Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit

{0} FDA U.S. FOOD &amp; DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ## I Background Information: A 510(k) Number K203292 B Applicant Gold Standard Diagnostics C Proprietary and Established Names Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | LSR | Class II | 21 CFR 866.3830 - Treponema Pallidum Treponemal Test Reagents | MI - Microbiology | ## II Submission/Device Overview: ### A Purpose for Submission: To obtain a substantial equivalence determination and FDA clearance for a new device and to obtain a substantial equivalence determination and FDA clearance for a modified intended use for a previously cleared medical device. This regulatory filing follows the FDA guidance document titled “Bundling Multiple Devices or Multiple Indications in a Single Submission”1. For these devices, bundling is appropriate since the device review presented scientific and regulatory issues that were most efficiently addressed during a single review. In determining whether a bundled submission was appropriate FDA considered that: (i) the supporting data are similar; (ii) primarily one review division/group will be involved; and (iii) the devices or indications for use are similar. ### B Measurand: Anti-Borrelia burgdorferi antibodies Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} C Type of Test: Enzyme-linked immunosorbent assay (ELISA) III Intended Use/Indications for Use: A Intended Use(s): See Indications for Use below. B Indication(s) for Use: Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit The Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit is intended as a qualitative test for the detection of IgG and IgM antibodies to Borrelia 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 confirmed through additional testing by one of the following methods: a) Standard two-tier test methodology (STTT) using an IgG and/or IgM blot testing following current interpretation guidelines, OR b) 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 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. Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test Kit The Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test Kit is intended as a qualitative test for the detection of IgG and IgM class antibodies to VlsE and OspC antigens from Borrelia burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of having Lyme disease. When used as the first-tier screening test, positive and equivocal results must be confirmed through additional testing by one of the following methods: a) Standard two-tier test methodology (STTT) using an IgG and/or IgM blot testing following current interpretation guidelines, OR b) Modified two-tier test methodology (MTTT) using one or more of the following three ELISA based assays: Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test, Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test, Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test. K203292 - Page 2 of 13 {2} The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with one or more of the following three ELISA based assays: Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test, Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test, Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA 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. ## C Special Conditions for Use Statement(s): Rx - For Prescription Use Only ## D Special Instrument Requirements: N/A ## IV Device/System Characteristics: ### A Device Description: The Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit is an Enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of IgG and IgM antibodies to Borrelia burgdorferi in human serum. 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/IgM antibodies conjugated with horseradish peroxidase is then added, which binds to the antigen-antibody 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/IgM 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. The kit includes 12 x 8 well Antigen Coated strips, Conjugate, Substrate, Stop Solution, Wash Buffer, Diluent, Negative Control, Positive Control, and a 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. K203292 - Page 3 of 13 {3} V Substantial Equivalence Information: A Predicate Device Name(s): Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit B Predicate 510(k) Number(s): K180264; K113847; K113846 C Comparison with Predicate(s): | Device & Predicate Device(s): | K203292 | K180264 | K113847 | K113846 | | --- | --- | --- | --- | --- | | Device Trade Name | Device 1: Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit Device 2: Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test Kit | Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit | Gold Standard Diagnostics Borrelia burgdorferi IgG Blot Test Kit | Gold Standard Diagnostics Borrelia burgdorferi IgM Blot Test Kit | | General Device Characteristic Similarities | | | | | K203292 - Page 4 of 13 {4} K203292 - Page 5 of 13 | Intended Use/Indications For Use | These are two ELISA assays for the qualitative detection of B. burgdorferi antibodies in human serum. For a complete description of the Intended Use please see Item III B. above. | The Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit is intended as a qualitative presumptive (first-step) test for the detection of IgG and IgM 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. | The Gold Standard Diagnostics Borrelia burgdorferi B31 IgG Line Blot Test Kit is intended for the qualitative detection of IgG antibodies to B. burgdorferi sensu stricto (B31) in human serum. This test is intended for use in testing human serum samples which have been found positive or equivocal using an ELISA or IFA test procedure to provide supportive evidence of infection with B. burgdorferi. | The Gold Standard Diagnostics Borrelia burgdorferi B31 IgM Line Blot Test Kit is intended for the qualitative detection of IgM antibodies to B. burgdorferi sensu stricto (B31) in human serum. This test is intended for use in testing human serum samples which have been found positive or equivocal using an ELISA or IFA test procedure to provide supportive evidence of infection with B. burgdorferi. | | --- | --- | --- | --- | --- | | Sample Matrix | Same | Serum | Same | Same | | Sample Processing | Same | Dilute samples 1:100 in Diluent | Same | Same | | Controls Provided | Same | Positive, Cutoff, Negative | Same | Same | | Assay Type | Same | Qualitative | Same | Same | | General Device Characteristic Differences | | | | | | Antigens | B. burgdorferi B31 strain, B. burgdorferi 2591 strain, B. burgdorferi recombinant VlsE, B31 strain Recombinant VlsE and OspC from B. burgdorferi strain B31 | B. burgdorferi B31 strain, B. burgdorferi 2591 strain, B. burgdorferi recombinant VlsE, B31 strain | B. burgdorferi B31 strain | B. burgdorferi B31 strain | {5} | Assay Format | Same | Antigen coated microtiter plate – 96 wells | Nitrocellulose strips | Nitrocellulose strips | | --- | --- | --- | --- | --- | | Technology | Same | ELISA | Immunoblot | Immunoblot | | Reagents Provided | Same | Diluent, Wash, Conjugate, Substrate, Stop Solution | Diluent/Wash, Conjugate, Substrate | Diluent/Wash, Conjugate, Substrate | | Volumes | Same | 100 μL sample, 50 μL substrate, 50 μL stop solution | 1500ul sample, 1500ul substrate, | 1500ul sample, 1500ul substrate, | | Incubation | Same | 15/15/15 minutes at room temperature | 30/30/10-13 minutes at room temperature | 30/30/10-13 minutes at room temperature | | Reported Results | Same | Positive, Equivocal, Negative | Positive, Negative | Positive, Negative | | Interpretation | Same | Optical density readings from Spectrophotometer | Visual | Visual | | Results interpretation | Same | Convert to units. Negative <9 Equivocal 9.0-11.0 Positive >11.0 | Compare to cutoff band | Compare to cutoff band | VI Standards/Guidance Documents Referenced: None. VII Performance Characteristics (if/when applicable): A Analytical Performance: Note: This clearance is for a modified use for a previously cleared IVD, the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test Kit (K180264). Representative analytical study data are presented below. 1. Precision/Reproducibility: To determine the precision of the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM 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. Table 1. Precision Study Results K203292 - Page 6 of 13 {6} | Sample | N | Mean Units | | Within-Run | Between-Run | Between-Day | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | Moderate Positive | 48 | 19.6 | SD | 0.815 | 1.534 | 1.472 | 1.737 | | | | | CV | 4.2% | 7.8% | 7.5% | 8.9% | | Low Positive | 48 | 12.1 | SD | 0.267 | 1.417 | 1.248 | 1.442 | | | | | CV | 2.2% | 11.7% | 10.3% | 11.9% | | High Negative | 48 | 6.1 | SD | 0.211 | 0.662 | 0.642 | 0.695 | | | | | CV | 3.4% | 10.8% | 10.5% | 11.4% | | Negative | 48 | 1.7 | SD | 0.113 | 0.164 | 0.151 | 0.199 | | | | | CV | 6.6% | 9.6% | 8.8% | 11.6% | 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 results are summarized in the following table. Table 2. Reproducibility Study Results | Sample | N | Mean Units | | Within-Run | Between-Run | Between-Day | Between-Sites | Total | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Moderate Positive | 90 | 21.1 | SD | 1.117 | 1.543 | 1.434 | 0.386 | 1.905 | | | | | CV | 5.3% | 7.3% | 6.8% | 1.8% | 9.0% | | Low Positive | 90 | 13.8 | SD | 0.591 | 1.155 | 1.026 | 0.404 | 1.297 | | | | | CV | 4.3% | 8.4% | 7.5% | 2.9% | 9.4% | | High Negative | 90 | 6.4 | SD | 0.323 | 0.756 | 0.715 | 0.237 | 0.822 | | | | | CV | 5.0% | 11.7% | 11.1% | 3.7% | 12.8% | | Negative | 90 | 1.6 | SD | 0.145 | 0.335 | 0.312 | 0.347 | 0.365 | | | | | CV | 9.1% | 21.1% | 19.6% | 21.8% | 23.0% | 2. Linearity: Not Applicable. 3. Analytical Specificity/Interference: Analytical Specificity: The analytical specificity was determined by testing 210 asymptomatic individuals' samples from endemic (Pennsylvania) and non-endemic (Arizona) regions. The Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test results are summarized in the following table. Table 3. Analytical Specificity Study | | Number of samples | Number Positive/Equivocal | Analytical Specificity | | --- | --- | --- | --- | | Endemic Region | 110 | 3 | 97.3% | K203292 - Page 7 of 13 {7} Non-endemic Region 100 2 98.0% Cross-reactivity Study: A study using 221 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/IgM ELISA Test. The results are summarized in the following table: Table 4. Cross-reactivity Study Results | Organism/Disease State | Samples Tested (N) | # Positive / (%) | | --- | --- | --- | | Tick borne Relapsing Fever | 26 | 2 / (7.7%) | | Treponemal Infections | 23 | 2* / (8.7%) | | Rickettsia | 10 | 0 / 0% | | Ehrlichiosis | 10 | 0 / 0% | | Babesiosis | 11 | 0 / 0% | | Leptospirosis | 11 | 0 / 0% | | Parvovirus B19 | 12 | 0 / 0% | | Influenza A&B | 10 | 0 / 0% | | Epstein-Barr Virus | 10 | 0 / 0% | | Cytomegalovirus | 19 | 0 / 0% | | H. pylori | 11 | 0 / 0% | | Fibromyalgia | 10 | 1 / 10% | | Rheumatoid Arthritis | 11 | 0 / 0% | | Herpes Simplex Virus 1&2 | 13 | 0 / 0% | | Varicella Zoster virus | 12 | 0 / 0% | | Autoimmune Disease | 22 | 0 / 0% | *Also positive on the predicate device Interfering Substances Study: The effect of potential interfering substances on samples using the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test was evaluated. Three samples, a negative, a low positive, and a moderate 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 EP7-A2" from the Clinical and Laboratory Standards Institute were used. The tested substances did not affect the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test. Table 5. Interference Testing | Substance | Concentration | Interference | | --- | --- | --- | | Albumin | 120 g/L | None Detected | | Bilirubin | 342 μmol/L | None Detected | | Cholesterol | 13 mmol/L | None Detected | | Hemoglobin | 2 g/L | None Detected | | Triglycerides | 37 mmol/L | None Detected | K203292 - Page 8 of 13 {8} 4. Assay Reportable Range: Not Applicable. 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): Not Applicable. 6. Detection Limit: Not Applicable. 7. Assay Cut-Off: The cut-off was determined by testing a total of 200 normal sera which consisted of 100 sera from an endemic region of Lyme disease and 100 sera from a non-endemic region. The mean plus two standard deviations was used to determine the assay cut-off. After the cut-off was determined 125 characterized Lyme disease samples were tested. An ROC analysis was then generated with the 325 samples (200 normal and 125 characterized samples) to verify the chosen cut-off. The analysis confirmed that the assay cut-off provided an optimal level of sensitivity and specificity. B Comparison Studies: 1. Method Comparison with Predicate Device: Gold Standard Diagnostics MTTT-IgG/IgM ELISA Method Comparison: The Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM 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/IgM ELISA followed by testing all positive and equivocal results on the predicate Gold Standard Diagnostics Borrelia burgdorferi IgG blot test and Gold Standard Diagnostics Borrelia burgdorferi IgM blot test. 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/IgM ELISA Test. A total of 54 positive and equivocal samples were obtained. In the STTT protocol the samples that were positive or equivocal (n=54) were tested with B. burgdorferi IgG and IgM blot tests. In the MTTT protocol the samples (n=54) were tested on a second ELISA, the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test. In the second-tier ELISA test, positive and equivocal results were considered positive. A summary of the two test methodologies is provided in the figures below. K203292 - Page 9 of 13 {9}  Figure 1. STTT-IgG/IgM Western Blot Algorithm (WB-STTT [IgG/IgM]) Figure 2. MTTT-IgG/IgM ELISA Algorithm (ELISA-MTTT [IgG/IgM])  Performance of the second-tier Gold Standard Diagnostics VlsE-OspC IgG/IgM ELISA was assessed by comparing results to second-tier western blot testing on only those samples positive by the first-tier Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA. Table 6. Second-tier Performance Summary First-tier Positives Only | | Predicate WB [IgG/IgM] | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Gold Standard Diagnostics VlsE-OspC IgG/IgM ELISA | Positive | 36 | 13 | 49 | | | Negative | 0 | 5 | 432 | | Total | | 36 | 18 | 481 | PPA: $100\%$ (36/36) $95\%$ CI: 90.3-100.0% NPA: $27.8\%$ (5/18) $95\%$ CI: 9.7-53.5% K203292 - Page 10 of 13 {10} 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: Table 7. Performance Summary - ELISA-MTTT [IgG/IgM] compared to WB-STTT [IgG/IgM] | | WB-STTT [IgG/IgM] | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Gold Standard Diagnostics ELISA-MTTT [IgG/IgM] | Positive | 36 | 13 | 49 | | | Negative | 0 | 432 | 432 | | Total | | 36 | 445 | 481 | PPA: 100% (36/36) 95% CI: 90.3-100.0% NPA: 97.1% (432/445) 95% CI: 95.1-98.4% The above performance table artificially inflates the negative percent agreement of the second-tier test since a large number of negatives are negative by the first-tier test. 2. Matrix Comparison: Not Applicable. C Clinical Studies: 1. Clinical Sensitivity: 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 algorithm and on the predicate STTT algorithm. The results are summarized in the following table: Table 8. Sensitivity Study Results - Comparison of ELISA-MTTT [IgG/IgM] and WB-STTT [IgG/IgM] algorithms | | N | Gold Standard Diagnostics ELISA-MTTT [IgG/IgM] | | Predicate WB-STTT [IgG/IgM] | | | --- | --- | --- | --- | --- | --- | | Disease Stage | | Positive/Eqv. | % Agreement with Clinical Diagnosis | Positive/Eqv. | % Agreement with Clinical Diagnosis | | Early | 62 | 38 | 61.3% | 39 | 62.9% | | Disseminated | 22 | 20 | 90.9% | 20 | 90.9% | | Late | 41 | 40 | 97.6% | 40 | 97.6% | K203292 - Page 11 of 13 {11} CDC Reference Panel: A panel of 280 positive and negative specimens from the Centers of Disease Control (CDC) for Lyme disease detection was tested using both the Gold Standard Diagnostics MTTT algorithm and on the predicate STTT algorithm. The results are summarized in the following table: Table 9. CDC Reference Panel Test Results – Comparison of ELISA-MTTT [IgG/IgM] and WB-STTT [IgG/IgM] algorithms | Sample Category | Gold Standard Diagnostics ELISA-MTTT [IgG/IgM] | | Predicate WB-STTT [IgG/IgM] | | | --- | --- | --- | --- | --- | | | Pos. | % Agreement with Clinical Diagnosis | Pos. | % Agreement with Clinical Diagnosis | | Early Lyme (N = 60) | 46 | 76.7% | 37 | 61.7% | | Cardiac Lyme (N = 3) | 2 | 66.7% | 2 | 66.7% | | Neurological Lyme (N = 7) | 6 | 85.7% | 6 | 85.7 | | Late Lyme (N = 20) | 20 | 100% | 20 | 100% | | Healthy Controls (N = 100) | 0 | 100% | 0 | 100% | | Disease Controls (N = 90) | 1 | 98.9% | 4 | 95.6% | 2. Clinical Specificity: N/A 3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable): N/A D Clinical Cut-Off: Not Applicable. E Expected Values/Reference Range: The range of values and positivity rate among different studies and population for the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test are as follows: K203292 - Page 12 of 13 {12} Table 10. Expected Values of the Gold Standard Diagnostics Borrelia burgdorferi IgG/IgM ELISA Test | Population | # Samples | Unit Results | | | Qualitative Results | | | --- | --- | --- | --- | --- | --- | --- | | | | Mean | Range | Std. Dev. | # Positive /Equivocal | % Positive /Equivocal | | Normal Endemic | 110 | 5.8 | 0.6 – 11.3 | 2.075 | 3 | 2.7% | | Normal Non-Endemic | 100 | 4.2 | 0.9 – 12.3 | 2.077 | 2 | 2.0% | | Prospective Study | 520 | 6.3 | 0.70 – 32.3 | 5.407 | 83 | 16.0% | | Sensitivity Study | 89 | 25.0 | 3.6 – 34.9 | 8.449 | 79 | 88.8% | ## VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. ## IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K203292 - Page 13 of 13