The iDart™ Lyme IgM ImmunoBlot Kit is an immunoblot assay intended for the in vitro qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum. The iDart™ Lyme IgM ImmunoBlot Kit is intended to detect antibodies to Lyme Screen Antigen (LSA) and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the iDart™ Lyme IgM ImmunoBlot Kit are supportive evidence for the presence of antibodies and exposure to B. burgdorferi. Negative results do not preclude infection with B. burgdorferi. iDart™ Lyme IgM ImmunoBlot Kit is intended to aid in the diagnosis of Lyme disease and the test kit should only be used on samples from patients with clinical history, signs and symptoms consistent with Lyme disease. The iDart™ Lyme IgM Immunoblot Kit is not intended as a screen for asymptomatic patients.

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

Device Description

The iDart™ Lyme IgM ImmunoBlot tests are line immunoblot assays. Antigenic proteins specific for Borrelia species that cause Lyme Disease are produced by recombinant DNA technology in Escherichia coli. The purified proteins are then applied as discrete lines on a nitrocellulose membrane along with two control proteins. The iDart™ Lyme IgM ImmunoBlot Kit contains IgM ImmunoBlot strips and the proteins are applied in the following order: C1 (IgG/IgM – conjugate control), C2 (Protein L – calibrator/serum control), P93, P41 (2 antigen bands), P39 (2 antigen bands), P23 (9 antigen bands), P31 (9 antigen bands), P34, C10 and LSA (a chimeric VlsE peptide termed the Lyme Screen Antigen).

AI/ML Overview

Here's an analysis of the acceptance criteria and study detailed in the provided FDA 510(k) clearance letter for the iDart™ Lyme IgM ImmunoBlot Kit.

Note: The provided text details the performance characteristics but does not explicitly state "acceptance criteria" in a separate section with numerical thresholds. Therefore, the "Acceptance Criteria" column in the table below is inferred from the achieved "Reported Device Performance" and common expectations for such devices (e.g., high agreement, specificity, and sensitivity). The "study that proves the device meets the acceptance criteria" refers to the clinical and analytical performance studies conducted.

1. Table of Acceptance Criteria and Reported Device Performance

Criteria Category	Acceptance Criteria (Inferred)	Reported Device Performance
Reproducibility	High agreement across sites, operators, and runs (e.g., ≥95% agreement)	100% agreement on all test results across runs, days, and sites for all panel samples (negative, high negative, low positive, moderate positives, high positive). (N=90 replicates per sample across 3 sites, 2 operators, 5 days, 2 runs/day).
Analytical Specificity (Endemic)	High Agreement (e.g., ≥95%) in endemic healthy population	99.44% specificity (1 false positive out of 177 samples) on samples from apparently healthy individuals in endemic areas (US, CDC, Bay Area Foundation NY, MA, WI).
Analytical Specificity (Non-Endemic)	High Agreement (e.g., ≥95%) in non-endemic healthy population	99.21% specificity (1 false positive out of 127 samples) on samples from apparently healthy individuals in non-endemic areas (US, CDC, IGeneX, Inc.).
Cross-Reactivity	High specificity (e.g., ≥95%) in samples with potentially interfering conditions	97.94% specificity (5 false positives out of 243 samples) in cross-reactivity study (various bacterial/viral infections, autoimmune diseases). Note: 2 Leptospira samples and 1 Mononucleosis, 1 H. pylori, 1 Parvovirus-19 sample were positive with iDart Lyme IgM. The Leptospira samples were also positive by STTT.
Interference (Endogenous Analytes)	No interference from common endogenous substances at specified concentrations	No interference observed from Bilirubin (1, 15mg/dL), Albumin (3.5, 5g/dL), Cholesterol (150, 250mg/dL), Triglycerides (150, 500mg/dL), Hemoglobin (10, 20g/dL) in positive, low positive, and negative Borrelia IgM samples.
Method Comparison (PPA)	Acceptable Positive Percent Agreement (PPA) with STTT (e.g., lower bound of 95% CI > 80-85%)	90.91% PPA (95% CI: 81.55%– 95.77%) against STTT in a cohort of 997 serum samples.
Method Comparison (NPA)	Acceptable Negative Percent Agreement (NPA) with STTT (e.g., lower bound of 95% CI > 95%)	98.07% NPA (95% CI: 96.96%– 98.77%) against STTT in a cohort of 997 serum samples.
Clinical Sensitivity (CDC Panel)	Acceptable sensitivity across disease stages compared to STTT (e.g., comparable or better performance)	Overall Sensitivity (CDC Panel): 65.8% compared to STTT's 55.7%. Stage I: 66.0% (STTT 56.0%), Stage II: 88.9% (STTT 77.8%), Stage III: 55.0% (STTT 45.0%). The iDart kit showed higher sensitivity than STTT across all stages in this particular panel.
Clinical Specificity (CDC Healthy Controls)	High specificity (e.g., ≥95%) in healthy controls for CDC panel	97.9% agreement (3.1% false positive rate) for healthy controls within the CDC panel (N=95). Note that the table reports "Agreement," which implies specificity here.
Clinical Specificity (CDC Disease Controls)	High specificity (e.g., ≥95%) in disease controls for CDC panel	98.9% agreement (1.1% false positive rate) for disease controls within the CDC panel (N=84). Note that the table reports "Agreement," which implies specificity here.
Fresh vs. Frozen Samples	Equivalent performance between fresh and frozen samples	Performance is equivalent between fresh and frozen samples. (N=63 fresh, N=63 frozen; same positive/negative counts).
Antibody Class Specificity	Specific detection of IgM antibodies (i.e., minimal cross-reactivity with IgG)	The kit specifically detected IgM; treatment with human IgM blocked reactivity, while treatment with human IgG did not affect reactivity in positive samples. Negative samples remained negative under all conditions.

Study Proving Device Meets Acceptance Criteria

The studies detailed in the "PERFORMANCE CHARACTERISTICS" section of the 510(k) summary are designed to prove that the iDart™ Lyme IgM ImmunoBlot Kit meets the necessary performance standards for its intended use. These include:

Reproducibility Study: Tested consistency across multiple sites, operators, and runs.
Analytical Specificity Studies: Evaluated performance in healthy endemic and non-endemic populations, and cross-reactivity with various non-Borrelia pathogens and autoantibodies.
Interference Study: Assessed impact of common endogenous substances.
Method Comparison with STTT: Compared the device's accuracy (PPA and NPA) against the Standard Two-Tier Test methodology, which is a recognized approach for Lyme disease diagnosis.
CDC Serum Panel Study: Evaluated sensitivity and specificity across different disease stages and in various control groups using a well-characterized reference panel.
Fresh and Frozen Samples Comparison Study: Demonstrated stability of samples under different storage conditions.
Antibody Class Specificity Study: Confirmed the specific detection of IgM antibodies.

Additional Information:

2. Sample size used for the test set and the data provenance:

Reproducibility: A panel of coded samples with different levels of anti-B. burgdorferi IgM (negative, high negative, low positive, moderate positives, and high positive samples). 90 replicates per sample were generated. The specific number of distinct samples in this panel is not explicitly stated beyond "a panel of coded samples".
Analytical Specificity (Endemic): 177 samples from apparently healthy individuals in the US from endemic areas (CDC and Bay Area Foundation NY, MA, WI).
Analytical Specificity (Non-Endemic): 127 samples from apparently healthy individuals in the US from non-endemic areas (CDC and IGeneX, Inc.).
Cross-Reactivity Study: 243 serum samples from patients with bacterial or viral infections, as well as sera from patients with diagnoses that could be confused with Lyme disease (CDC, IGeneX, Inc., New York Biologics (NY), Kamineni Life Sciences Pvt. Ltd, Hydrabad (India), Warde Medical Laboratory (MI)).
Interference Study: One positive, one low positive, and one negative Borrelia IgM samples (tested in singlicate with various spiked interfering agents).
Method Comparison with STTT: 997 prospectively collected serum samples procured from IGeneX, Inc.
CDC Serum Panel: 258 serum samples from the CDC panel. These included patients with Lyme disease at different stages and Lyme disease look-alike infections, and healthy controls.
Fresh and Frozen Samples Comparison Study: 63 fresh samples and 63 frozen samples.
Antibody Class Specificity: 8 previously tested patient samples (4 negatives, 4 positives).

Data Provenance: The data appears to be a mix of retrospective and prospective. Samples from "apparently healthy individuals" and "patients with bacterial or viral infections" can be either. The "Method Comparison with STTT" explicitly states "Prospectively collected serum samples" from IGeneX, Inc. The CDC Serum Panel is a reference panel, usually well-characterized retrospectively. Locations include the US (CDC, Bay Area Foundation NY, MA, WI, IGeneX, Inc., New York Biologics (NY), Warde Medical Laboratory (MI)) and India (Kamineni Life Sciences Pvt. Ltd, Hydrabad).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
The document does not explicitly state the number of experts or their qualifications for establishing ground truth for the clinical study samples. For the "Method Comparison with comparators (STTT)", the comparison is against "FDA-cleared EIA and immunoblot as part of the standard two-tier test methodology (STTT)". This implies the ground truth for these samples was established by the results of the STTT, which is a standardized and accepted diagnostic algorithm, rather than by individual expert interpretation. For the CDC Panel, the samples are "from patients diagnosed with Lyme Disease at different stages" or "Lyme disease look-like infections", suggesting that the CDC provided established diagnoses based on their protocols, which would implicitly involve expert clinical and laboratory evaluation.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set:
The document does not describe an explicit adjudication method (like 2+1 or 3+1) for establishing ground truth for the clinical test sets. The ground truth for the method comparison study was the result of the "FDA-cleared EIA and immunoblot as part of the standard two-tier test methodology (STTT)". For the CDC panel, it was based on the provided "diagnosis".

5. 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 MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) kit for laboratory use (an ImmunoBlot assay, specifically evaluating IgM antibodies) rather than an AI-powered image analysis system or a device requiring human-in-the-loop interpretation that would typically necessitate such a study. The "reading" of the immunoblot strips is performed visually but the interpretation criteria are clearly defined, not requiring subjective interpretive discretion usually addressed by MRMC studies.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
This question is not directly applicable in the terms typically used for AI/software devices. The iDart™ Lyme IgM ImmunoBlot Kit is a lab-based immunoassay. Its performance characteristics (sensitivity, specificity, reproducibility) were evaluated as a standalone device (without external assistance like AI or human readers for interpretation, other than the standard visual reading of the bands per the kit's instructions), as demonstrated by the analytical and clinical studies. The results are generated by the chemical reaction on the strip and interpreted based on predefined criteria. It's an "algorithm only" in the sense of the assay's biochemical process and visual interpretation rules, operating without additional "human-in-the-loop" modifications to its fundamental mechanism or output.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):
The ground truth used primarily appears to be:

Standard Two-Tier Test (STTT) results: For the majority of the clinical method comparison study (N=997), the device was compared against the results from "FDA-cleared EIA and immunoblot as part of the standard two-tier test methodology".
Clinical Diagnosis from CDC: For the CDC reference panel, samples were from patients "diagnosed with Lyme Disease at different stages" or "Lyme disease look-like infections". This implies a ground truth established through a combination of clinical evaluation, laboratory tests, and expert judgment according to CDC protocols.

8. The sample size for the training set:
Not applicable/Not specified. This is an in vitro diagnostic kit (ImmunoBlot assay), not a machine learning or AI-based device that typically requires a distinct "training set" for model development. The antigens used are recombinant proteins, and the assay's interpretation criteria are rule-based, not learned from a dataset.

9. How the ground truth for the training set was established:
Not applicable. As stated above, this device does not utilize a training set in the context of machine learning. The design and validation of immunoblots are based on known antigen-antibody interactions and assay development methodologies.

Summary

U.S. Food & Drug Administration 510(k) Clearance Letter

Page 1

U.S. Food & Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993
www.fda.gov

Doc ID # 04017.07.05

June 12, 2025

ID-FISH Technology, Inc.
Jyotsna Shah
Vice President of Research and Development
556 Gibraltar Drive
Milpitas, California 95035

Re: K242872
Trade/Device Name: iDart Lyme IgM ImmunoBlot Kit
Regulation Number: 21 CFR 866.3830
Regulation Name: Treponema Pallidum Treponemal Test Reagents
Regulatory Class: Class II
Product Code: LSR
Dated: May 19, 2025
Received: May 20, 2025

Dear Jyotsna Shah:

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 (the 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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

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K242872 - Jyotsna Shah Page 2

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 Part 801 and Part 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-devices/medical-device-safety/medical-device-reporting-mdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-

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K242872 - Jyotsna Shah Page 3

assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Noel J. Gerald -S

Noel J. Gerald, Ph.D.
Deputy Division Director
Division of Microbiology Devices
OHT7: Office of In Vitro Diagnostics
Office of Product Evaluation and Quality
Center for Devices and Radiological Health

Enclosure

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FORM FDA 3881 (8/23) Page 1 of 1 PSC Publishing Services (301) 443-6740 EF

DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration

Indications for Use

Form Approved: OMB No. 0910-0120
Expiration Date: 07/31/2026
See PRA Statement below.

510(k) Number (if known): K242872

Device Name: iDart Lyme IgM ImmunoBlot Kit

Indications for Use (Describe)

The iDart™ Lyme IgM ImmunoBlot Kit is an immunoblot assay intended for the in vitro qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum. The iDart Lyme IgM ImmunoBlot Kit is intended to detect antibodies to Lyme Screen Antigen (LSA) and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the iDart Lyme IgM ImmunoBlot Kit are supportive evidence for the presence of antibodies and exposure to B. burgdorferi. Negative results do not preclude infection with B. burgdorferi. iDart™ Lyme IgM ImmunoBlot Kit is intended to aid in the diagnosis of Lyme disease and the test kit should only be used on samples from patients with clinical history, signs and symptoms consistent with Lyme disease. The iDart Lyme IgM Immunoblot Kit is not intended as a screen for asymptomatic patients.

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

Type of Use (Select one or both, as applicable)

☒ Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

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"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

Page 5

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION SUMMARY

I. BACKGROUND INFORMATION:

A. 510(k) Number

K242872

B. Applicant

ID-FISH Technology, Inc.
556 Gibraltar Drive
Milpitas CA 95035 USA

C. Contact Person

Dr. Jyotsna Shah
VP of Research and Development
regulatory@idfishtechnology.com
1-650-269-8610

D. Date Prepared

May 20, 2025

E. Proprietary and Established Names

iDart™ Lyme IgM ImmunoBlot Kit

F. Regulatory Information

Product Code(s): LSR – Reagents, Borrelia Serological Reagent
Classification: Class II
Regulation Section: 21 CFR 866.3830 - Treponema Pallidum Treponemal Test Reagents
Panel: MI - Microbiology

II. SUBMISSION/DEVICE OVERVIEW:

A. Purpose for Submission:

To obtain a substantial equivalence determination for a new device.

B. Measurand:

IgM antibodies to Borrelia burgdorferi (B. burgdorferi)

C. Type of Test:

ImmunoBlot Assay

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III. INTENDED USE/INDICATIONS FOR USE:

A. Intended Use(s):

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

B. Indication(s) for Use:

iDart™ Lyme IgM ImmunoBlot Kit should be used for diagnosing Lyme Disease in patients with a clinical history, and signs and symptoms consistent with Lyme Disease. It is not intended for use in asymptomatic patients.

C. Special Conditions for Use Statement(s):

For in vitro diagnostic use only
For professional use only
For prescription use only

IV. DEVICE CHARACTERISTICS:

A. Device Description:

B. Principle of procedures:

The iDart™ Lyme IgM ImmunoBlot test is a line blot assay. The recombinant Borrelial proteins, along with 2 controls proteins are dispensed onto a nitrocellulose membrane by spraying.

During the test procedure, if antibodies to Borrelia burgdorferi infection are present in the human serum sample, they will bind to the antigens sprayed onto the nitrocellulose strips. After removing serum and unbound antibodies by washing, the nitrocellulose strip is incubated with an anti-human IgM antibody conjugated with Alkaline Phosphatase.

After removing the unbound conjugated antibody by a washing step, visualization of the antigen-antibody complex is accomplished by the addition of a substrate 5-bromo, 4-chloro, 3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) which forms a strong bluish purple reaction product by the action of alkaline phosphatase. The reaction is stopped by washing the nitrocellulose strip with distilled or deionized water. Depending on the observed band pattern one can interpret the presence or absence of specific IgM antibodies to B. burgdorferi antigens.

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V. SUBSTANTIAL EQUIVALENCE INFORMATION:

A. Predicate Device Name(s):

Viramed Borrelia All-In-One ViraChip Test Kit

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

K220016

C. Comparison with Predicate(s):

Item	Device: iDart™ Lyme IgM ImmunoBlot Kit	Predicate: Viramed Borrelia All-In-One ViraChip Test Kit (K220016)

Similarities

| Intended Use | The iDart™ Lyme IgM ImmunoBlot Kit is an immunoblot assay intended for the in vitro qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum. The iDart™ Lyme IgM ImmunoBlot Kit is intended to detect antibodies to Lyme Screen Antigen (LSA) and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the iDart™ Lyme IgM ImmunoBlot Kit are supportive evidence for the presence of antibodies and exposure to B. burgdorferi. Negative results do not preclude infection with B. burgdorferi. iDart™ Lyme IgM ImmunoBlot Kit is intended to aid in the diagnosis of Lyme disease and the test kit should only be used on samples from patients with clinical history, signs and symptoms consistent with Lyme disease. The iDart™ Lyme IgM Immunoblot Kit is not intended as a screen for asymptomatic patients. Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures. For in vitro diagnostic use only For professional use only For prescription use only | The Viramed Biotech AG Borrelia All-In-One ViraChip is an in vitro qualitative microarray assay for the detection of IgM and IgG antibodies to Borrelia burgdorferi in human serum. The assay is intended for testing serum samples from symptomatic patients or those suspected of Lyme Disease. It is intended to detect antibodies to VlsE and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the Viramed Biotech AG Borrelia All-In-One ViraChip are supportive evidence for the presence of antibodies and exposure to B. burgdorferi, the causative agent for Lyme disease. Negative results do not preclude infection with B. burgdorferi. Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures as an aid in diagnosis of Lyme disease. The Viramed Biotech AG Borrelia All-In-One ViraChip Test must be used with a ViraChip Reader and the ViraChip Software. |
| Specimen Type | Serum | Serum |
| Antibodies Detected | IgM | IgM and IgG |
| Controls | Positive Control serum, Negative Control Serum | Positive Control serum, Negative Control Serum |
| Method | Qualitative | Qualitative |

Differences

Item	Device	Predicate
Assay Technology	ImmunoAssay	Antigen Coated wells (Microarrays)

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| Antigens | Recombinant antigenic proteins - LSA antigen (chimeric VlsE peptide termed the Lyme Screen Antigen), C10, 93 kD, 41 kD, 39 kD, 34 kD, 31 kD and 23 kD | VlsE, 93 kD, 58 kD, 45 kD, 39 kD, 30 kD, 23 kD, 21 kD, 19 kD, 18 kD, and 17 kD antigens of B.burgdorferi |
| Procedure | Line Blot Assay Borrelia IgM antibodies to specific antigen bands. Wash between sample and conjugate incubation steps, incubate with substrate | Wash after Sample and Conjugate Step |
| Sample Volume | 20ul neat serum in 1000ml sample diluent | Samples diluted 1:76 and 100 µL added per well |
| Reagents | Sample diluent, Wash Buffer, Milk powder, Conjugate Buffer, Substrate solution | 10X Wash Buffer, Sample Buffer, Chromogen/Substrate Solution |
| Result Generation | Manual reading | Automated with ViraChip Reader |

VI. STANDARDS/GUIDANCE DOCUMENTS REFERENCED:

Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Antibodies to Borrelia burgdorferi - Guidance for Industry and FDA Staff - MARCH 2013

VII. PERFORMANCE CHARACTERISTICS (IF/WHEN APPLICABLE):

A. Analytical Performance:

1. Reproducibility:

The iDart™ Lyme IgM ImmunoBlot Kit was tested in a blind study to evaluate reproducibility across 3 separate sites each with 2 operators over 5 days, 2 runs a day, using a panel of coded samples containing different levels of anti-B. burgdorferi IgM: negative, high negative, low positive, moderate positives, and high positive samples. This study generated 90 replicates per sample. There was 100% agreement on all test results across runs, days and sites (See Table 1).

Table 1. Reproducibility Study Summary - Overall – All Sites – 6 operators/5 days/ 3 replicates

Sample #	Sample Type	IgM	# of Samples (+)	Expected Result	% samples matched to expected result
MA	High Positive	P	90/90	P	100%
MB	Moderate Positive	P	90/90	P	100%
MC	Moderate Positive	P	90/90	P	100%
MD	Low Positive	P	90/90	P	100%
ME	High Negative	N	0/90	N	100%
MF	Negative	N	0/90	N	100%

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2. Analytical Specificity/Interference:

Analytical Specificity

Table 2 and 3 below show the results of testing iDart™ Lyme IgM ImmunoBlot Kit on samples collected from a population in the US of 177 apparently healthy individuals from endemic areas resulted with 99.44% specificity and 127 samples collected from healthy individuals in non-endemic areas with 99.21% specificity.

Table 2: iDart™ IgM ImmunoBlot Kit - Analytical Specificity (Endemic) (N=177)

Sample Source	N	IgM	% Positive
CDC	50	1	2.00%
BAY AREA FOUNDATION (NY, MA, WI)	127	0	0.00%
TOTAL	177	1	0.56%
Agreement			99.44%

Table 3: iDart™ IgM ImmunoBlot Kit - Analytical Specificity (Non- Endemic) (N=127)

Sample Source	N	IgM	% Positive
CDC	45	1	2.22%
IGeneX, Inc.	82	0	0.00%
TOTAL	127	1	0.79%
Agreement			99.21%

Cross-Reactivity Study

A cross-reactivity study was performed on specimens known to contain potentially cross-reactive antibodies to Lyme infection. A total of 243 serum samples from patients with bacterial or viral infections, as well as sera from patients with diagnoses that could be confused with Lyme disease, were tested. Based on the data presented in Table 4, the iDart™ Lyme IgM ImmunoBlot Kit demonstrated 97.94% specificity to samples containing antibodies to non-Borrelia pathogens or autoimmune diseases.

Table 4: iDart™ Lyme Borrelia IgM ImmunoBlot Kit - Analytical Specificity

Source	Disease State	N	IgM Positive	% Cross-reactivity
CDC	Fibromyalgia	15	0	0%
	Mononucleosis	15	1	6.67%
	Multiple sclerosis	15	0	0%
	Rheumatoid arthritis	11	0	0%
	Severe periodontitis	14	0	0%
	Syphilis	14	0	0%
	Leptospira	10	2*	20.00%*
IGeneX, Inc.	Rheumatoid Factor	5	0	0%
	ANA	5	0	0%
	Bartonella	11	0	0%
	Bartonella & Anaplasma	1	0	0%
	Bartonella &TBRF Borrelia	1	0	0%
	Babesiosis	8	0	0%

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| | Babesiosis & Tick-Borne Relapsing Fever | 2 | 0 | 0% |
| | Babesiosis & Rickettsiosis | 1 | 0 | 0% |
| | Tick Borne Relapsing Fever | 10 | 0 | 0% |
| | Tick-Borne Relapsing Fever & Anaplasma | 1 | 0 | 0% |
| | Tick-Borne Relapsing Fever & Ehrlichiosis | 1 | 0 | 0% |
| | Tick-Borne Relapsing Fever & Rickettsia | 1 | 0 | 0% |
| | Anaplasmosis | 8 | 0 | 0% |
| | Anaplasmosis & Ehrlichiosis | 1 | 0 | 0% |
| | Ehrlichiosis | 4 | 0 | 0% |
| | Rickettsiosis | 11 | 0 | 0% |
| New York Biologics (NY) | HIV | 6 | 0 | 0% |
| | HCV | 5 | 0 | 0% |
| | HSV1 | 7 | 0 | 0% |
| | CMV | 11 | 0 | 0% |
| | EBV | 9 | 0 | 0% |
| Kamineni Life Sciences Pvt. Ltd, Hydrabad (India) | Pregnant women | 11 | 0 | 0% |
| | H. pylori | 9 | 1 | 11.11% |
| Warde Medical Laboratory (MI) | Parvovirus-19 | 10 | 1 | 10.00% |
| | Varicella-zoster virus | 10 | 0 | 0% |
| False Positive | | 5 | | |
| Agreement | | 243 | | 97.94% |

*Two Leptospira samples positive by iDart Lyme IgM testing were also positive for IgM with STTT.

Interference from Endogenous Analytes

The potential interfering effect of endogenous substances in patient samples when using the iDart Lyme IgM ImmunoBlot was evaluated using one positive, one low positive and one negative Borrelia IgM samples. Samples were spiked with the endogenous substances at the final concentrations listed in the table below. All samples were tested in singlicate. No interference was observed in the tested samples.

Table 5. Effect of Interference Substances on iDart™ Lyme IgM ImmunoBlot Kit

Agent	Concentration in serum	iDart™ Lyme IgM ImmunoBlot Kit result			Effect on iDart™ Lyme IgM ImmunoBlot Kit
		High Pos (mA)	Low Pos (mB)	Negative (mC)
Bilirubin	1mg/dL (low)	Positive	Positive	Negative	No effect
Bilirubin	15mg/dL (high)	Positive	Positive	Negative	No effect
Albumin	3.5g/dL (low)	Positive	Positive	Negative	No effect
Albumin	5g/dL (high)	Positive	Positive	Negative	No effect
Cholesterol	150mg/dL (low)	Positive	Positive	Negative	No effect
Cholesterol	250mg/dL (high)	Positive	Positive	Negative	No effect
Triglycerides	150mg/dL (low)	Positive	Positive	Negative	No effect
Triglycerides	500mg/dL (high)	Positive	Positive	Negative	No effect
Hemoglobin	10g/dL (low)	Positive	Positive	Negative	No effect
Hemoglobin	20g/dL (high)	Positive	Positive	Negative	No effect

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3. Assay Reportable Range:

Not applicable.

4. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

Not applicable.

5. Detection Limit:

Not applicable.

6. Assay Cut-Off:

Not applicable.

B. Clinical Studies:

1. Method Comparison with comparators (STTT):

The performance of the iDart™ Lyme IgM ImmunoBlot Kit for detection of Borrelial-specific antibodies was compared to FDA-cleared EIA and immunoblot as part of the standard two-tier test methodology (STTT). Results are summarized below. A total of 997 serum samples were procured from IGeneX, Inc. and tested at three clinical sites. Table 6 bellow summarizes the distribution of samples per testing site.

Table 6. Sample distribution by clinical site and cohort.

	Number of Samples	Sample Type	Vendor Providing Samples
Site 1	304	Prospectively collected serum samples	IGeneX Inc.
Site 2	357	Prospectively collected serum samples	IGeneX Inc.
Site 3	336	Prospectively collected serum samples	IGeneX Inc.

All samples were blinded, re-coded, and tested at the respective clinical sites as per the instructions for use for the iDart™ Lyme IgM ImmunoBlot Kit. Overall performance is summarized in Table 7.

Table 7. Performance Summary. iDart™ Lyme IgM ImmunoBlot Kit vs STTT

N=997

	STTT
	Positive (+)	Negative (-)
iDart™ Lyme IgM ImmunoBlot Kit Positive (+)	60	18
iDart™ Lyme IgM ImmunoBlot Kit Negative (-)	6	913
Total	66	931

PPA (95% CI): 90.91% (81.55%– 95.77%)
NPA (95% CI): 98.07% (96.96%– 98.77%)

2. Clinical Sensitivity/Specificity:

CDC Serum Panel:

A reference Pre-Marketing Panel of 258 serum samples were received from CDC. These samples were from patients diagnosed with Lyme Disease at different stages (Stages 1, 2, and 3), Lyme disease look-like infections (infectious mononucleosis, multiple sclerosis, rheumatoid arthritis, fibromyalgia and severe periodontitis), and from healthy controls living in endemic and non-endemic regions of Lyme disease. Results are analyzed according to disease stages and compared to STTT (See Table 8).

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Table 8. Performance on CDC Pre-Marketing Panel with respect to different Disease Stages. iDart Lyme IgM ImmunoBlot Kit vs STTT (N=258)

Disease Stage	Stage I	Stage II	Stage III	Overall	Healthy controls	Disease Controls
N	50	9	20	79	95	84
Test Kits	iDart	STTT	iDart	STTT	iDart	STTT
Positive	33	28	8	7	11	9
Negative	17	22	1	2	9	11
Sensitivity	66.0%	56.0%	88.9%	77.8%	55.0%	45.0%
Agreement

3. Other Clinical Supportive Data:

Fresh and Frozen Samples Comparison Study

The Clinical Performance of iDart™ Lyme IgM ImmunoBlot Kit is equivalent between fresh and frozen samples. This is supported by the evidence shown in this study by which samples were tested fresh and after frozen. This study demonstrates that freezing does not alter the performance of iDart™ Lyme IgM ImmunoBlot Kit in comparison to testing of fresh samples.

Table 9: Fresh and Frozen Samples Tested with iDart™ Lyme IgM ImmunoBlot Kit

Sample Type	Tested	N	Interpretation Criteria - POSITIVE: LSA positive or indeterminant and one or more bands from at least TWO of the following groups are present – LSA, P41, P39, P23, P31 and P34 are present. NEGATIVE: If the band pattern does not meet the positive criteria.
			IgM	Neg
Fresh Samples	Within 2 weeks of collection. (stored refrigerated)	63	21	42
Frozen Samples	After being Frozen (2-22 (days)	63	21	42

Antibody Class Specificity

This study was conducted to demonstrate the antibody class specificity of the goat anti-human IgM Conjugate used in the iDart™ Lyme IgM ImmunoBlot Kit. 8 previously tested patient samples that includes 4 negatives and 4 positives were included in the study. These samples were tested in iDart™ Lyme Diluent provided with the test kit.

3 sets of anti-human IgM conjugate were prepared and tested.

Control – no additives
1ug/ml of human IgG
1ug/ml of human IgM

The iDart™ Lyme IgM ImmunoBlot Kit is intended to detect IgM antibodies to B. burgdorferi antigens from patients with clinical history, signs and symptoms consistent with Lyme disease. This study demonstrates that iDart™ Lyme IgM ImmunoBlot Kit is specific in detecting IgM antibodies in Lyme patient samples.

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Table 12: iDart™ Lyme IgM ImmunoBlot Kit - Antibody Specificity Study Summary

Sample #	Sample Type #	No treatment (a); goat anti-human IgM conjugate treated with human IgG (b) or with human IgM (c).	iDart™ Lyme IgM ImmunoBlot Kit							LSA	Band Groups	IgM
			LSA Positive or Indeterminant AND one or more bands from at least TWO of the following groups are present – P41, P39, P23, P31 and P34 are present; and NEGATIVE if the Lyme ImmunoBlot IgM band pattern does not meet the positive criteria.
			P41	P39	P23	P31	P34	LSA

| A | High Positive | No treatment | P | P | P | P | 3+ | 3+ | P | P | P |
| | | Human IgG | P | P | P | P | 3+ | 3+ | P | P | P |
| | | Human IgM | | | | | | | N | N | N |

| B | Moderate Positive | No treatment | P | P | P | P | 3+ | 3+ | P | P | P |
| | | Human IgG | P | P | P | P | 3+ | 3+ | P | P | P |
| | | Human IgM | | | | | | | N | N | N |

| C | Moderate Positive | No treatment | P | | P | P | 3+ | 3+ | P | P | P |
| | | Human IgG | P | | P | P | 3+ | 3+ | P | P | P |
| | | Human IgM | | | | | | | N | N | N |

| D | High Negative | No treatment | | | | | 1+ | | P | N | N |
| | | Human IgG | | | | | 1+ | | P | N | N |
| | | Human IgM | | | | | | | N | N | N |

| E | Low Positive | No treatment | P | | P | | I | 2+ | P | P | P |
| | | Human IgG | P | | P | | I | 2+ | P | P | P |
| | | Human IgM | | | | | | | N | N | N |

| F | Negative | No treatment | | | | | | | N | N | N |
| | | Human IgG | | | | | | | N | N | N |
| | | Human IgM | | | | | | | N | N | N |

| G | Negative | No treatment | | | | | | | N | N | N |
| | | Human IgG | | | | | | | N | N | N |
| | | Human IgM | | | | | | | N | N | N |

| H | Negative | No treatment | | | | | | | N | N | N |
| | | Human IgG | | | | | | | N | N | N |
| | | Human IgM | | | | | | | N | N | N |

C. Clinical Cut-Off:

Not applicable.

D. Expected Values/Reference Range:

Not applicable.

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VIII. PROPOSED LABELING:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10

IX. CONCLUSION:

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

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).