near cut-off, low positive, and moderate and high positive. To assess reproducibility, on each day of testing, each sample was diluted twice and then each dilution was run in triplicate. This was done twice per day by two different technicians, and was repeated for five days.

PanelMember	SampleN	MeanAU/mL	Within-Run		Within-Day		Between-Run		Between-Site		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
VlsE-1 Negative	180	31	4.5	14.7	5.5	17.9	3.7	12.1	5.9	18.8	6.3	19.7
VlsE-1 Near Cut-off	180	110.4	12.8	11.6	14	12.6	7.5	6.7	15.2	13.3	16.6	13.5
VlsE-1 Low Positive	180	136.7	13.8	20.2	15.6	11.4	9.1	6.7	16.8	11.5	16.8	12.4
VlsE-1 Moderate Positive	180	312.7	24.5	7.7	32.7	10.2	26.5	8.2	43.4	9.7	49.8	10.2
VlsE-1 High Positive	180	1869	103.1	5.5	105.6	5.6	37.5	2	107.6	5.4	112.8	5.3
pepC10 Negative	180	23.6	1.9	7.8	2.3	9.8	7.5	6.4	3.4	10.2	3.7	11.1
pepC10 Near Cut-off	180	108.9	10	9	11	10	5.4	4.9	13	10.3	13	10.4
pepC10 Low Positive	180	150.8	10.5	6.9	15.2	9.9	12.7	8.1	17.9	9.5	20.3	10

Table 6. Summary Of Reproducibility

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pepC10 Moderate Positive	180	192.3	12.8	6.8	15.6	8.1	10.7	5.5	23	8	124.3	8.1
pepC10 High Positive	180	1222.0	58.1	4.7	70.8	5.7	49	3.8	93.4	5.9	129.3	6.2

Precision

Assay repeatability was evaluated at the manufacturer site. The study was conducted as follows: six samples were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte assay. Selected samples were negative, high negative, near cut-off, low positive, and moderate and high positive. On each day of testing, the samples were diluted twice and tested. This was repeated in a second run on the same day by a different technologist for a total of twelve days. This study is summarized in iAU to assess detailed individual bead performance.

Table 7. Summary of Repeatability

PanelMember	SampleN	MeanAU/mL	Within Run		Within Day		Total
			SD	%CV	SD	%CV	SD	%CV
VIsE-1Negative 1	48	37.9	4.5	11.4	9.2	25.1	5.7	29.7
VISE-1 high negative	48	91.7	6.4	7.1	7.1	7.7	10.5	11.4
near cut-off	48	119.6	8.1	6.6	9.9	8.3	13.1	11.0
low pos	48	129.8	8.2	6.4	11.6	9.0	14.4	11.1
mod pos	48	157.3	11.3	7	11.1	6.9	15.1	9.6
high pos	48	2031	111.1	5.4	119.8	5.9	139.7	6.9
pepC10 Negative 1	48	36.8	3.9	10.5	4.1	11.0	5.1	13.1
pepC10high neg	48	95.2	6.3	6.3	8.9	9.4	11.3	11.8
pepC10 near cut-off	48	119.4	7.4	6.1	7.9	6.6	11.4	9.5
pepC10 low pos	48	130.4	9.7	7.4	10.5	8.0	12.2	9.4
pepC10 mod pos	48	295.4	28.4	9.5	34.0	11.5	41.1	13.9
pepC10 high pos	48	1207.2	38.4	3.2	52.8	4.4	65	5.4

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Image /page/13/Picture/1 description: The image shows the address for the Food and Drug Administration. The address is 10903 New Hampshire Avenue, Document Mail Center - WO66-0609, Silver Spring, MD 20993-0002. The text is left-aligned and uses a serif font.

Zeus Scientific, Inc. C/O Ewa K. Nadolczak Manager, Clinical Affairs P.O. Box 38 Raritan, NJ 08869

JUL 0 6 2019

Re: K100728

Trade/Device Name: AtheNA Multi-Lyte Borrelia VIsE-1/pepC10 Plus Test System Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema pallidum treponemal test reagents Regulatory Class: Class II Product Code: LSR Dated: March 10, 2010 Received: April 9, 2010

Dear Ms. Nadolczak:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA), You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of

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Page 2-Ewa K. Nadolczak

medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office

of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Sally Hojvat, M.Sc., h.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indications for Use

2 100 728 510(k) Number (if known): Device Name: AtheNA Multi-Lyte® Borrelia VLSe-1/pepC10 Plus Test System

Indications for Use:

The Zeus Scientific, Inc AtheNA Multi-Lyte Borrelia VisE-1/ pepC10 Plus Test System is a multiplexed sandwich immunoassay for the qualitative detection of IgG class antibody to recombinant VlsE-1 and the IgM class of antibody to synthetic pepC10 in human serum. The AtheNA Multi-lyte Borrelia VlsE-1/pepC10 Plus Test System is intended for use with the Luminex® 200 IS and the AtheNA Multi-Lyte data management package in testing serum samples from symptomatic patients or those with a history of Lyme Borreliosis. All positive specimens should be tested with a second-tier test such as Western Blot which if positive, is supportive evidence of infection with B.burgdorferi. Diagnosis of Borreliosis should be made based on the presence of B.burgdorferi antibodies, history, symptoms and other laboratory data. Negative first or second tier results should not be used to exclude Borreliosis. This kit is for in vitro diagnostic use only.

Prescription Use X (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use (21 CFR 807 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

Division Sign-Off

ice of In Vitro Diagnostic Device aluation and Safety

Page 1 of -

510(k) k 100728

Regulation Number and Section

§ 866.3830

Treponema pallidum treponemal test reagents.(a)
Identification. Treponema pallidum treponemal test reagents are devices that consist of the antigens, antisera and all control reagents (standardized reagents with which test results are compared) which are derived from treponemal sources and that are used in the fluorescent treponemal antibody absorption test (FTA-ABS), theTreponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies toTreponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).