The QualiCode™ B. burgdorferi IgG Western Blot Kit is an in vitro qualitative assay for the detection of human IgG antibodies reactive with Borrelia burgdorferi antigens present on a membrane strip. The QualiCode™ B. burgdorferi IgG Western Blot Kit is intended for supplemental testing of human serum specimens which yield positive or equivocal results on B. burgdorferi ELISA or IFA screening assays: QualiCode test results can provide additional, specific evidence of infection with B. burgdorferi which may be useful in the diagnosis of Lyme disease. The OualiCode™ B, burgdorferi IgG Western Blot Kit can be used to test human sera at any time following onset of symptoms, and when (1) only IgM antibodies were originally detected: (2) IgG antibodies were detected previously, but were not considered significant by Western Blot; or (3) previously seronegative patients become positive by ELISA or IFA screening tests.

Device Description

The device is a Western Blot assay. Proteins and other antigenic components of the Borrelia spirochete are fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecvl sulfate. The separated proteins are electrophoretically transferred from the gel to nitrocellulose membranes, which are subsequently blocked to minimize non-specific binding and cut into strips. These nitrocellulose strips with resolved Borrelia burgdorferi antigens are then reacted with diluted serum and controls (positive and negative sera of defined reactivity).

During the incubation period, human antibodies specific to the B. burgdorferi antigens, if present in the sample or control, will bind to the antigen to which they have affinity. Unbound serum and non-specific antibodies are washed from the strip. Detection of bound IgG antibodies is accomplished by reacting and incubating the strips with a solution containing anti-human IgG antibodies conjugated with alkaline phosphatase. Unbound conjugate antibodies are removed by washing. The qualitative assessment of the detected IgG antibodies is then accomplished by the reaction of the alkaline phosphatase with a chemical substrate, which is cleaved into a colored, insoluble product that can be visualized. The determination of the reactivity of each unknown specimen is accomplished by comparison of the identified, visualized bands to the Band Identifying and Band Intensity Controls.

AI/ML Overview

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Acceptance Criteria and Study to Prove Device Meets Criteria:

1. Table of Acceptance Criteria and Reported Device Performance:

The document does not explicitly state pre-defined acceptance criteria for the device's performance metrics (sensitivity, specificity, reproducibility). Instead, it presents the "Summary of Performance" as the observed outcomes from the clinical trials. Given this, the table below reflects the reported performance as if they were the benchmarks the device met.

Metric	Acceptance Criteria (Implied by Study Results)	Reported Device Performance
Specificity
Normal - endemic region	≥ 95% Negative	97% Negative (95-99% CI)
Normal - non-endemic region	≥ 92% Negative	96% Negative (92-98% CI)
Normal - overall	≥ 95% Negative	97% Negative (95-98% CI)
Cross-reactive Panel	≥ 88% Negative	93% Negative (88-96% CI)
Sensitivity
Unknown Draw Time	≥ 69% Positive	81% Positive (69-91% CI)
<1 Month Draw Time	≥ 33% Positive	44% Positive (33-56% CI)
1-2 Months Draw Time	≥ 33% Positive	54% Positive (33-72% CI)
3-12 Months Draw Time	≥ 59% Positive	80% Positive (59-93% CI)
>12 Months Draw Time	≥ 17% Positive	39% Positive (17-63% CI)
Reproducibility
Inter-lot Agreement	≥ 90%	90%
Inter-run Agreement	≥ 92%	92%
Inter-reader Agreement	≥ 97%	97%

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

Test Set Sample Sizes:
- Specificity (Normal Control): 430 samples (279 from endemic regions, 151 from non-endemic regions).
- Specificity (Cross-Reactive Panel): 172 samples.
- Sensitivity (Lyme Disease Panel): 199 samples (further categorized by draw time).
- Reproducibility (Panel): 20 specimens.
- CDC Lyme Disease Serum Panel: Used for "Substantial Equivalence" but specific sample size for this panel within the clinical trials is not explicitly stated.
Data Provenance:
- The samples were obtained from patients with a clinical diagnosis of Lyme Disease and from normal donors from endemic and non-endemic regions.
- "Each of the clinical trial sites provided specimens that were well characterized by the site." This implies data was collected from multiple clinical trial sites.
- The data appears to be retrospective, as it refers to "archived Lyme Disease specimens" and samples that "had been drawn at different times after onset of disease."

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

The document does not explicitly state the number of experts used to establish ground truth for the test set.
Qualifications of Experts: Not specified. However, the ground truth for "clinical diagnosis of Lyme Disease" was based on a "clinical diagnosis… in accordance with the CDC case definition," and some infections were "confirmed by culture of Borrelia from biopsies." This suggests that medical professionals (e.g., physicians, pathologists) were involved in the initial diagnosis and characterization of these samples.

4. Adjudication Method for the Test Set:

The document does not explicitly mention an adjudication method (e.g., 2+1, 3+1).
For Inter-reader reproducibility, it states, "each strip interpreted by two independent readers." While this involves multiple readers, it describes a reproducibility assessment rather than a consensus for establishing a definitive ground truth for individual cases.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:

No, a MRMC comparative effectiveness study comparing human readers with and without AI assistance was NOT done. This device is a diagnostic kit (Western Blot assay), not an AI-powered image analysis or decision support system that would assist human readers in interpretation. The "Inter-reader reproducibility" section assesses consistency between human readers interpreting the device's results, not the impact of an AI on human performance.

6. Standalone Performance Study:

Yes, a standalone performance study was done. The entire "Summary of Performance" section details the direct performance of the QualiCode™ B. burgdorferi IgG Western Blot Kit (an "algorithm" in a broader sense of a diagnostic protocol) in detecting IgG antibodies against a defined ground truth (Lyme disease diagnosis, normal controls, cross-reactive conditions). The performance metrics (sensitivity, specificity, reproducibility) are reported for the device itself.

7. Type of Ground Truth Used:

Clinical Diagnosis: For the Lyme disease panel, the ground truth was "clinical diagnosis of Lyme Disease in accordance with the CDC case definition, i.e. based on the presence of EM (erythema migrans) or the presentation of late Lyme clinical manifestations (e.g., arthritic, cardiac, or neurological symptoms)."
Pathology/Culture: Infection was "confirmed by culture of Borrelia from biopsies in many examples." This provides a strong, direct form of ground truth for a subset of the Lyme disease cases.
Screening Assay Results: Specimens were "required to have tested positive or indeterminate on a B. burgdorferi screening assay, typically an ELISA." This indicates a level of pre-screening was used to select the Lyme disease panel.
Normal Donor Status: For the specificity studies, ground truth was based on samples from "Normal population" (from endemic and non-endemic regions) and "samples from patients with diseases other than Lyme disease that may be cross - reactive." The assumption here is that these samples are truly negative for Lyme disease.

8. Sample Size for the Training Set:

The document does not explicitly mention a separate "training set" for the device. As a Western Blot kit, it's a biochemical assay rather than a machine learning model that typically requires a distinct training phase. The "clinical trials" described here are designed for validation and performance evaluation of the developed assay, not for training it.

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

Since a separate training set is not indicated in the context of this type of device, this question is not applicable. The device's components (antigens, reagents) are developed based on scientific understanding of B. burgdorferi and immunology, not through a data-driven training process with a specific ground truth dataset like an AI model.

Summary

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and the comments of the comments of the comments of

510(k) Safety and Effectiveness Summary

Submitted by:

Immunetics. Inc. 63 Rogers Street Cambridge, MA 02142

Contact Person:

Andrew E. Levin, Ph.D. Scientific Director

(617) 492 - 5416

Date of Preparation:

September 9, 1999

1. Name and Address of Owner/Operator and Manufacturer:

Immunetics, Inc. 63 Rogers Street Cambridge, MA 02142

2. Product Name:

Trade Name: QualiCode™ B. burgdorferi IgG Western Blot Kit

Common Name: B. burgdorferi IgG Western Blot Kit

3. Claim of Substantial Equivalence

The characterized samples used for the establishment of Substantial Equivalence were obtained from patients with a clinical diagnosis of Lyme Disease in accordance with the CDC case definition, i.e. based on the presence of EM (erythema migrans) or the presentation of late Lyme clinical manifestations (e.g., arthritic, cardiac, or neurological symptoms). Infection was confirmed by culture of Borrelia from biopsies in many examples.

Each of the clinical trial sites provided specimens that were well characterized by the site using Lyme-specific serological analyses, including EIA and/or Western Blot testing. In particular, specimens selected for the trial were required to have tested positive or indeterminate on a B. burgdorferi screening assay, typically an ELISA, in accordance with the two-tier testing protocol recommended by CDC/ASTPHLD (CDC (1995) "Recommendations for test performance and interpretation from the Second National

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Conference on Serologic Diagnosis of Lyme Disease", Morbid. Mort. Weekly Rep. 44:590-591.).

Substantial equivalence of this device is based on the assessment of performance of the device in these clinical trials in which the well-characterized, archived Lyme Disease specimens, the Centers for Disease Control Lyme Disease Serum Panel, normal donor specimens (from endemic and non-endemic regions), and samples from diverse disease conditions were analyzed.

4. Description

റ് Intended Use

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6. Summary of Performance

From a summary of the clinical trial data, the following performance characteristics are described:

Expected values

Three investigational sites, including Immunetics and two independent off-site investigators assaved samples from the following patient populations:

1. Normal population (n=430) comprised of samples from Lyme disease endemic (n=279) and non-endemic (n=151) regions.
1. Cross Reactive Panel (n=172) comprised of samples from patients with diseases other than Lyme disease that may be cross - reactive.
1. Lyme Disease Panel (n=199) comprised of samples from patients with a clinical diagnosis of Lyme disease, based on presence of erythema migrans or one or more symptoms of late Lyme disease, and which tested positive or equivocal on ELISA or other screening assays for B. burgdorferi antibodies.

Specificity

Specificity of the device was determined from analysis of testing normal donor specimens from endemic and non-endemic regions and potentially cross reactive disease specimens (602 total samples). The specificity values derived from testing each population are shown in the table below in the "% Negative" column:

Sample Type	n	% Positive	% Negative	95% CI
Normal - endemic region	279	3	97	95-99 %
Normal - non-endemic region	151	4	96	92-98%
Normal - overall	430	3	97	95-98 %
Cross-reactive Panel	172	7	93	88-96 %

Sensitivity

Sensitivity of the device was determined by testing a total of 199 well-characterized sera from patients with Lyme disease, which had been drawn at different times after onset of disease. Sensitivity for each time after onset category is shown in the table below in the "% Positive" column:

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Sensitivity of the Immunetics, Inc. QualiCode™ B. burgdorferi IgG Western Blot Kit vs. Time after Onset

Draw Time (months)	n	% Positive	% Negative	95% CI
Unknown	54	81%	19%	69-91 %
<1	81	44%	56%	33-56 %
1-2	26	54%	46%	33-72 %
3-12	20	80%	20%	59-93 %
>12	18	39%	61%	17-63 %

Reproducibility

Inter-lot reproducibility was determined by assaying a panel of 20 specimens, including positive, weakly reactive and negative sera, on three lots of the Immunetics QualiCode™ kit. There was 90% agreement between interpretations from the three kit lots. The reproducibility of scoring of individual bands between the three lots varied from 60% to 95%, averaging 77%.

Inter-run reproducibility was determined by assaying the 20 specimen panel twice, on separate days, at each of three sites. There was 92% agreement overall between interpretations from the two runs averaged over all three sites. The reproducibility of individual band scoring between the two runs averaged over all three sites varied between 77% and 95%, with an average of 88% over all ten bands.

Inter-reader reproducibility was determined by assaying the 20 specimen panel twice, on separate days, at each of three sites, with each strip interpreted by two independent readers. There was 97% agreement overall between interpretations from the two readers averaged over the three sites. Band scoring reproducibility varied between 83% and 100% averaged over the three sites, with an average of 91.5% over all ten bands.

Study	n	% Interpretation agreement
Inter-lot	20	90
Inter-run	20	92
Inter-reader	20	97

7. Conclusions

Based on performance in the clinical trial, this device has been shown to be safe and effective for the intended use in the qualitative detection of human immunoglobulin G (IgG) antibodies in serum or plasma to Borrelia burgdorferi antigens, and as a supplemental, more specific, test to aid in the diagnosis of infection by or exposure to Borrelia burgdorferi, the causative agent of Lyme disease.

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Image /page/4/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged around the perimeter. Inside the circle is a stylized image of three human profiles forming a wing-like shape.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

SEP 2 1 1999

Andrew E. Levin, Ph.D. Scientific Director Immunetics, Inc. 63 Rogers Street Cambridge, Massachusetts 02142

Re: K991063

Trade Name: QualiCode™ B. burgdorferi IgG Western Blot Kit Regulatory Class: II Product Code: LSR Dated: July 8, 1999 Received: July 9, 1999

Dear Dr. Levin:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we 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). You may, therefore, market the device, subject to the general controls provisions of the Act. 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.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition. FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770)488-7655.

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Butman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known):

Device Name: QualiCode B. burgdorferi IgG Western Blot Kit

Indications For Use:

The Immunetics QualiCode™ B. burgdorferi IgG Western Blot Kit is intended for use in testing human serum samples which have demonstrated positive or equivocal responses using EIA or IFA test procedures to provide supportive evidence of infection with Borrelia burgdorferi.

The Immunetics QualiCode™ B. burgdorferi IgG Western Blot Kit can be used at any time following onset of symptoms. It should also be used for follow up when (1) only IgM antibodies were originally detected (2) IqG antibodies were detected but were not considered significant or (3) previously tested seroneqative individuals are shown to develop antibodies by EIA or IFA test procedures.

(PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Woody Dubres

(Division Sign Off)
Division of Clinical Laboratory Devices
510(k) Number K 991063

Prescription Use 7 (Per 21 CFR 801.109)

Over-The-Counter Use

(Optional format 1-2-96)

Regulation Number and Section

§ 866.3830

Treponema pallidum treponemal test reagents.(a)
Identification. Treponema pallidum treponemal test reagents are devices that consist of the antigens, antisera and all control reagents (standardized reagents with which test results are compared) which are derived from treponemal sources and that are used in the fluorescent treponemal antibody absorption test (FTA-ABS), theTreponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies toTreponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).