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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.

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 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.

This document describes the validation of the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit, including a new proposed Modified Two-tier Testing (MTTT) methodology. The provided text primarily focuses on comparative studies against predicate devices and existing standard methods, rather than defining explicit acceptance criteria in a quantitative table format. However, implied acceptance criteria can be derived from the performance percentages presented.

Based on the provided information, here's a structured response addressing the requested points:

1. Table of Acceptance Criteria (Implied) and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical cutoffs in the document. However, they can be inferred from the reported "Percent Agreement" values in the comparison studies. The studies aim to demonstrate substantial equivalence to established predicate devices and methodologies.

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

• Determination of Assay Cutoff (Nonclinical):

  • Sample Size: 210 normal sera (105 endemic, 105 non-endemic) + 194 samples (114 Lyme disease stages, 8 healthy negative, 72 negative Lyme disease with other conditions). Total = 404 samples.
  • Data Provenance: Not explicitly stated, but "endemic region of Lyme disease" and "non-endemic region of Lyme disease" suggest geographical diversity. Retrospective, as these are "tested" samples for cutoff determination.

• Precision (Nonclinical):

  • Sample Size: 48 runs for each of 4 panel members (negative, high negative, low positive, moderate positive) + kit controls. This is a repetitive testing design, not individual patient samples.

• Reproducibility (Nonclinical):

  • Sample Size: 90 runs (3 sites x 2 runs/day x 5 days x 3 replicates) for each of 4 panel members (negative, high negative, low positive, moderate positive) + kit controls (30 runs for Pos/Neg control, 60 runs for Cutoff control).

• Analytical Specificity:

  • Sample Size: 208 asymptomatic individuals (103 from endemic regions, 105 from non-endemic regions).
  • Data Provenance: Endemic and non-endemic regions. Retrospective (asymptomatic individuals' samples).

• Cross Reactivity:

  • Sample Size: 377 samples.
  • Data Provenance: Samples obtained from "serum vendors who confirmed their positivity for each respective marker," implying retrospective, controlled samples.

• Comparison/Prospective Study (Clinical):

  • STTT Comparison: 523 serum samples.
  • MTTT Comparison: 481 serum samples for the initial screening. 38 positive/equivocal samples were carried forward for second-tier testing comparison.
  • Data Provenance: "Prospective samples submitted for Lyme serology testing" from "three sites (one internal and two external reference laboratories)." This indicates a prospective study design with samples from potentially diverse geographical locations (clinical labs).

• Sensitivity Study (Clinical):

  • STTT: 114 clinically characterized samples.
  • MTTT: 125 clinically characterized samples.
  • Data Provenance: "Clinically characterized samples" from unspecified sources; likely retrospective collection of known patient cohorts.

• CDC Panel (Clinical):

  • Sample Size: 280 positive and negative specimens.
  • Data Provenance: From the "Center of Disease Control (CDC)." These are reference panels, typically collected and characterized over time, so retrospective.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the test sets.

• For "clinically characterized samples" and "CDC panel" samples, the implication is that these samples have a pre-defined and reliable "clinical diagnosis" or "reference characterization" based on established medical criteria. This often involves consensus from clinical experts (e.g., infectious disease specialists) and/or a combination of clinical presentation, symptoms, and other laboratory findings, but the specifics are not detailed here.
• For the "Second-Tier Testing" comparison, the "FDA cleared IgG blot assay" and "predicate B. burgdorferi IgG blot test" serve as the ground truth. These are approved diagnostic methods, and their interpretation would follow established guidelines.

4. Adjudication Method for the Test Set

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for resolving discrepancies in the test results or for establishing the initial ground truth diagnoses. The comparisons are based on the results of the different assays and against established clinical characterizations or CDC panels.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC study was performed or described. This device is an ELISA test kit, a laboratory diagnostic assay for detecting antibodies, not an AI or imaging diagnostic tool that involves human readers interpreting images. Therefore, the concept of "human readers improving with AI assistance" is not applicable.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the primary performance evaluation is standalone. The Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit operates as a standalone diagnostic assay. Its performance (sensitivity, specificity, agreement) is measured as the output of the kit itself (optical density readings converted to units and qualitative results) without direct human interpretation of complex visual patterns or AI assistance. The results are quantitative and then interpreted qualitatively (positive, equivocal, negative) based on predefined cutoffs.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

The ground truth used varies by study:

• Predicate Devices/Methodologies: For the STTT and MTTT comparison studies, the ground truth for the device's performance is a comparison against the results from legally marketed predicate devices (e.g., Trinity Biotech MarDx Borrelia burgdorferi EIA IgG Test Kit) and/or a FDA cleared IgG blot assay. This is a form of comparative ground truth against established methods.
• Clinical Diagnosis/Characterization: For the Sensitivity Study and CDC Panel, the ground truth is "clinically characterized samples" and "positive and negative specimens from the Centers of Disease Control (CDC)." This implies a clinical diagnosis or consensus diagnosis based on comprehensive patient data (history, symptoms, other laboratory findings) or a reference panel with established disease status.
• Analytical Ground Truth: For non-clinical studies like analytical specificity and cross-reactivity, the ground truth is often the confirmed absence or presence of specific conditions in the tested samples, usually based on prior laboratory testing or sample vendor claims.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning. The term "training set" is usually associated with the development of AI algorithms. For an ELISA kit, development involves:

• Antigen Selection and Optimization: Which antigens to use (e.g., B31 lysate, 2591 lysate, recombinant VlsE).
• Reagent Formulation: Optimizing conjugate, substrate, buffers, etc.
• Cutoff Determination: "The cutoff was determined by testing a total of 210 normal sera..." and "An additional 194 samples..." This process of determining the cutoff could be considered analogous to a 'calibration' or 'training' phase for a traditional diagnostic test, where a dataset is used to define the diagnostic thresholds. In this sense, a total of 404 samples (210 normal + 194 other known samples) were used for cutoff determination.

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

As discussed above, for establishing the cutoff (analogous to training/calibration):

• Normal Sera: The 210 normal sera were likely verified as "normal" through standard clinical practice or donor screening, indicating the absence of target antibodies/disease in a healthy population.
• Known Positive/Negative/Other Disease Samples: The additional 194 samples included "different phases of Lyme disease," "negative healthy samples," and "negative Lyme disease samples but do have other diseases that may cause serologic cross-reactivity." The "ground truth" for these samples would be their known clinical status or disease classification, typically established through comprehensive clinical evaluation, follow-up, and/or panels from disease banks. The ROC analysis was used to confirm the chosen cutoff provides the best compromise between sensitivity and specificity, indicating an analytical optimization process based on these known samples.

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The Gold Standard Diagnostics Borrelia burgdorferi 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.

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. 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.

The provided document is a 510(k) summary for the Gold Standard Diagnostics Borrelia burgdorferi IgG ELISA Test Kit, a medical device for diagnosing Lyme disease. It describes the device's technical specifications and the nonclinical and clinical studies performed to demonstrate its substantial equivalence to a legally marketed predicate device.

Here's the breakdown of the acceptance criteria and the study that proves the device meets them:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly present acceptance criteria in a formal table with pass/fail thresholds. Instead, it demonstrates the device's performance through various studies and compares it to a predicate device. The implicit acceptance criteria are that the new device performs comparably to the predicate device and demonstrates acceptable precision, specificity, and sensitivity.

Here's a summary of the reported device performance for key metrics:

2. Sample sizes used for the test set and the data provenance

• Comparison with Predicate Device: 523 serum samples.
  • Provenance: Prospective samples submitted for Lyme serology testing. Conducted at three sites (one internal and two external reference laboratories). The specific country of origin is not noted, but given the FDA submission, it's likely primarily US-based or at least includes US data.
• Cutoff Determination: 210 normal sera (105 from an endemic region, 105 from a non-endemic region). An additional 194 samples (114 Lyme disease, 8 negative healthy, 72 negative Lyme disease with other potential cross-reactive diseases).
  • Provenance: Retrospective (these seem to be characterized samples). Specific regions/countries not detailed.
• Precision Study: 48 replicates for each of the 6 panel members (negative, high negative, low positive, moderate positive sample, positive control, cutoff control, negative control).
  • Provenance: In-house study.
• Reproducibility Study: 90 replicates for each of 4 panel members (moderate positive, low positive, high negative, negative). 30 or 60 replicates for controls (positive, cutoff, negative).
  • Provenance: Tested at three different sites.
• Analytical Specificity: 208 asymptomatic individual samples (103 from an endemic region, 105 from a non-endemic region).
  • Provenance: Retrospective (asymptomatic individuals). Specific regions/countries not detailed.
• Cross-Reactivity: 377 samples from various infection/disease conditions.
  • Provenance: Obtained from serum vendors, confirmed for positivity for respective markers or clinical diagnosis. Retrospective.
• Clinical Sensitivity (Clinically characterized samples): 114 clinically characterized samples (58 early, 17 disseminated, 39 late stages of Lyme disease).
  • Provenance: Retrospective (clinically characterized samples).
• CDC Panel: 280 positive and negative specimens from the CDC.
  • Provenance: Retrospective. The CDC panel implies a well-characterized, reference sample set.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

This is an ELISA diagnostic kit, not an AI/imaging device. Therefore, the concept of "experts establishing ground truth" in the sense of radiologists reading images is not directly applicable.

For this type of device:

• Ground Truth: The ground truth for the test samples (e.g., "Lyme positive/negative," "specific infection") is established through clinical characterization, confirmed diagnosis, or established reference panels (like the CDC panel), potentially involving a combination of clinical symptoms, other laboratory tests (e.g., Western blot, PCR), and expert clinical judgment.
• The document states that for the cross-reactivity study, samples were obtained from "serum vendors who confirmed their positivity for each respective marker or clinical diagnosis," implying that the ground truth for these samples was based on established laboratory methods or clinical records, not necessarily adjudicated by human experts in a reading study.
• For the "Clinically characterized samples" in the sensitivity study, the ground truth ("early, disseminated, and late stages of Lyme disease") would have been established by clinicians based on the patients' medical history, symptoms, and other diagnostic findings. The number and specific qualifications of these clinicians are not provided in this summary.

4. Adjudication method for the test set

Not applicable in the context of an ELISA kit where pre-characterized biological samples define the "ground truth." The "adjudication" is inherent in the characterization of the biological samples used for testing, often through a combination of clinical presentation and other established laboratory methods (e.g., a two-tiered testing algorithm for Lyme disease).

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This is an in vitro diagnostic (IVD) kit, not an AI-assisted diagnostic imaging device that involves human readers.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, this entire study is a "standalone" performance evaluation of the ELISA kit. The device itself (the ELISA kit) provides a quantitative and qualitative result (positive, equivocal, negative) based on the optical density reading, without direct human cognitive input shaping that specific result beyond the laboratory technician performing the assay according to instructions. The interpretation of "positive/equivocal/negative" is based on pre-defined cutoffs.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth for the various studies includes:

• Clinical Diagnosis/Characterization: For the sensitivity study (early, disseminated, late Lyme disease), the ground truth was based on the clinical status of the patients.
• Reference Panels: The CDC panel specimens serve as a well-characterized reference (positive and negative) for Lyme disease.
• Other Laboratory Confirmatory Tests: For the comparison study, all positive and equivocal samples from both the subject and predicate ELISA were tested by an FDA-cleared IgG Western blot assay, which serves as a second-tier confirmatory ground truth for these specific samples.
• Confirmed Positivity/Clinical Diagnosis from Serum Vendors: For cross-reactivity studies, samples were sourced with confirmed positivity for specific markers or clinical diagnoses.
• Healthy/Asymptomatic Status: For cutoff determination and analytical specificity, ground truth was derived from healthy, asymptomatic individuals from endemic and non-endemic regions.

8. The sample size for the training set

This document describes a premarket notification for a traditional ELISA diagnostic kit, not a machine learning/AI algorithm. Therefore, there is no explicit "training set" in the context of supervised machine learning. The term "training set" is usually associated with AI model development.

For an ELISA kit, development involves:

• Assay Design/Optimization: This is an iterative process using various internal samples to optimize antigen/antibody concentrations, incubation times, buffer compositions, etc.
• Cutoff Determination: As stated, 210 normal sera and 194 other characterized samples were used to determine and confirm the assay cutoff. This set functions somewhat like a "calibration" or "training" set for defining the interpretation thresholds.

9. How the ground truth for the training set was established

Again, this is not a machine learning model. For the samples used in cutoff determination (which is analogous to a "training" or "calibration" phase), the ground truth was established by:

• Normal Sera: Defined as "normal" based on clinical health and potentially geographic origin (endemic/non-endemic for Lyme disease).
• Lyme Disease Samples: "114 samples from different phases of Lyme disease" implies these were clinically characterized Lyme patient samples.
• Other Disease Samples: "72 negative Lyme disease samples but do have other diseases that may cause serologic cross-reactivity," implying a clinical diagnosis of the other disease and a confirmed negative status for Lyme.

The goal of this "cutoff determination" phase is to establish a threshold that optimizes sensitivity and specificity against a diverse set of known samples.

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