Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit

K200025 · Gold Standard Diagnostics · LSR · Apr 6, 2020 · Microbiology

Device Facts

• Record ID: K200025
• Device Name: Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit
• Applicant: Gold Standard Diagnostics
• Product Code: LSR · Microbiology
• Decision Date: Apr 6, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3830
• Device Class: Class 2

Intended Use

The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test kit is intended as a qualitative presumptive (first step) test for the detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. Positive and equivocal results must be supplemented by testing with a second-step Western blot assay.

Device Story

The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit is an in vitro diagnostic assay used in clinical laboratories. It detects IgG antibodies to B. burgdorferi sensu stricto in human serum. The device uses antigen-coated microtiter plates (B. burgdorferi B31 lysate, 2591 lysate, and recombinant VlsE protein). Patient serum is added to wells; if specific antibodies are present, they bind to the antigens. After washing, a goat anti-human IgG-horseradish peroxidase conjugate is added, followed by a TMB chromogenic substrate. The enzymatic reaction produces a blue color, which turns yellow upon addition of a stop solution. The intensity is measured photometrically at 450nm. Results are interpreted against a cutoff control to determine if the sample is positive, equivocal, or negative. The test serves as a first-tier presumptive screen; positive or equivocal results must be confirmed by a second-tier Western blot assay to support a clinical diagnosis of Lyme disease.

Clinical Evidence

Clinical performance evaluated via prospective study (n=523) and sensitivity study (n=114). Prospective study showed 90.2% PPA and 99.6% NPA compared to predicate. Sensitivity study across disease stages (early, disseminated, late) showed improved detection over predicate, particularly in early (46.6% vs 27.6%) and disseminated (82.4% vs 52.9%) stages. CDC panel testing confirmed performance across characterized samples. Bench testing included precision, reproducibility, analytical specificity, cross-reactivity, and interference studies.

Technological Characteristics

ELISA-based immunoassay. Antigens: B. burgdorferi sensu stricto strain B31 lysate, strain 2591 lysate, and recombinant VlsE protein. Detection: Goat anti-human IgG-HRP conjugate with TMB substrate. Readout: Photometric microplate reader at 450nm. Manual procedure.

Indications for Use

Indicated for qualitative presumptive detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or those suspected of Lyme disease infection. Requires second-tier Western blot confirmation for positive/equivocal results.

Regulatory Classification

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), the Treponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies to Treponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genus Treponema and provides epidemiological information on syphilis.

Predicate Devices

• Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test Kit (K894224)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

April 21, 2020

Gold Standard Diagnostics Napoleon Monce Director, Product Development 2851 Spafford St. Davis, California 95618

## Re: K200025

Trade/Device Name: Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema Pallidum Treponemal Test Reagents Regulatory Class: Class II Product Code: LSR Dated: December 27, 2019 Received: January 6, 2020

## Dear Napoleon Monce:

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. 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 located at https://www.accessdata.fda.gov/scripts/cdrh/ofdocs/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.

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 of medical device-related adverse events) (21 CFR

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803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-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 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/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-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE(@fda.hhs.gov) or phone [phone] or 301-796-7100).

Sincerely,

Steven R. Gitterman -S

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

Enclosure

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# Indications for Use

#### 510(k) Number (if known) K200025

#### Device Name

Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit

#### Indications for Use (Describe)

The Gold Standard Diagnostics Borrelia burgdorfer igG ELISA Test Kit is intended as a qualitative presumptive (first-step) test for the detection of IgM antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients of infection. Positive and equivocal results must be supplemented by testing with a second-step Western blot assay.

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

| <div style="display:inline-block;"><b>X</b></div> Prescription Use (Part 21 CFR 801 Subpart D) |
|------------------------------------------------------------------------------------------------|
| <div style="display:inline-block;"></div> Over-The-Counter Use (21 CFR 801 Subpart C) |

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Image /page/3/Picture/0 description: The image shows the logo for Gold Standard Diagnostics. The logo features the words "GOLD STANDARD" in large, bold, black letters. To the left of the words is a gold circle with the letters "GSD" inside. Below the words "GOLD STANDARD" is the word "DIAGNOSTICS" in smaller, black letters.

# 510(k) Summary

This 510(k) Summary is being submitted in accordance with the requirement of SMDA 1990 and 21 CFR 807.92.

- 1) Submitter's Name: Gold Standard Diagnostics Address: 2851 Spafford St. Davis, CA. 95618 Phone Number: 530-759-8000 Contact Person: Napoleon Monce Date: December 27, 2019
- 2) Product and Trade Name: Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit

Common Name: Lyme ELISA Test

Regulation Section: 21 CFR 866.3830; Treponema pallidum treponemal test reagents.

Classification: Class II

Product Code: LSR; Reagent, Borrelia Serological Reagent

- 3) Legally Marketed Device to Which the Submitter Claims Equivalence: Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test Kit (K894224).
# 4) Description of the Device:

The kit includes 12 x 8 well Antigen Coated strips, Conjugate, Substrate, Stop Solution, Wash Buffer, Diluent, Negative Control, Positive Control, and Cutoff Control. The controls are provided to determine if the assay is functioning properly and to determine the antibody level. The reagents are sufficient for 96 determinations.

During the test procedure, antibodies to B. burgdorferi (sensu stricto) if present in the human serum sample will bind to the antigens coated onto the wells forming antigen-antibody complexes. Excess antibodies are removed by washing. A conjugate of goat anti-human IgG antibodies conjugated with horseradish peroxidase is then added, which binds to the antigenantibody complexes. Excess conjugate is removed by washing. This is followed by the addition of a chromogenic substrate, tetramethylbenzidine (TMB). If specific antibodies to the antigen are present in the patients' serum, a blue color will develop. The enzymatic reaction is then stopped with a stopping solution causing the contents of the well to turn yellow. The wells are read photometrically with a microplate reader at 450mm.

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The antigens used in the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test kit is a combination of B. burgdorferi sensu stricto strain B31 lysate, B. burgdorferi sensu stricto strain 2591 lysate, and a recombinant VlsE protein from B. burgdorferi sensu stricto strain B31. The lysates use spirochetes growing in BSK-H complete medium until mid-exponential phase. The recombinant VlsE protein is produced in E. coli SURE2 cells and purified by affinity chromatography.

# 5) Intended Use of the Device:

The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test kit is intended as a qualitative presumptive (first step) test for the detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. Positive and equivocal results must be supplemented by testing with a second-step Western blot assay.

# 6) Comparison with the Predicate Device:

The tables below provide a comparison of the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test kit with the Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test kit (predicate device: K894224).

| Similarities | | |
|--------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Item | Subject Device: Gold Standard<br>Diagnostics Borrelia burgdorferi<br>IgG ELISA Test Kit | Predicate Device: Trinity Biotech<br>MarDx Borrelia burgdorferi EIA IgG<br>Test Kit |
| Intended Use | The Gold Standard Diagnostics<br>Borrelia burgdorferi IgG ELISA<br>Test kit is intended as a qualitative<br>presumptive (first step) test for the<br>detection of IgG antibodies to B.<br>burgdorferi sensu stricto in human<br>serum from symptomatic<br>patients or people suspected of<br>infection. Positive and equivocal<br>results must be supplemented by<br>testing with a second-step Western<br>blot assay. | Trinity Biotech MarDx Borrelia<br>burgdorferi EIA IgG Test System is a<br>qualitative test intended for use in the<br>presumptive detection of human IgG<br>antibodies to Borrelia burgdorferi in<br>human serum. This EIA system should<br>be used to test serum from patients with<br>a history and symptoms of infection with<br>B. burgdorferi. All positive and<br>equivocal specimens should be retested<br>with a highly specific, second-tier test<br>such as Western blot. Positive second-<br>tier results are supportive evidence of<br>infection with B. burgdorferi. The<br>diagnosis of Lyme disease should be<br>made based on history and symptoms<br>(such as erythema migrans), and other<br>laboratory data, in addition to the<br>presence of antibodies to B. burgdorferi.<br>Negative results (either first or second-<br>tier) should not be used to exclude Lyme<br>disease. |
| Assay Format | Antigen coated microtiter plate –<br>96 wells. | Same |
| Technology | ELISA | Same |

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| Sample Matrix | Human serum | Same |
|-------------------|---------------------------------|------|
| Sample Processing | Dilute Samples 1:100 in Diluent | Same |
| Controls Provided | Positive, Cutoff, Negative | Same |
| Reagents Provided | Diluent, Wash, Conjugate, | Same |
| Reagents Provided | Substrate, Stop Solution | Same |
| Reported Results | Positive, Equivocal, Negative | Same |
| Assay Output | Optical density readings from | Same |
| Assay Output | Spectrophotometer | Same |

| Differences | | |
|------------------------|--------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|
| Item | Subject Device: Gold Standard<br>Diagnostics Borrelia burgdorferi<br>IgG ELISA Test Kit | Predicate Device: Trinity Biotech<br>MarDx Borrelia burgdorferi EIA<br>IgG Test Kit |
| Volumes | 100ul sample, 50ul substrate, 50ul stop solution | 100ul sample, 100ul substrate, 100ul stop solution |
| Incubation | 15/15/15 minutes at room<br>temperature | 30/30/10 minutes at room<br>temperature |
| Antigens | <i>B. burgdorferi</i> B31 strain,<br><i>B. burgdorferi</i> 2591 strain,<br><i>B. burgdorferi</i> recombinant VlsE strain | <i>B. burgdorferi</i> B31 strain |
| Results Interpretation | Convert to units.<br>Negative <9<br>Equivocal 9.0-11.0<br>Positive >11.0 | Convert to units.<br>Negative <0.80<br>Equivocal 0.80-1.19<br>Positive ≥1.2 |

#### 6(b1): Nonclinical Studies:

#### Determination of the Assay Cutoff

The cutoff was determined by testing a total of 210 normal sera which consisted of 105 sera from an endemic region of Lyme disease and 105 sera from a non-endemic region of Lyme disease. The mean plus two standard deviations was used to determine the assay cutoff. A known positive sample was then diluted to produce a ready to use cutoff control. An additional 194 samples consisting of 114 samples from different phases of Lyme disease, 8 negative healthy samples, 72 negative Lyme disease samples but do have other diseases that may cause serologic cross-reactivity, were tested. A receiver operating characteristics (ROC) analysis was performed to evaluate the performance of the assay and confirm that the chosen cutoff provided the best compromise between sensitivity and specificity.

#### Precision

To determine the precision of the Borrelia burgdorferi IgG ELISA Test, a within-lab precision study was conducted. A precision panel consisting of a negative sample, a high negative sample, a low positive sample, and a moderate positive sample, along with the kit controls, was tested in-house. The sample panel was masked and randomized. Each of the panel

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| Sample | N | Mean<br>Units | | Within-Run | Between-Run | Between-Day | Total |
|----------------------|----|---------------|----|------------|-------------|-------------|-------|
| Moderate<br>Positive | 48 | 20.3 | SD | 1.488 | 1.312 | 1.257 | 1.439 |
| | | | CV | 7.3% | 6.5% | 6.2% | 7.1% |
| Low<br>Positive | 48 | 11.5 | SD | 0.845 | 0.718 | 0.718 | 0.816 |
| | | | CV | 7.4% | 6.2% | 6.2% | 7.1% |
| High<br>Negative | 48 | 8.3 | SD | 0.880 | 0.646 | 0.615 | 0.857 |
| | | | CV | 10.6% | 7.8% | 7.4% | 10.4% |
| Negative | 48 | 0.8 | SD | 0.116 | 0.049 | 0.076 | 0.113 |
| | | | CV | 14.2% | 6.5% | 10.0% | 14.8% |
| Positive<br>Control | 48 | 17.2 | SD | 0.947 | 0.649 | 0.739 | 0.932 |
| | | | CV | 5.5% | 3.8% | 4.3% | 5.4% |
| Cutoff<br>Control | 48 | 10.1 | SD | 0.241 | 0.115 | 0.285 | 0.264 |
| | | | CV | 2.7% | 1.1% | 2.8% | 2.6% |
| Negative<br>Control | 48 | 0.4 | SD | 0.052 | 0.424 | 0.144 | 0.051 |
| | | | CV | 12.9% | 10.6% | 11.0% | 12.7% |

members was tested in duplicate, twice per day, for 12 days. The results are summarized in the following table:

# Reproducibility

A reproducibility panel consisting of a negative sample, a high negative sample, a low positive sample, and a moderate positive sample, along with the kit controls, was tested at three different sites. The sample panel was masked and randomized. Each of the panel members was tested in triplicate, twice per day, for five days. The Within-Run, Between-Run, Between-Days, and Between-Sites Standard Deviation and Coefficients of Variation (CV) were calculated. The sample panel was masked and randomized. The results are summarized in the following table:

| Sample | N | Mean<br>Units | | Within-<br>Run | Between-<br>Run | Between-<br>Day | Between-<br>Site | Total |
|----------------------|----|---------------|----|----------------|-----------------|-----------------|------------------|-------|
| Moderate<br>Positive | 90 | 21.0 | SD | 1.54 | 0.40 | 1.07 | 0.91 | 1.29 |
| | | | CV | 7.3% | 1.9% | 5.1% | 4.3% | 6.1% |
| Low<br>Positive | 90 | 13.7 | SD | 0.72 | 0.34 | 1.09 | 1.24 | 1.28 |
| | | | CV | 5.5% | 2.6% | 8.0% | 9.1% | 9.3% |
| High<br>Negative | 90 | 6.6 | SD | 0.76 | 0.27 | 0.46 | 0.68 | 0.67 |
| | | | CV | 11.7% | 4.1% | 7.0% | 10.3% | 10.2% |
| Negative | 90 | 3.0 | SD | 0.33 | 0.56 | 0.49 | 0.56 | 0.55 |
| | | | CV | 21.1% | 18.7% | 16.4% | 18.8% | 18.3% |
| Positive<br>Control | 30 | 19.1 | SD | 0.65 | 0.67 | 0.67 | 0.63 | 0.62 |
| | | | CV | 3.5% | 3.5% | 3.5% | 3.3% | 3.2% |

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| Cutoff<br>Control | 60 | 10.0 | SD | 0.25 | 0.22 | 0.23 | 0.22 | 0.22 |
|---------------------|----|------|----|-------|-------|-------|------|------|
| | | | CV | 2.4% | 2.2% | 2.3% | 2.2% | 2.2% |
| Negative<br>Control | 30 | 0.5 | SD | 0.08 | 0.06 | 0.06 | 0.50 | 0.50 |
| | | | CV | 11.0% | 11.0% | 11.0% | 9.5% | 9.6% |

## Analytical Specificity

The analytical specificity was determined by testing 208 asymptomatic individuals' samples from endemic and non-endemic regions. The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test results are summarized in the following table:

| | Number of Samples | Number<br>Positive/Equivocal | Analytical Specificity |
|--------------------|-------------------|------------------------------|------------------------|
| Endemic Region | 103 | 4 | 96.1% |
| Non-endemic Region | 105 | 0 | 100% |

## Cross Reactivity

A study using 377 samples was conducted to evaluate potential cross reactivity from different infections and disease conditions. The samples were obtained from serum vendors who confirmed their positivity for each respective marker or clinical diagnosis. The samples were tested on the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test. The results are summarized in the following table:

| Infection / Diagnosis | Number of<br>Sera Tested | # Positive / (%) |
|--------------------------------|--------------------------|------------------|
| Tick-borne Relapsing Fever IgG | 21 | 0 / (0%) |
| Treponemal Infections (TPPA) | 23 | 0 / (0%) |
| Rickettsiosis IgG | 25 | 6 / (24%) |
| Ehrlichiosis IgG | 10 | 2 / (20%) |
| Babesiosis IgG | 12 | 0 / (0%) |
| <i>H. pylori</i> IgG | 11 | 0 / (0%) |
| Parvovirus B19 IgG | 12 | 0 / (0%) |
| Influenza A&B IgG | 12 | 0 / (0%) |
| Epstein-Barr Virus IgG | 34 | 1 / (3%) |
| Cytomegalovirus IgG | 31 | 0 / (0%) |
| Herpes Simplex Virus IgG | 21 | 0 / (0%) |
| Varicella Zoster Virus | 16 | 1 / (6%) |
| Fibromyalgia | 32 | 0 / (0%) |
| Rheumatoid Arthritis | 12 | 0 / (0%) |
| Autoimmune Disease | 59 | 0 / (0%) |
| Multiple Sclerosis | 23 | 0 / (0%) |
| Severe Periodontitis | 23 | 0 / (0%) |

# Interfering Substances

The effect of potential interfering substances on samples using the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test was evaluated. Three samples, a high negative, an equivocal and a low positive were spiked with high levels of interferants and were tested along

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with serum without spiked interferants. The recommended concentrations from the guideline "Interference Testing in Clinical Chemistry" EP07-A3 from the Clinical and Laboratory Standards Institute were used (see table below). The tested substances did not affect the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test.

| Substance | Concentration | Interference |
|---------------|---------------|---------------|
| Albumin | 60 mg/ml | None detected |
| Bilirubin | 0.4 mg/ml | None detected |
| Cholesterol | 4.0 mg/ml | None detected |
| Hemoglobin | 10 mg/ml | None detected |
| Triglycerides | 15 mg/ml | None detected |

# 6(b2): Clinical Studies:

# Comparison with Predicate Device

Comparison studies were conducted at three sites (one internal and two external reference laboratories) using prospective samples submitted for Lyme serology testing. Five hundred twenty three (523) serum samples were tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test and on the predicate B. burgdorferi IgG ELISA Test. The results are summarized in the following table:

| | Predicate IgG ELISA | | | | |
|--------------------------------------------------------------------------------|---------------------|------------|----------|-------|-----|
| | Positive | Equivocal* | Negative | Total | |
| Gold Standard Diagnostics<br><i>Borrelia burgdorferi IgG</i><br>ELISA Test Kit | Positive | 40 | 5 | 2 | 47 |
| | Equivocal* | 3 | 7 | 0 | 10 |
| | Negative | 2 | 4 | 460 | 466 |
| | Total | 45 | 16 | 462 | 523 |

*Equivocal samples counted as positive

| Positive percent agreement = 90.2% (55/61) | 95% CI (79.8% - 96.3%) |
|----------------------------------------------|------------------------|
| Negative percent agreement = 99.6% (460/462) | 95% CI (98.5% - 99.9%) |

Second Tier Testing: All positive and equivocal samples by the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test and by the Predicate IgG ELISA were tested by an FDA cleared IgG Western blot assay. The results are summarized in the following table:

| | Tier 1 Positive<br>or Equivocal | IgG Blot<br>Positive | IgG Blot<br>Negative |
|----------------------------------------------------------------------------|---------------------------------|----------------------|----------------------|
| Predicate IgG ELISA | 61 | 37 | 24 |
| Gold Standard<br>Diagnostics Borrelia<br>burgdorferi IgG<br>ELISA Test Kit | 57 | 37 | 20 |

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| Predicate IgG ELISA<br>+<br>Gold Standard Diagnostics Borrelia<br>burgdorferi IgG<br>ELISA Test Kit | 55 | 37 | 18 |
|-----------------------------------------------------------------------------------------------------------------|----|----|----|
|-----------------------------------------------------------------------------------------------------------------|----|----|----|

| 2nd Tier Percent Agreement | | |
|----------------------------|------------------------|-------|
| 2nd Tier PPA<br>(95% CI) | 100%<br>(92.2% - 100%) | 37/37 |

# Clinical Sensitivity

## Sensitivity Study

A sensitivity study was performed on 114 clinically characterized samples. The samples encompass early, disseminated, and late stages of Lyme disease. The samples were tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test and on the predicate B. burgdorferi IgG ELISA Test. The results are summarized in the following table:

| Disease<br>Stage | n | Gold Standard<br>Diagnostics Borrelia<br>burgdorferi IgG ELISA<br>Test Kit | Predicate<br>IgG ELISA |
|------------------|----|----------------------------------------------------------------------------|------------------------|
| Early | 58 | 46.6% (27/58) | 27.6% (16/58) |
| Disseminated | 17 | 82.4% (14/17) | 52.9% (9/17) |
| Late | 39 | 97.4% (38/39) | 97.4% (38/39) |

# CDC Panel

A panel of 280 positive and negative specimens from the Center of Disease Control (CDC) for Lyme disease detection was tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test and on the predicate device. The results are presented as a means to convey further information on the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test with a masked characterized serum panel. This does not imply an endorsement of the assay by the CDC. The results are summarized in the following table:

| Disease Stage | n | Gold Standard Diagnostics<br><i>Borrelia burgdorferi</i> IgG<br>ELISA Test Kit | | Predicate<br>IgG ELISA | |
|---------------|-----|--------------------------------------------------------------------------------|-------------------------------------------|-----------------------------|-------------------------------------------|
| | | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis |
| Healthy | 100 | 1 | 99.0% | 1 | 99.0% |
| Early Lyme | 60 | 41 | 68.3% | 21 | 35.0% |

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| Cardiac Lyme | 3 | 2 | 66.7% | 3 | 100% |
|-----------------------|----|----|-------|----|-------|
| Neurological Lyme | 7 | 6 | 85.7% | 3 | 42.9% |
| Late | 20 | 20 | 100% | 20 | 100% |
| Look-alike<br>Disease | 90 | 11 | 87.8% | 10 | 88.9% |

## Expected Values

The range of values and positivity rate among different studies and population for the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test are as follows:

| Population | # Samples | Unit Results | | | Qualitative Results | |
|-----------------------|-----------|--------------|------------|--------------|-----------------------------|-----------------------------|
| | | Mean | Range | Std.<br>Dev. | #<br>Positive/<br>Equivocal | %<br>Positive/<br>Equivocal |
| Normal<br>Endemic | 103 | 3.7 | 0.5 – 14.3 | 2.452 | 4 | 3.9% |
| Normal<br>Non-Endemic | 105 | 3.9 | 0.6 – 8.9 | 2.002 | 0 | 0.0% |
| Prospective<br>Study | 523 | 4.3 | 0.1 – 22.2 | 4.409 | 57 | 10.9% |
| Sensitivity<br>Study | 114 | 13.6 | 0.9 – 40.4 | 7.994 | 81 | 71.1 % |

Note: It is recommended that each laboratory determine its own normal range based on the population.

# 7) Conclusion:

From the comparison data, we conclude that the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test is substantially equivalent to the Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test kit (predicate device: K894224).