Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit
Device Facts
| Record ID | K203296 |
|---|---|
| Device Name | Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit |
| Applicant | Gold Standard Diagnostics |
| Product Code | LSR · Microbiology |
| Decision Date | Mar 22, 2021 |
| 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 test for the detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing by one of the following methods: · Standard two-tier test methodology (STTT) using an IgG blot test following current interpretation guidelines, OR • Modified two-tier test methodology (MTTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test. The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test as the first-tier screening test. Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
Device Story
ELISA test kit detects IgG antibodies to B. burgdorferi in human serum; utilizes antigen-coated microtiter plates (B. burgdorferi B31/2591 lysates and recombinant VlsE). Procedure involves sample incubation, washing, addition of HRP-conjugated goat anti-human IgG, and chromogenic TMB substrate. Enzymatic reaction produces color change (blue to yellow after stop solution); measured photometrically at 450 nm. Used in clinical laboratories by trained personnel. Results support Lyme disease diagnosis when combined with clinical history and symptoms. Supports both Standard Two-Tier Testing (STTT) and Modified Two-Tier Testing (MTTT) algorithms. MTTT utilizes a second ELISA as a confirmatory step, potentially improving sensitivity in early and disseminated disease stages compared to traditional Western blot confirmation.
Clinical Evidence
Clinical sensitivity evaluated on 125 characterized samples (early, disseminated, late Lyme) and 280 CDC reference panel samples. In sensitivity study, MTTT algorithm showed higher agreement with clinical diagnosis (Early: 48.4% vs 8.1%; Disseminated: 81.8% vs 22.7%) compared to STTT. CDC panel results confirmed improved sensitivity in early Lyme (60.0% vs 33.3%) and neurological Lyme (85.7% vs 14.3%). Negative percent agreement with STTT was 96.7% (95% CI: 94.7-98.2%).
Technological Characteristics
ELISA-based immunoassay; 96-well microplate format. Antigens: B. burgdorferi B31/2591 lysates and recombinant VlsE protein. Detection: Photometric measurement at 450 nm using TMB substrate. Reagents: Conjugate (HRP-labeled goat anti-human IgG), wash buffer, stop solution. Standalone diagnostic test; no specialized software or connectivity required beyond standard microplate reader.
Indications for Use
Indicated for qualitative detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or those suspected of Lyme disease infection.
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)
- Gold Standard Diagnostics Borrelia burgdorferi IgG Blot Test Kit (K113847)
Related Devices
- K200025 — Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit · Gold Standard Diagnostics · Apr 6, 2020
Submission Summary (Full Text)
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March 22, 2021
Gold Standard Diagnostics Jennifer Roth Vice President, Product Development 2851 Spafford St. Davis, California 95618
Re: K203296
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: November 4, 2020 Received: November 9, 2020
Dear Jennifer Roth:
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/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.
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
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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 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 medical devices and radiation-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 (1-800-638-2041 or 301-796-7100).
Sincerely,
Maria Ines Garcia, Ph.D. Branch Chief 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) K203296
#### Device Name
Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit
#### Indications for Use (Describe)
The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit is intended as a qualitative test for the detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing by one of the following methods:
· Standard two-tier test methodology (STTT) using an IgG blot test following current interpretation guidelines, OR
• Modified two-tier test methodology (MTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/ IgM ELISA Test.
The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test as the first-tier screening test.
Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
| Type of Use (Select one or both, as applicable) | | | |
|-------------------------------------------------|---------------------------------------------|--|--|
| X Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) | | |
| AAUFILIP ALL APRIRIFE BLAF IF LIFFAFA | | | |
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# 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: 620 Cantrill Drive Davis, CA. 95618 Phone Number: 530-759-8000 Contact Person: Jennifer Roth Date: November 3, 2020
- 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
- Note: This clearance is for a modified use (Modified Two-tier Testing or MTTT use) for the previously cleared IVD test, the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit (K200025). The information and study data for the modified use is presented under the heading of "MTTT Comparison" below. With the exception of the intended use, all other information and data remain the same.
### 3) Legally Marketed Device to Which the Submitter Claims Equivalence:
- a. STTT Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test Kit (K894224).
- b. MTTT Gold Standard Diagnostics Borrelia burgdorferi IgG Blot Test Kit (K113847)
### 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
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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 450nm.
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 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. The purity of each antigen is assayed by SDS-PAGE followed by Coomassie staining and/or Western blotting.
### 5) Intended Use of the Device:
The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit is intended as a qualitative test for the detection of IgG antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing bv one of the following methods:
- Standard two-tier test methodology (STTT) using an IgG blot test following current ● interpretation guidelines, OR
- . Modified two-tier test methodology (MTTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test.
The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test as the first-tier screening test.
Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
### 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 Diagnostics <i>Borrelia burgdorferi</i> IgG ELISA Test Kit | Predicate Device: Trinity Biotech MarDx <i>Borrelia burgdorferi</i> EIA IgG Test Kit |
| Intended Use | The Gold Standard Diagnostics <i>Borrelia burgdorferi</i> IgG ELISA | Trinity Biotech MarDx <i>Borrelia burgdorferi</i> EIA IgG Test System is a |
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| | 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 patients<br>or people suspected of infection.<br>Positive and equivocal results<br>must be supplemented by testing<br>with a second-step Western blot<br>assay. | 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. burdorferi. All positive and equivocal<br>specimens should be retested with a<br>highly specific, second-tier test such as<br>Western blot. Positive second-tier<br>results are supportive evidence of<br>infection with B. burdorferi. 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. burdorferi.<br>Negative results (either first or second-<br>tier) should not be used to exclude Lyme | | |
|-------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|--|
| Assay Format | Antigen coated microtiter plate -<br>96 wells. | Same | | |
| Technology | ELISA | Same | | |
| 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,<br>Substrate, Stop Solution | Same | | |
| Reported Results | Positive, Equivocal, Negative | Same | | |
| Interpretation | Optical density readings from<br>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<br>stop solution | 100ul sample, 100ul substrate,<br>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<br>B31 strain | <i>B. burgdorferi</i> B31 strain |
| Results Interpretation | Convert to units.<br>Negative <9<br>Equivocal 9.0-11.0 | Convert to units.<br>Negative <0.80<br>Equivocal 0.80-1.19 |
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| | Positive >11.0 | Positive >1.2 |
|--|----------------|---------------|
|--|----------------|---------------|
#### 6(b1): Nonclinical Studies:
#### Determination of the Assav 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 members was tested in duplicate. twice per day, for 12 days. The results are summarized in the following table:
| 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 | .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% |
#### Reproducibility
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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% |
| 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 | 100.0% |
### Cross Reactivity
A study using 377 samples was conducted to evaluate potential cross reactivity from different disease conditions. The samples were obtained from serum vendors who confirmed their positivity for each respective marker. 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 /<br>(%) |
|--------------------------------|--------------------------|---------------------|
| Tick-borne Relapsing Fever IgG | 21 | 0 / (0%) |
| Treponemal Infections (TPPA) | 23 | 0 / (0%) |
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| Rickettsiosis IgG | 25 | 6 / (24%) |
|--------------------------|----|-----------|
| Ehrlichiosis IgG | 10 | 2 / (20%) |
| Babesiosis IgG | 12 | 0 / (0%) |
| H. pylori 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 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:
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| | | Predicate IgG ELISA | | | |
|-------------------------------------------------------------------------|------------|---------------------|------------|----------|-------|
| | | Positive | Equivocal* | Negative | Total |
| Gold Standard Diagnostics<br>Borrelia burgdorferi IgG<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 a FDA cleared IgG 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 |
| Predicate IgG ELISA<br>+<br>Gold Standard<br>Diagnostics Borrelia<br>burgdorferi IgG<br>ELISA Test Kit | 55 | 37 | 18 |
| Percent Agreement with Predicate Device | | |
|-----------------------------------------|----------------------------|-------|
| 2nd Tier PPA<br>(95% CI) | 100.0%<br>(92.2% - 100.0%) | 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:
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| 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:
| | | Gold Standard Diagnostics<br>Borrelia burgdorferi IgG<br>ELISA Test Kit | | Predicate<br>IgG ELISA | |
|-----------------------|-----|-------------------------------------------------------------------------|-------------------------------------------|-----------------------------|-------------------------------------------|
| Disease Stage | n | 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% |
| Cardiac Lyme | 3 | 2 | 66.7% | 3 | 100.0% |
| Neurological Lyme | 7 | 6 | 85.7% | 3 | 42.9% |
| Late | 20 | 20 | 100.0% | 20 | 100.0% |
| 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. Dev. | # Positive/ Equivocal | % Positive/ 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 - 9.0 | 2.002 | 0 | 0.0% |
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| 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).
# MTTT Comparison:
### Comparison with the Predicate Device - MTTT:
The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit, when used as the firststep or second-step test in combination with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test Kit in the Modified Two-tier Testing (MTTT) method, was compared to the Standard Two-tier testing (STTT) method using the predicates Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA (k200025) Test Kit as the first-step test followed by testing all the positive and equivocal results on the Gold Standard Diagnostics Borrelia burgdorferi IgG Blot Test (k113847). Below are tables comparing the two devices.
| Similarities | | |
|--------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Item | Subject Device: Gold Standard<br>Diagnostics Borrelia burgdorferi IgG<br>ELISA Test Kit | Predicate Device: Gold Standard<br>Diagnostics Borrelia burgdorferi<br>IgG Blot (k113847) |
| Intended Use | The Gold Standard Diagnostics Borrelia<br>burgdorferi IgG ELISA Test Kit is<br>intended as a qualitative test for the<br>detection of IgG antibodies to B.<br>burgdorferi sensu stricto in human serum<br>from symptomatic patients or people<br>suspected of infection. When used as the<br>first-tier screening test, positive and<br>equivocal results must be supplemented<br>through additional testing by one of the<br>following methods:<br>•Standard two-tier test methodology<br>(STTT) using an IgG blot test following<br>current interpretation guidelines, OR<br>•Modified two-tier test methodology<br>(MTTT) using the Gold Standard<br>Diagnostics Borrelia burgdorferi VlsE-<br>OspC IgG/IgM ELISA Test.<br>The assay can also be used as a second-<br>tier confirmation test using the MTTT | The Gold Standard Diagnostics<br>Borrelia burgdorferi B31 IgG Line<br>Blot Test Kit is intended for the<br>qualitative detection of IgG antibodies<br>to B. burgdorferi sensu stricto (B31)<br>in human serum. This test is intended<br>for use in testing human serum<br>samples which have been found<br>positive or equivocal using an ELISA<br>or IFA test procedure to provide<br>supportive evidence of infection with<br>B. burgdorferi. |
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| | methodology when used with the Gold<br>Standard Diagnostics Borrelia<br>burgdorferi VlsE-OspC IgG/IgM ELISA | |
|-------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|
| | Test as the first-tier screening test. | |
| | Positive test results by either the STTT<br>or MTTT methodology are supportive<br>evidence for the presence of antibodies<br>and exposure to Borrelia burgdorferi, the<br>cause of Lyme disease. A diagnosis of<br>Lyme disease should be made based on<br>the presence of Borrelia burgdorferi<br>antibodies, history, symptoms, and other<br>laboratory findings. | |
| Antigens | B. burgdorferi B31 strain, | Same |
| Sample Matrix | Human serum | Same |
| Controls Provided | Positive, Cutoff, Negative | Same |
| Sample Processing | Dilute Samples 1:100 | Same |
| Assay Type | Qualitative | Same |
| Differences | | |
|------------------------|-----------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|
| Item | Subject Device: Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit | Predicate Device: Gold Standard Diagnostics Borrelia burgdorferi IgG Blot (k113847) |
| Assay Format | Antigen coated microtiter plate – 96 wells. | Nitrocellulose Strips |
| Reagents Provided | Diluent, Wash, Conjugate, Substrate, Stop Solution | Diluent/Wash, Conjugate, Substrate |
| Volumes | 100ul sample, 50ul substrate, 50ul stop solution | 1500ul sample, 1500ul substrate, |
| Incubation | 15/15/15 minutes at room temperature | 30/30/10-13 minutes at room temperature |
| Interpretation | Optical density readings from Spectrophotometer | Visual |
| Results Interpretation | Convert to units.<br>Negative <9<br>Equivocal 9.0-11.0<br>Positive >11.0 | Compare to cutoff band |
| Reported Results | Positive, Equivocal, Negative | Positive, Negative |
# Method Comparison MTTT – IgG
The following studies were conducted to determine the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test as a first-tier or second-tier assay in the modified two-tier testing (MTTT) methodology.
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Gold Standard Diagnostics MTTT-IgG ELISA Method Comparison: The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test was utilized in a MTTT (2-ELISA) protocol with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test. The MTTT (2-ELISA) results were compared to the standard two-tier testing (STTT) using the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA followed by testing all positive and equivocal results on the predicate Gold Standard Diagnostics Borrelia burgdorferi IgG blot test.
# Prospective Study
Comparison studies were conducted at three sites (one internal and two external reference laboratories) using prospective samples submitted for Lyme serology testing. Four hundred eighty-one (481) serum samples were tested on the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test. A total of 38 positive and equivocal samples were obtained.
In the STTT protocol the samples that were positive or equivocal (n=38) were tested with the predicate B. burgdorferi IgG blot test. In the MTTT protocol the samples (n=38) were tested on a second ELISA, the Gold Standard Diagnostics Borrelia burgdorferi VIsE-OspC IgG/IgM ELISA Test. In the second-tier ELISA test, positive and equivocal results were considered positive.
The results of the second-tier test of the STTT when compared to the second-tier test of the MTTT, including only the samples that were positive in the first tier, are summarized in the following table:
| | | Predicate IgG Immunoblot | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------|----------------------------------------------------|----------|-------|
| | | Positive | Negative | Total |
| Gold Standard Diagnostics | Positive | 23 | ો ર | 38 |
| Borrelia burgdorferi IgG | Negative | 0 | 0 | 0 |
| ELISA | Total | 23 | 15 | 38 |
| Positive percent agreement = 100.0%<br>AT - - - Linne - un - - use - - use<br>1 - 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - | | 95% CI (85.2% - 100.0%)<br>000/ CT /0 00/ 01 00/ \ | | |
Negative percent agreement = 0.0%
95% Cl (0.0% - 21.8%)
The results of the MTTT when compared to the STTT, including all samples that were part of the prospective study (n=481), are summarized in the following table:
| | | Predicate STTT- IgG | | |
|----------------------------------------|----------|---------------------|----------|-------|
| | | Positive | Negative | Total |
| Gold Standard Diagnostics<br>MTTT- IgG | Positive | 23 | 15 | 38 |
| | Negative | 0 | 443 | 443 |
| | Total | 23 | 458 | 481 |
Positive percent agreement = 100.0% Negative percent agreement = 96.7%
95% CI (85.2% - 100.0%) 95% CI (94.7% - 98.2%)
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# Sensitivity Study
A sensitivity study was performed on 125 clinically characterized samples. The samples encompass early, disseminated, and late stages of Lyme disease. The samples were tested on both the Gold Standard Diagnostics MTTT-IgG and on the predicate STTT-IgG. The results are summarized in the following table:
| Disease<br>Stage | n | Gold Standard Diagnostics<br>MTTT - IgG | | Predicate<br>STTT - IgG | |
|------------------|----|-----------------------------------------|-------------------------------------------|-----------------------------|-------------------------------------------|
| | | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis |
| Early | 62 | 30 | 48.4% | 5 | 8.1% |
| Disseminated | 22 | 18 | 81.8% | 5 | 22.7% |
| Late | 41 | 40 | 97.6% | 39 | 95.1% |
### CDC Reference Panel
A panel of 280 positive and negative specimens from the Centers of Disease Control (CDC) for Lyme disease detection was tested on both the Gold Standard Diagnostics MTTT-IgG and on the predicate STTT-IgG. The results are summarized in the following table:
| | n | Gold Standard Diagnostics<br>MTTT-IgG | | Predicate STTT- IgG | |
|---------------------|-----|---------------------------------------|-------------------------------------------|-----------------------------|-------------------------------------------|
| Disease Stage | | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis | Positive<br>or<br>Equivocal | % Agreement<br>with Clinical<br>Diagnosis |
| Healthy | 100 | 0 | 100.0% | 0 | 100.0% |
| Early Lyme | 60 | 36 | 60.0% | 12 | 33.3% |
| Cardiac Lyme | 3 | 2 | 66.7% | 1 | 33.3% |
| Neurological Lyme | 7 | 6 | 85.7% | 1 | 14.3% |
| Late | 20 | 20 | 100.0% | 20 | 100.0% |
| Look-alike Disease* | 90 | 0 | 100.0% | 0 | 100.0% |
*infectious mononucleosis, fibromyalgia, multiple sclerosis, rheumatoid arthritis, syphilis and severe periodontitis
### 8) Conclusion:
From the comparison data, we conclude that the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test is substantially equivalent to the Gold Standard Diagnostics Borrelia burgdorferi IgG Blot Test Kit (K113847) when used for the Modified Two-tier Testing (MTTT) Lyme testing.
