BioPlex 2200 Syphilis Total & RPR, BioPlex 2200 Syphilis Total & RPR Calibrator Set, BioPlex 2200 Syphilis Total & RPR Control Set

K170413 · Bio-Rad Laboratories · LIP · May 11, 2017 · Microbiology

Device Facts

• Record ID: K170413
• Device Name: BioPlex 2200 Syphilis Total & RPR, BioPlex 2200 Syphilis Total & RPR Calibrator Set, BioPlex 2200 Syphilis Total & RPR Control Set
• Applicant: Bio-Rad Laboratories
• Product Code: LIP · Microbiology
• Decision Date: May 11, 2017
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3830
• Device Class: Class 2

Intended Use

The BioPlex Syphilis Total & RPR kit is a multiplex flow immunoassay intended for the qualitative detection of total (IgG/IgM) antibodies to Treponema pallidum and the qualitative detection and/or titer determination of non-treponemal reagin antibodies in human serum or plasma. The Syphilis Total or RPR assays may be used to supplement a previously determined reactive treponemal or non-treponemal test. The test system should be used in conjunction with other laboratory tests and clinical findings to aid in the diagnosis of syphilis infection. The BioPlex 2200 Syphilis Total assay is not intended for use in screening blood or plasma donors. The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System. The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the calibration of the BioPlex 2200 Syphilis Total & RPR Reagent Pack. The BioPlex 2200 Syphilis Total & RPR Control Set is intended for use as an assayed quality control to monitor the performance of the BioPlex 2200 Instrument and BioPlex 2200 Syphilis Total & RPR assay in the clinical laboratory. The performance of the BioPlex 2200 Syphilis Total & RPR Control Set has not been established with any other Syphilis Total & RPR assays.

Device Story

Multiplex flow immunoassay; uses fluoromagnetic beads coated with recombinant T. pallidum rTP47/rTP17 fusion protein and cardiolipin antigen. Input: human serum or plasma. Process: two-step assay; sample/diluent/beads incubated at 37°C; wash cycle; phycoerythrin-conjugated anti-human IgG/IgM added; incubation; wash; detection via fluorescence. Output: antibody index (AI) for Syphilis Total and RPR; RPR titer determination via on-board dilution. Used in clinical laboratories; operated by trained personnel. Output aids clinicians in syphilis diagnosis. Benefits: automated, high-throughput, multiplexed detection of both treponemal and non-treponemal antibodies.

Clinical Evidence

Clinical performance evaluated using 2008 samples (prospective and retrospective). Prospective study (n=1001) showed 92.45% PPA and 97.86% NPA for Syphilis Total vs. comparator algorithm; 81.52% PPA and 96.48% NPA for RPR vs. predicate. Retrospective study (n=546) showed 99.59% PPA and 100% NPA for Syphilis Total. Subgroup analysis performed for pregnant women and HIV-positive patients. Bench testing included precision (CLSI EP05-A3), reproducibility (CLSI EP15-A3), interference (CLSI EP07-A2), and cross-reactivity.

Technological Characteristics

Multiplex flow immunoassay; fluoromagnetic beads coated with rTP47/rTP17 fusion protein or cardiolipin. Energy source: BioPlex 2200 System (fluorescence detection). Materials: MOPS buffer, bovine/murine proteins, ProClin 300/sodium benzoate/sodium azide preservatives. Connectivity: LIS integration via ASTM protocols. Software: Version 4.3, Windows-based, controls instrument modules, data analysis, and QC management.

Indications for Use

Indicated for qualitative detection of total (IgG/IgM) antibodies to Treponema pallidum and qualitative detection/titer determination of non-treponemal reagin antibodies in human serum or plasma. Used as a supplement to previously determined reactive treponemal or non-treponemal tests to aid in syphilis diagnosis. Not for blood/plasma donor screening. Intended for use with BioPlex 2200 System.

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

• LIAISON Treponema Assay (k061247)
• BD Macro-Vue RPR Card Tests (Pre-amendment)

Submission Summary (Full Text)

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609

Silver Spring, MD 20993-0002

May 11, 2017

BIO-RAD Laboratories Juang Wang Regulatory Affairs Representative, Clinical Immunology Division 5500 E. 2nd Street Benicia, CA 94510

Re: K170413

Trade/Device Name: Bioplex 2200 Syphilis Total & RPR Kit Bioplex 2200 Syphilis Total & RPR Calibrator Set Bioplex 2200 Syphilis Total & RPR Control Set Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema pallidum treponemal test reagents Regulatory Class: II Product Code: LIP, GMQ, JIT, JJX Dated: February 9, 2017 Received: February 10, 2017

Dear Mr. Wang:

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. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

# Steven R. Gitterman -S

for Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K170413

Device Name BioPlex 2200 Syphilis Total & RPR BioPlex 2200 Syphilis Total & RPR Control Set BioPlex 2200 Syphilis Total & RPR Calibrator Set

Indications for Use (Describe)

The BioPlex Syphilis Total & RPR kit is a multiplex flow immunoassay intended for the qualitative detection of total (IgG/IgM) antibodies to Treponema pallidum and the qualitative detection and/or titer determination of non-treponemal reagin antibodies in human serum or plasma. The Syphilis Total or RPR assays may be used to supplement a previously determined reactive treponemal or non-treponemal test. The test system should be used in conjunction with other laboratory tests and clinical findings to aid in the diagnosis of syphilis infection.

The BioPlex 2200 Syphilis Total & RPR kit is not intended for use in screening blood or plasma donors

The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System.

The BioPlex 2200 Syphilis Total & RPR Control Set is intended for use as an assayed quality control to monitor the performance of the BioPlex 2200 Instrument and BioPlex 2200 Syphilis Total & RPR assay in the clinical laboratory. The performance of the BioPlex 2200 Syphilis Total & RPR Control Set has not been established with any other Syphilis Total & RPR assays.

The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the BioPlex 2200 Syphilis Total & RPR Reagent Pack.

| Type of Use (Select one or both, as applicable) | |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| <div> <span> <svg height="16" width="16"> <rect fill="white" height="16" stroke="black" stroke-width="1" width="16"></rect> <path d="M2,2 L14,14 M14,2 L2,14" stroke="black" stroke-width="1"></path> </svg> </span> <span>Prescription Use (Part 21 CFR 801 Subpart D)</span> </div> | <div> <span> <svg height="16" width="16"> <rect fill="white" height="16" stroke="black" stroke-width="1" width="16"></rect> </svg> </span> <span>Over-The-Counter Use (21 CFR 801 Subpart C)</span> </div> |

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# BioPlex 2200 Syphilis Total & RPR 510(k) Summary

Bio-Rad Laboratories hereby submits this 510(k) in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. This summary of 510(k) safety and effectiveness information provides detail as a basis for a determination of substantial equivalence for the BioPlex 2200 Syphilis Total & RPR kit.

#### Applicant/Sponsor I.

Bio-Rad Laboratories, INC Clinical Immunology Division 5500 E. Second Street Benicia, CA 94510

Contact Person: Juang Wang, PhD, RAC Tel: [phone] FAX: [phone] [e-mail]

Date Prepared: May 09, 2017

#### Device Name II.

Proprietary Name: BioPlex 2200 Syphilis Total & RPR BioPlex 2200 Syphilis Total & RPR Calibrator BioPlex 2200 Syphilis Total & RPR Control

# Classification Name:

- Enzyme Linked Immunoabsorption Assay, Treponema pallidum
- Antigens, Nontreponemal, All
- Calibrator, Secondary
- Single (specified) Analyte Controls (Assayed and Unassayed)

#### Regulatory Information: III.

| Product Code | Classification | Regulation Section | Panel |
|--------------------------------------------------------------------------------|----------------|-----------------------------------------------------------------------------|----------------------|
| Enzyme linked<br>immunoabsorption assay,<br><i>Treponema pallidum</i><br>(LIP) | Class II | 21 CFR §866.3830 – <i>Treponema pallidum</i> treponemal test<br>reagents | Microbiology<br>(83) |
| Antigens, Nontreponemal,<br>All (GMQ) | Class II | 21 CFR §866.3820 – <i>Treponema pallidum</i> nontreponemal test<br>reagents | Microbiology<br>(83) |

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| Product Code | Classification | Regulation Section | Panel |
|-------------------------------------------------------------------------|----------------|--------------------------------------------------------------------------|-----------------------|
| Calibrator, Secondary(JIT) | Class II | 21 CFR § 862.1150 – Calibrator | Clinical<br>Chemistry |
| Single (specified) analyte<br>controls (assayed and<br>unassayed) (JJX) | Class I | 21 CFR § 862.1660-Quality<br>control Material (Assayed and<br>Unassayed) | Clinical<br>Chemistry |

#### IV. Predicate Devices

LIAISON Treponema Assay, k061247 BD Macro-Vue RPR Card Tests, Pre-amendment prior to May 28, 1976

#### V. Device Description:

BioPlex 2200 Syphilis Total & RPR kit includes the following components:

- One (1) 10 mL vial, containing dyed beads coated with recombinant Syphilis ● rTP47/rTP17 fusion protein, a cardiolipin antigen, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB) in MOPS (3-[N-Morpholino] propanesulfonic acid) buffer containing bovine proteins with protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
- One (1) 5 mL vial, containing phycoerythrin conjugated murine monoclonal antihuman IgG and murine monoclonal anti-human IgM, and phycoerythrin conjugated murine monoclonal anti-human FXIII antibody in phosphate buffer supplemented with murine and bovine protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
- One (1) 10 mL vial, containing bovine and murine protein stabilizers in MOPS (3-IN-Morpholino] propanesulfonic acid) buffer. ProClin 300 (< 0.3%), sodium benzoate (< 0.1%) and sodium azide (< 0.1%) are added as preservatives

BioPlex 2200 Syphilis Total & RPR Calibrator Set: Four (4) 0.5 mL vials, containing T. pallidum and reagin antibodies in a human serum matrix made from defibrinated plasma, and one (1) 0.5 mL vial containing human serum matrix made from defibrinated plasma for a total of five (5) calibrator vials. All calibrators contain ProClin 300 (≤ 0.3%), sodium benzoate (< 0.1%) and sodium azide (< 0.1%) as preservatives

BioPlex 2200 Syphilis Total & RPR Control Set: Two sets of three (3) control vials. Each set contains two (2) 1.5 mL Positive Control vials with antibodies to T. pallidum and reagin in a human serum matrix made from defibrinated plasma and one (1) 1.5 mL vial of Negative Control in a human serum matrix made from defibrinated plasma. ProClin 300 (≤ 0.3%) sodium benzoate (< 0.1%) and sodium azide (< 0.1%) are added as preservatives for all controls.

Additional materials required but not supplied include BioPlex 2200 Sheath Fluid containing Phosphate Buffered Saline (PBS), ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives; and BioPlex 2200 Wash Solution containing Phosphate Buffered Saline (PBS) and Tween 20. ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives.

#### VI. Intended Use:

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The BioPlex Syphilis Total & RPR kit is a multiplex flow immunoassay intended for the qualitative detection of total (IgG/IgM) antibodies to Treponema pallidum and the qualitative detection and/or titer determination of non-treponemal reagin antibodies in human serum or plasma. The Syphilis Total or RPR assays may be used to supplement a previously determined reactive treponemal or non-treponemal test. The test system should be used in conjunction with other laboratory tests and clinical findings to aid in the diagnosis of syphilis infection.

The BioPlex 2200 Syphilis Total kit is not intended for use in screening blood or plasma donors.

The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System.

The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the calibration of the BioPlex 2200 Syphilis Total & RPR Reagent Pack.

The BioPlex 2200 Syphilis Total & RPR Control Set is intended for use as an assayed quality control to monitor the performance of the BioPlex 2200 Instrument and BioPlex 2200 Syphilis Total & RPR assay in the clinical laboratory. The performance of the BioPlex 2200 Syphilis Total & RPR Control Set has not been established with any other Syphilis Total & RPR assays.

#### VII. Test Principle and Procedure

The BioPlex 2200 Syphilis Total & RPR kit employs Treponema pallidum fusion protein (rTP47/rTP17) and cardiolipin antigen-coated fluoromagnetic beads with unique fluorescent signatures to identify the presence of IgG and IgM antibodies to Treponema pallidum and reagin in a two-step assay format.

Dyed beads are coated with recombinant T. pallidum rTP47/rTP17 fusion protein or cardiolipin antigen.

The BioPlex 2200 System combines an aliquot of patient sample, sample diluent, and bead reagent into a reaction vessel. The mixture is incubated at 37℃. After a wash cycle, a mixture of murine monoclonal anti-human IgG and murine monoclonal antihuman IgM antibody conjugated to phycoerythrin (PE), is added to the dyed beads, and this mixture is incubated at 37℃. The excess conjugate is removed in another wash cycle and the beads are re-suspended in wash buffer. The bead mixture then passes through the detector. The identity of the dyed beads is determined by the fluorescence signature of the dyes, and the amount of antibody captured by the antigen is determined by the fluorescence intensity of the attached PE. Raw data is calculated in relative fluorescence intensity (RFI).

Two additional dyed beads, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB) are present in each reaction mixture to verify detector response and the presence of serum or plasma in the reaction vessel. Refer to the BioPlex 2200 System Operation Manual for more information.

The system is calibrated using a set of five (5) distinct calibrator vials, supplied separately by Bio-Rad Laboratories. One vial containing negative sample, and four

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vials containing human Treponema pallidum or human reagin antibodies, are used for qualitative calibration of assays. The results are expressed in antibody index (AI). The Syphilis Total assay results are reported as nonreactive (≤ 0.8 AI), equivocal (0.9, 1.0 AI) or reactive (≥ 1.1 AI); while the RPR assay results are reported as nonreactive (< 1.0 AI) or reactive (≥ 1.0 AI).

#### Technological Characteristics and Substantial Equivalence VIII.

The following tables summarize the similarities and differences between the BioPlex 2200 Syphilis Total & RPR kit and the predicate devices used in comparative studies with the BioPlex 2200 Syphilis Total & RPR kits.

| Characteristics | New Device<br>BioPlex 2200 Syphilis Total<br>& RPR Kit | Predicate Device<br>LIAISON Treponema<br>Assay, k061247 |
|---------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | Multiplex flow immunoassay<br>intended for the qualitative<br>detection of Total (IgG/IgM)<br>antibodies to Treponema<br>pallidum in human serum or<br>plasma | Chemiluminescent<br>immunoassay intended<br>for the qualitative<br>determination of total<br>antibodies directed<br>against Treponema<br>pallidum in human<br>serum |
| Indications for Use | Used in conjunction with<br>other serological tests and<br>clinical findings to aid in the<br>diagnosis of syphilis<br>infection. | Same |
| Measured Analyte | Total antibodies (IgG/IgM)<br>to T. pallidum | Same |
| Assay Type | Qualitative | Same |
| Solid Phase | Antigen-coated paramagnetic<br>microbeads | Antigen coated<br>magnetic particles |
| Cut-off Index | 1.0 Antibody Index (AI) | Index 1.0 |
| Equivocal Zone | 0.9 - 1.0 | 0.9 - 1.1 |
| Standardization | The calibrator is referenced<br>to an internal reference<br>material | The calibrator<br>concentrations are<br>referenced to an in-<br>house antibody<br>preparation |
| Controls | 2 (Negative and Positive) | Same |

| Device Differences | | |
|--------------------|------------------------------------------|-------------------------------------|
| Characteristics | New Device | Predicate Device |
| | BioPlex 2200 Syphilis Total &<br>RPR Kit | LIAISON Treponema<br>Assay, k061247 |

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| Characteristics | New Device<br>BioPlex 2200 Syphilis Total &<br>RPR Kit | Predicate Device<br>LIAISON Treponema<br>Assay, k061247 |
|------------------------------------------|---------------------------------------------------------------------------------------------------------|---------------------------------------------------------|
| Assay Technology | Automated multiplex flow<br>immunoassay | Sandwich<br>chemiluminescence<br>immunoassay (CLIA) |
| Antigen | Recombinant fusion TP antigen:<br>rTP17/rTP47 | DNA-Tp17<br>Recombinant antigen |
| Conjugate | Phycoerythrin conjugated<br>murine monoclonal anti-human<br>IgG and murine monoclonal<br>anti-human IgM | Conjugated to an<br>Isoluminol derivative |
| Signal Detection | Fluorescence | Chemiluminescent |
| Sample Matrix | Serum or Plasma | Serum |
| Calibrator(s) | 4 calibrator levels (sold<br>separately) | Two positive<br>calibrators |
| Open Pack Stability | 60 days | 4 weeks |
| Reagent Pack<br>Calibration<br>Frequency | Every 30 days | Every 14 days |
| Instrumentation | Bio-Rad BioPlex 2200 System | DiaSorin LIAISON<br>Analyzer |

| Characteristics | New Device<br>BioPlex 2200 Syphilis Total &<br>RPR Kit | Predicate Device<br>BD Macro-Vue RPR<br>CARD TEST,<br>Pre-amendment |
|------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|
| Intended Use | Multiplex flow immunoassay<br>intended for the qualitative<br>detection and/or titer<br>determination of non- Treponema<br>pallidum reagin antibodies in<br>human serum or plasma | A non-treponemal<br>testing procedure for<br>the serological<br>detection of syphilis<br>in human serum or<br>plasma |
| Measured Analyte | Non-Treponema pallidum reagin<br>antibodies | Same |
| Antigen | Cardiolipin/lecithin/cholesterol | Same |
| Sample matrix | Serum or plasma | Same |

| Characteristics | New Device<br>BioPlex 2200 Syphilis Total &<br>RPR Kit | Predicate Device<br>BD Macro-Vue RPR<br>CARD TEST,<br>Pre-amendment |
|------------------|--------------------------------------------------------|---------------------------------------------------------------------|
| Assay Technology | Automated multiplex flow<br>immunoassay | Macroscopic<br>flocculation |
| Solid phase | Antigen-coated paramagnetic | Antigen carbon particle |

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| Characteristics | New Device<br>BioPlex 2200 Syphilis Total &<br>RPR Kit | Predicate Device<br>BD Macro-Vue RPR<br>CARD TEST,<br>Pre-amendment |
|------------------------------------------|---------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------|
| | microbeads | suspension |
| Conjugate | Phycoerythrin conjugated<br>murine monoclonal anti-human<br>IgG and murine monoclonal<br>anti-human IgM | None |
| Calibrator(s) | 2 levels – negative and positive | None |
| Control(s) | 2 (Negative and Positive) | 3 (Negative and 2<br>Positive) |
| Standardization | The calibrator is referenced to<br>an internal reference material | None |
| Cut- off Index | 1.0 Antibody Index (AI) | None |
| Signal Detection | Fluorescence | Flocculation by naked<br>eye |
| Reagent Pack<br>Calibration<br>Frequency | Every 30 days | None |
| Instrumentation | Bio-Rad BioPlex 2200 System | Card Test (Manual) |

#### IX. Performance Characteristics

- 1. Analytical Performance:
- a. Precision/Reproducibility:

### Internal Precision (CLSI EP05-A3)

Precision testing of the BioPlex 2200 Syphilis Total & RPR kit on the BioPlex 2200 instrument was performed in accordance with CLSI EP05-A3 guideline. A human serum panel consisting of 6 frozen samples spanning the measuring range was assayed in duplicate per run on two runs daily over 20 days (N=80) on one reagent lot. Two levels of the BioPlex Syphilis Total & RPR controls were also included. The data were analyzed for within-run, between-run, between-day. The total precision and the mean (AI), standard deviation (AI) and percent coefficient of variation (%CV) are summarized below:

| Serum<br>Panel | N | Mean AI | Within Run | | Between Run | | Between Day | | Total Precision | |
|---------------------|----|---------|------------|-------|-------------|------|-------------|-------|-----------------|-------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 80 | 0.3 | 0.03 | 10.4% | 0.00 | 0.0% | 0.03 | 8.4% | 0.04 | 13.4% |
| Sample 2 | 80 | 0.5 | 0.04 | 6.5% | 0.02 | 3.5% | 0.03 | 5.6% | 0.05 | 9.3% |
| Sample 3 | 80 | 0.9 | 0.03 | 3.3% | 0.02 | 2.2% | 0.02 | 2.7% | 0.04 | 4.8% |
| Sample 4 | 80 | 1.5 | 0.06 | 3.7% | 0.02 | 1.0% | 0.12 | 7.8% | 0.13 | 8.7% |
| Sample 5 | 80 | 1.8 | 0.08 | 4.3% | 0.00 | 0.0% | 0.19 | 10.3% | 0.20 | 11.2% |
| Sample 6 | 80 | 3.2 | 0.08 | 2.5% | 0.04 | 1.4% | 0.09 | 2.9% | 0.13 | 4.1% |
| Positive<br>Control | 80 | 2.6 | 0.13 | 4.7% | 0.01 | 0.4% | 0.05 | 1.8% | 0.13 | 5.1% |

BioPlex 2200 Syphilis Total -Precision (CLSI EP5-A3)

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| Serum<br>Panel | N | Mean AI | Within Run | | Between Run | | Between Day | | Total Precision | |
|---------------------|----|---------|------------|-------|-------------|------|-------------|-------|-----------------|-------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 80 | 0.2 | 0.03 | 11.2% | 0.02 | 6.5% | 0.04 | 16.2% | 0.05 | 20.7% |
| Sample 2 | 80 | 0.7 | 0.04 | 6.6% | 0.06 | 9.0% | 0.05 | 7.3% | 0.09 | 13.4% |
| Sample 3 | 80 | 0.9 | 0.04 | 4.2% | 0.06 | 6.5% | 0.05 | 5.1% | 0.09 | 9.3% |
| Sample 4 | 80 | 1.0 | 0.05 | 4.7% | 0.08 | 7.5% | 0.08 | 7.8% | 0.12 | 11.8% |
| Sample 5 | 80 | 1.9 | 0.10 | 5.2% | 0.10 | 5.3% | 0.08 | 4.2% | 0.16 | 8.6% |
| Sample 6 | 80 | 3.4 | 0.09 | 2.6% | 0.16 | 4.8% | 0.12 | 3.5% | 0.22 | 6.5% |
| Positive<br>Control | 80 | 2.0 | 0.05 | 2.4% | 0.11 | 5.4% | 0.08 | 4.0% | 0.14 | 7.1% |

BioPlex 2200 RPR-Precision (CLSI EP05-A3)

## Reproducibility (CLSI EP15-A3)

The reproducibility was also evaluated in accordance with CLSI EP15-A3 guideline "User Verification of Precision and Estimation of Bias, Third Edition".

Reproducibility testing was performed at each of three (3) US testing facilities using a single lot of the BioPlex 2200 Syphilis Total & RPR reagent lot. A serum panel consisting of 5 samples spanning the measuring range were assayed in 4 replicates per run, two runs per day over 5 days (4 reps x 2 runs x 5 days x 3 sites = 120 total data points). The QC Controls were also included. The data were analyzed for within-run, between run, between day, between site/instrument and total precision and the mean AI, standard deviation and percent coefficient of variation (%CV) are summarized below:

| Syphilis Total | | | Within Run | | Between Run | | Between Day | | Between Site/Instrument | | Total | |
|------------------|-----|-----------------|------------|------|-------------|------|-------------|------|-------------------------|-------|-------|-------|
| Sample | N | Grand Mean (AI) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 120 | 0.5 | 0.02 | 5.1% | 0.02 | 4.1% | 0.00 | 0.9% | 0.08 | 16.9% | 0.08 | 18.2% |
| Sample 2 | 120 | 1.0 | 0.04 | 3.9% | 0.02 | 1.6% | 0.02 | 1.9% | 0.08 | 7.5% | 0.09 | 8.8% |
| Sample 3 | 120 | 1.5 | 0.05 | 3.5% | 0.00 | 0.0% | 0.02 | 1.5% | 0.08 | 5.5% | 0.10 | 6.7% |
| Sample 4 | 120 | 2.2 | 0.08 | 3.4% | 0.03 | 1.5% | 0.03 | 1.1% | 0.01 | 0.5% | 0.09 | 3.9% |
| Sample 5 | 120 | 6.8 | 0.22 | 3.2% | 0.11 | 1.7% | 0.17 | 2.6% | 0.00 | 0.0% | 0.30 | 4.4% |
| Positive Control | 119 | 2.7 | 0.15 | 5.7% | 0.07 | 2.7% | 0.00 | 0.0% | 0.08 | 3.1% | 0.19 | 7.0% |

BioPlex 2200 Syphilis Total - Reproducibility (CLSI EP15-A3)

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| RPR | | | Within Run | | Between Run | | Between Day | | Between Site/Instrument | | Total | |
|------------------|-----|-----------------------|------------|------|-------------|------|-------------|------|-------------------------|-------|-------|-------|
| Sample | N | Grand<br>Mean<br>(AI) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 120 | 0.8 | 0.05 | 5.9% | 0.01 | 1.7% | 0.04 | 5.4% | 0.03 | 4.0% | 0.07 | 9.1% |
| Sample 2 | 120 | 1.0 | 0.05 | 4.7% | 0.01 | 0.9% | 0.04 | 3.4% | 0.04 | 3.5% | 0.07 | 6.8% |
| Sample 3 | 120 | 2.0 | 0.08 | 4.0% | 0.09 | 4.5% | 0.09 | 4.7% | 0.21 | 10.7% | 0.26 | 13.2% |
| Sample 4 | 120 | 3.0 | 0.08 | 2.8% | 0.10 | 3.2% | 0.07 | 2.3% | 0.07 | 2.4% | 0.16 | 5.4% |
| Sample 5 | 120 | 7.4 | 0.26 | 3.6% | 0.27 | 3.6% | 0.24 | 3.3% | 0.23 | 3.2% | 0.51 | 6.9% |
| Positive Control | 120 | 2.7 | 0.10 | 3.5% | 0.04 | 1.5% | 0.05 | 1.9% | 0.05 | 1.9% | 0.13 | 4.7% |

BioPlex 2200 RPR - Reproducibility (CLSI EP15-A3)

## RPR Titer On-board Dilution Reproducibility

The BioPlex 2200 System has a feature for the determination of an end point RPR titer result. All reactive RPR samples can be diluted on board at 1:4, 1:8, 1:16, 1:32, and 1:64. Four reactive RPR samples, negative and positive controls selected to evaluate the titer precision were tested in two runs per day in duplicate per run for 5 days for a total of 20 data points. The results are summarized below.

| | End Point Titer Results | | | | | | | | % Agreement |
|-------------------------------|-------------------------|------|------|-----|-----|------|------|-------|----------------------------------|
| Sample Reactivity | Non-<br>Reactive | Neat | <1:4 | 1:4 | 1:8 | 1:16 | 1:32 | >1:64 | within $\pm$ 1 titer<br>(95% CI) |
| Negative Control | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 100%<br>(83.9 - 100%) |
| Positive Control | 0 | 20 | 20 | 0 | 0 | 0 | 0 | 0 | 100%<br>(83.9 - 100%) |
| Low Reactive (1:8) | 0 | 0 | 0 | 1 | 19 | 0 | 0 | 0 | 100%<br>(83.9 - 100%) |
| Moderately Reactive<br>(1:16) | 0 | 0 | 0 | 0 | 0 | 20 | 0 | 0 | 100%<br>(83.9 - 100%) |
| High Reactive (>1:64) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 20 | 100%<br>(83.9 - 100%) |
| High Reactive (>1:64) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 20 | 100%<br>(83.9 - 100%) |

### b. Traceability:

Value Assignment:

Calibrator assignment is established for the matched lot of BioPlex 2200 Syphilis Total & RPR kit using the internal reference standards. For each calibrator level, three vials are tested in replicates of five on three BioPlex 2200 analyzers for a total of 45 data points. The mean values obtained for each kit calibrator level are verified and must fall within specified acceptable range as shown below.

| Calibrator Set | Assay | Range (AI) |
|----------------|---------------------------|------------|
| Vial 1 | Syphilis Total<br>and RPR | 0.0 - 0.2 |
| Vial 2 | Syphilis Total | 1.4 - 2.2 |
| Vial 3 | Syphilis Total | 2.8 - 4.2 |
| Vial 4 | Syphilis Total | 4.8 - 7.2 |

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| Calibrator Set | Assay | Range (AI) |
|----------------|-------|------------|
| Vial 5 | RPR | 1.4 – 2.6 |

### Calibrator

There is no international or certified reference material available. The BioPlex Syphilis Total & RPR Calibrators are traceable to internal standard. The internal reference standard is manufactured by spiking high positive plasma into non-reactive immunodepleted human plasma. The internal reference calibrators are frozen at ≤ -80℃.

The BioPlex Syphilis Total assay is calibrated using a set of four levels of distinct serum based calibrators whereas the BioPlex RPR assay is calibrated with a set of two levels of calibrators.

### Controls

The BioPlex Syphilis & RPR Control Set contains one negative control, one positive control for Syphilis Total and one positive control for RPR. Positive controls are made from the human disease state serum containing anti-T. pallidum and reagin antibodies. The control set is provided in a human serum matrix stabilized with ≤ 0.3% ProClin 300, ≤0.1% sodium benzoate and <0.1% sodium azide.

For each control level, three vials are tested in multiple replicates using multiple reagent lots on three BioPlex 2200 analyzers for a minimum of 45 replicates per reagent lot. The minimum number of replicates for each control level is 90 when two reagent lots are used and 135 when three reagent lots are used. For each control level. the mean values were derived from replicate analyses and should fall within the corresponding ranges as shown below.

| Control Set | Range (AI) |
|---------------------------------|------------|
| Negative Control | 0.0 – 0.5 |
| Syphilis Total Positive Control | 1.8 - 3.8 |
| RPR Positive Control | 1.8 - 3.8 |

The manufacturing target ranges of the Control Sets are listed below.

#### Stability: C.

Stability studies have been performed to support the following claims:

### Calibrator and Control:

BioPlex 2200 Syphilis Total & RPR Calibrator set is stable for 60 days after opening the vial the first time; Control Open Vial Stability (2 to 8°C), 60 days from first opening; Onboard Calibration Curve Stability, 30

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days; Calibrators and Controls Real Time Stability (2 to 8°C), 24 month claim or until the date of expiration; Calibrators and Controls Accelerated Stability at 25℃, 2 years predicted. Calibrators and controls freeze-thaw (-20℃ and -80℃), 5 freeze-thaw cycles.

### Kit Stability:

BioPlex 2200 Syphilis Total & RPR Kit: Real Time (unopened) Kit Stability, 24 month claim or until the date of expiration when stored unopened on the instrument or at 2 to 8°C. The open kit claim is 60 days.

### Sample Stability:

Sample stability studies were also performed: Sample stability fresh (2 to 8°C), 7 days; Sample stability frozen (-20 or -80°C), 24 months claimed; Sample Freeze-thaw (-20 or -80°C), up to 5-freeze thaw cycles at -20°C and 5-freeze thaw cycles at -80℃ acceptable.

- d. Analytical specificity:
### Interfering Substances

An interfering substances study was conducted to evaluate the potential interference of specific endogenous and exogenous substances with the BioPlex 2200 Syphilis Total & RPR kit according to CLSI EP7-A2 guideline.

No interference was observed with any of the substances tested. The substances and the maximum levels tested are shown in the table below:

| Substance | Concentration |
|--------------------------|----------------|
| Hemoglobin | ≤500 mg/dL |
| Bilirubin (unconjugated) | ≤20 mg/dL |
| Bilirubin (conjugated) | ≤30 mg/dL |
| Triglycerides | ≤3300 mg/dL |
| Total Protein | ≤12 g/dL |
| Cholesterol | ≤450 mg/dL |
| Ascorbic Acid | ≤3 mg/dL |
| Heparin Sodium | <8000 units/dL |
| EDTA | <800 mg/dL |

### Cross-Reactivity:

A study was conducted to determine if samples containing various cross reactants interfere with test results when tested with the BioPlex 2200 Syphilis Total & RPR kit. A panel of at least ten (10) specimens positive for each potential cross reactant was evaluated for possible cross reactivity with the BioPlex 2200 Syphilis Total & RPR kit for Syphilis Total and RPR assays.

Most of the samples evaluated were high positive (2x cut-off) for each cross reactant. The potential cross reactant samples were tested with

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commercially available predicate kits in order to confirm the negative status for the analytes intended to be measured. The potentially cross reactive samples were assayed in a single point. Cross reactivity, expressed as percent negative agreement is calculated by the ratio of the number of negative results to the total number of samples assayed for each cross reactant sample set. The results of each potential cross reactant are listed below:

| Cross Reactant | Number | BioPlex Syphilis Total | BioPlex RPR |
|----------------------------------------------------|--------|------------------------|----------------------|
| | Tested | % Negative Agreement | % Negative Agreement |
| Anti-HBs | 12 | 100.0% | 100.0% |
| Anti-Cardiolipin IgG | 10 | 90.0% (9/10) | 100.0% |
| Anti-Cardiolipin IgM | 14 | 100.0% | 100.0% |
| Anti-Cardiolipin IgA | 10 | 100.0% | 90.0% (9/10) |
| Anti-nuclear antibody (ANA) | 10 | 100.0% | 100.0% |
| B.burgdorferi IgG<br>(US Strain) | 10 | 100.0% | 100.0% |
| B.burgdorferi IgG<br>(EU Strain) | 10 | 100.0% | 100.0% |
| B.burgdorferi IgM<br>(EU Strain) | 12 | 100.0% | 100.0% |
| Cytomegalovirus<br>(anti-CMV IgG positive) | 11 | 100.0% | 100.0% |
| Cytomegalovirus<br>(anti-CMV IgM positive) | 12 | 100.0% | 100.0% |
| E. coli | 12 | 100.0% | 100.0% |
| Epstein-Barr Virus<br>(EBV IgG positive) | 13 | 100.0% | 100.0% |
| Epstein-Barr Virus<br>(EBV IgM positive) | 12 | 100.0% | 100.0% |
| HBsAg | 13 | 100.0% | 100.0% |
| HCV | 13 | 100.0% | 100.0% |
| HIV | 10 | 100.0% | 100.0% |
| HSV<br>(anti-HSV-2 IgG positive) | 16 | 100.0% | 100.0% |
| Hyper gamma-globulinemia<br>IgG | 10 | 100.0% | 100.0% |
| Hyper gamma-globulinemia<br>IgM | 13 | 100.0% | 100.0% |
| Leptospirosis | 12 | 100.0% | 100.0% |
| Pregnancy | 17 | 100.0% | 100.0% |
| Rheumatoid Factor (RF) | 13 | 100.0% | 100.0% |
| Rubella IgG | 11 | 100.0% | 100.0% |
| Rubella IgM | 12 | 100.0% | 100.0% |
| Systemic Lupus<br>Erythematosus (SLE) | 12 | 100.0% | 91.7% (11/12) |
| Toxoplasma IgG positive | 10 | 100.0% | 100.0% |
| Toxoplasma IgM positive | 10 | 100.0% | 100.0% |
| Varicella Zoster Virus (anti-<br>VZV IgG positive) | 12 | 100.0% | 100.0% |
| Total | 332 | 99.7% (331/332) | 99.4 %(330/332) |

#### Assay cut-off: e.

The cutoff values were established in the feasibility phase of BioPlex 2200 Syphilis Total & RPR assay development using native human samples from apparently healthy subjects, patients sent to the laboratory

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for syphilis testing and patients diagnosed with syphilis infection. Based on the Receiver Operating Characteristics (ROC) analysis using predicate results as standard, calibrator values were adjusted such that the cut-off value was equal to 1.0 AI. Further confirmation of the cutoff values were compared the BioPlex Syphilis Total and RPR results from patient samples to those from the commercially available assays. Subsequently, clinical studies conducted at three sites in the US provided the final validation of the cut-off value.

For the Syphilis Total assay an equivocal range of ±10% was then set around the cut-off value. Thus, results above or equal to 1.1 AI are considered reactive and results below 0.9 AI are considered nonreactive. The 10% range was established based on the precision of the assay.

For RPR assay, no equivocal range was set around the cut-off value. Thus results <1.0AI are considered non-reactive and results ≥1.0 AI are considered reactive.

#### 2. Comparison studies:

- a. Method comparison with predicate device:
The results of the BioPlex 2200 Syphilis Total and RPR assays were compared to a composite comparator and RPR card test for Syphilis Total and RPR assays, respectively. The composite comparator is based on an algorithm of results obtained from three commercially available syphilis assays: (a) Treponemal chemiluminescent immunoassay (predicate). (b) a nontreponemal assay (RPR Card Test), and (c) a second treponemal assay (Treponema Pallidum Particle Agglutination (TP-PA). Additional details of how the final comparator result was determined are provided below in the "Clinical Studies" section.

### b. Matrix comparison:

The effect of anticoagulants in the samples on the performance of the BioPlex 2200 Syphilis Total and RPR assays was determined by comparing values obtained from the native serum samples spiked with anti-T. pallidum and reagin antibodies to the values of the matched donor's plasma containing the following anti-coagulants:

- Dipotassium EDTA ●
- Tripotassium EDTA
- Lithium heparin ●
- Sodium heparin

A total of 113 serum/plasma pairs, screened to be non-reactive using the BioPlex 2200 Syphilis Total & RPR kit, were spiked with an equal amount of a highly reactive syphilis and RPR sample to obtain concentrations that span the assay measuring range.

{15}------------------------------------------------

Each sample was tested in duplicate on one BioPlex 2200 instrument and the mean of the results for each matrix type was compared to the mean of the respective serum control sample by using Passing-Bablok analysis method to generate regression parameters (slope, intercept and correlation) along with 95% Confidence Interval. Sample results that exceeded the measuring range were excluded from the data analysis. Acceptance criteria for the regression analysis were: slope 0.8 to 1.2, intercept -0.2 to 0.2 and correlation >0.95.

| Matrix | BioPlex<br>Assay | Slope<br>(95% CI) | Intercept<br>(95% CI) | Correlation<br>(r) | N |
|--------------------|-------------------|------------------------------|---------------------------------|--------------------|-----|
| Lithium<br>heparin | Syphilis<br>Total | 0.8421<br>(0.8148 to 0.8694) | 0.0316<br>(-0.0312 to 0.0944) | 0.9899 | 89 |
| | RPR | 0.8684<br>(0.8333 to 0.9035) | 0.0908<br>(0.0625 to 0.1191) | 0.9893 | 113 |
| Sodium<br>heparin | Syphilis<br>Total | 0.8333<br>(0.8148 to 0.8519) | 0.0500<br>(0.0050 to 0.0950) | 0.9924 | 89 |
| | RPR | 0.8353<br>(0.8095 to 0.8611) | 0.1147<br>(0.0833 to 0.1461) | 0.9849 | 113 |
| K2EDTA | Syphilis<br>Total | 1.1923<br>(1.1538 to 1.230) | -0.0923<br>(-0.1538 to -0.0308) | 0.9943 | 83 |
| | RPR | 1.0930<br>(1.0709 to 1.1151) | -0.0314<br>(-0.0663 to 0.0035) | 0.9864 | 112 |
| K3EDTA | Syphilis<br>Total | 1.1667<br>(1.1290 to 1.2043) | -0.0625<br>(-0.1073 to -0.0177) | 0.9915 | 82 |
| | RPR | 1.0776<br>(1.0500 to 1.1052) | -0.0233<br>(-0.0484 to 0.0018) | 0.9810 | 113 |

The resulting data support the package insert claim that serum, lithium heparin, sodium heparin, K2 EDTA, and K3EDTA specimens are acceptable for use with the BioPlex Syphilis Total and RPR assays.

#### Clinical studies: 3.

The clinical Performance of the BioPlex 2200 Syphilis Total & RPR kit was evaluated in three testing sites from November 2016 using a total of 2008 samples prospectively and retrospectively collected from multiple commercial suppliers (approximately 91% in the US and 9% from outside of the US). There were 6 samples that generated no values due to repeat instrument error flags. The tested specimens consisted of 1347 prospective samples (301 apparently healthy subjects, 201 syphilis and 200 RPR test ordered for a total of 401 test ordered samples, 295 pregnant women and 45 pregnant women with STD, 305 HIV positive individuals) and 661 retrospective samples (412 RPR/VDRL positive, 160 clinically diagnosed syphilis, 32 pregnant syphilis positive, 57 HIV/Syphilis dual positive).

The 2008 specimens enrolled in the multiple site study were collected in the following areas:

- . US : 90.7%
- 23.6% Northeast (Maryland, Massachusetts, New York, Pennsylvania)

{16}------------------------------------------------

- -18.7% Southwest (California, Hawaii, New Mexico)
- 28.4% Southeast (Florida, Georgia) -
- 9.2% Midwest -
- -10.8% Unknown
- Outside US: 9.3% ●
- -3.5% Argentina
- -3.2% France/Europe
- -1.4% China
- 1.2% Others -

Performance of the BioPlex Syphilis Total assay was evaluated by calculating positive percent agreement and negative percent agreement of the assay with the final comparator results based on an algorithm of results from three commercially available assays: treponemal chemiluminescence immunoassay (Predicate), RPR Card Test, and a second treponemal assay (TP-PA). The final comparator results were determined using a "2 out of 3" rule (TP-CLIA, RPR Card and TP-PA) as shown below. Results with indeterminate final comparator results would be excluded from the data analysis.

Performance of the BioPlex RPR assay was also evaluated by calculating positive percent agreement and negative percent agreement against a commercially available RPR Card Test (Predicate).

{17}------------------------------------------------

| Treponemal<br>IgG/IgM<br>(Predicate) | Non-treponemal<br>(Predicate) | 2nd Treponemal<br>(TP-PA)<br>(Predicate) | Final Comparator<br>Algorithm Result |
|--------------------------------------|-------------------------------|------------------------------------------|--------------------------------------|
| Negative | Non-reactive | Reactive | Negative |
| | | Non-reactive | Negative |
| | | Inconclusive | Negative |
| Negative | Reactive | Reactive | Positive |
| | | Non-reactive | Negative |
| | | Inconclusive | Negative |
| Positive | Reactive | Reactive | Positive |
| | | Non-reactive | Positive |
| | | Inconclusive | Positive |
| Positive | Non-reactive | Reactive | Positive |
| | | Non-reactive | Negative |
| | | Inconclusive | Positive |
| Equivocal | Non-reactive | Reactive | Positive |
| | | Non-reactive | Negative |
| | | Inconclusive | Indeterminate |
| Equivocal | Reactive | Reactive | Positive |
| | | Non-reactive | Negative |
| | | Inconclusive | Indeterminate |

# a. Clinical Performance in Prospectively Collected Specimens in the Intended Use Population

The 1001 prospectively collected specimens in the intended use population evaluated with the BioPlex 2200 Syphilis Total & RPR kit consisted of 401 specimens sent for routine syphilis or RPR testing (194 females, 207 males,7 - 96 years old), 295 pregnant women (15 -42 years old), and 305 HIV positive individuals (97 females, 208 males, 17 -75 years old).

The comparison between the BioPlex Syphilis Total assay result and the final comparator result for the prospectively-collected specimens in the intended use population is shown below.

{18}------------------------------------------------

| Prospective Samples | Final Comparator Algorithm Result | | | | | |
|--------------------------|-----------------------------------------------------|-------|-------|--------------------------------|----------------------------------|----------------------------------|
| | R (+) | NR(-) | Total | % Pos<br>Agreement<br>(95% CI) | % Neg<br>Agreement<br>(95% CI) | |
| Syphilis<br>Test Ordered | Pos (+) | 26 | 3 | 29 | 89.7% (26/29)<br>73.6%-96.4% | 98.3% (169/172)<br>95.0%-99.4% |
| | EQ | 1 | 0 | 1 | | |
| | Neg (-) | 2 | 169 | 171 | | |
| | Total | 29 | 172 | 201 | | |
| RPR Test Ordered | Pos (+) | 31 | 0 | 31 | 96.9% (31/32)<br>84.3%-99.4% | 100% (168/168)<br>97.8%-100% |
| | EQ | 0 | 0 | 0 | | |
| | Neg (-) | 1 | 168 | 169 | | |
| | Total | 32 | 168 | 200 | | |
| HIV Positive | Pos (+) | 90 | 11 | 101 | 91.8% (90/98)<br>84.7% - 95.8% | 93.7%(194/207)<br>89.6% - 96.3% |
| | EQ | 2 | 2 | 4 | | |
| | Neg (-) | 6 | 194 | 200 | | |
| | Total | 98 | 207 | 305 | | |
| Pregnant Women | Pos (+) | 0 | 2 | 2 | N/A | 99.3% (293/295)<br>97.6% - 99.8% |
| | EQ | 0 | 0 | 0 | | |
| | Neg (-) | 0 | 293 | 293 | | |
| | Total | 0 | 295 | 295 | | |
| Overall | Pos (+) | 147 | 16 | 163 | 92.5% (147/159)<br>87.3% - 95.6% | 97.9% (824/842)<br>96.6% - 98.6% |
| | EQ | 3 | 2 | 5 | | |
| | Neg (-) | 9 | 824 | 833 | | |
| | Total | 159 | 842 | 1001 | | |
| | BioPlex 2200 Syphilis Total Assay (Treponemal Test) | | | | | |

R- Reactive; EQ-Equivocal; NR-Non-reactive; Pos – Positive; Neg- Negative

The comparison between the BioPlex RPR assay result and the predicate RPR result is shown below.

| | Predicate RPR Result | | | | | | |
|-------------------------------------------------|--------------------------|--------|-------|--------------------------------|--------------------------------|--------------------------------|--------------------------------|
| Prospective Samples | R (+) | NR (-) | Total | % Pos<br>Agreement<br>(95% CI) | % Neg<br>Agreement<br>(95% CI) | | |
| BioPlex 2200 RPR Assay<br>(Non-Treponemal Test) | Syphilis Test<br>Ordered | R (+) | 19 | 5 | 24 | 79.2% (19/24)<br>59.5%-90.8% | 97.2% (172/177)<br>93.6%-98.8% |
| | | NR (-) | 5 | 172 | 177 | | |
| | | Total | 24 | 177 | 201 | | |
| RPR Test<br>Ordered | R (+) | 27 | 2 | 29 | 77.1% (27/35)<br>61.0%-87.9% | 98.8% (163/165)<br>95.7%-99.7% | |
| | NR (-) | 8 | 163 | 171 | | | |
| | Total | 35 | 165 | 200 | | | |
| HIV Positive | R (+) | 29 | 21 | 50 | 87.9% (29/33)<br>72.7%-95.2% | 92.3% (251/272)<br>88.5%-94.9% | |
| | NR (-) | 4 | 251 | 255 | | | |
| | Total | 33 | 272 | 305 | | | |

{19}------------------------------------------------

| | R (+) | | | | | |
|-------------------|--------|----|-----|------|-------------------------------|---------------------------------|
| Pregnant<br>Women | NR (-) | 0 | 4 | 4 | N/A | 98.6% (291/295)<br>96.6%-99.5% |
| | Total | 0 | 291 | 291 | | |
| | | 0 | 295 | 295 | | |
| Overall | R(+) | 75 | 32 | 107 | | |
| | NR (-) | 17 | 877 | 894 | 81.5% (75/92)<br>72.4% -88.1% | 96.5%(877/909)<br>95.1% - 97.5% |
| | Total | 92 | 909 | 1001 | | |

### b. Clinical Performance in Retrospective known Syphilis Positive Samples

Clinical performance in the retrospective known syphilis positive samples was evaluated by testing a total of 544 samples including 412 known RRP/VDRL positive (106 females, 305 males, 1 unknown gender, <1 - 89 years old), 32 pregnant positive women (18- 36 years old), 45 pregnant women with a history of STD (20 - 39 years old), and 57 known HIV/Syphilis dual positive (24 females, 33 males, 20 - 67 years old). Two (2) samples from RPR/VDRL positive cohort obtained no values due to repeated instrument errors were excluded from analysis. The comparison between the BioPlex Syphilis Total results and the final comparator results is shown below.

| Retrospective Samples | Final Comparator Algorithm Result | | | | | |
|-------------------------------------------------|-----------------------------------|-------|-------|--------------------------------|--------------------------------|------------------------------|
| | R (+) | NR(-) | Total | % Pos<br>Agreement<br>(95% CI) | % Neg<br>Agreement<br>(95% CI) | |
| RPR/VDRL Positive<br>Pregnant Positive<br>Women | Pos (+) | 404 | 0 | 404 | 100% (404/404)<br>99.1%-100% | 100% (6/6)<br>61.0%-100% |
| | EQ | 0 | 0 | 0 | | |
| | Neg (-) | 0 | 6 | 6 | | |
| | Total | 404 | 6 | 410 | | |
| Pregnant Women<br>with STD | Pos (+) | 31 | 0 | 31 | 100% (31/31)<br>89.0%-100% | 100% (1/1)<br>20.7%-100% |
| | EQ | 0 | 0 | 0 | | |
| | Neg (-) | 0 | 1 | 1 | | |
| | Total | 31 | 1 | 32 | | |
| HIV/<br>Syphilis Dual<br>Positive | Pos (+) | 1 | 0 | 1 | 100% (1/1)<br>20.7% - 100% | 100% (44/44)<br>92.0% - 100% |
| | EQ | 0 | 0 | 0 | | |
| | Neg (-) | 0 | 44 | 44 | | |
| | Total | 1 | 44 | 45 | | |
| All known<br>positive<br>RPR/VDRL | Pos (+) | 50 | 0 | 50 | 96.2%(50/52)<br>87.0% - 98.9% | 100% (5/5)<br>56.6% - 100% |
| | EQ | 1 | 0 | 1 | | |
| | Neg (-) | 1 | 5 | 6 | | |
| | Total | 52 | 5 | 57 | | |

{20}------------------------------------------------

| All known positive | Pos (+) | 486 | 0 | 486 | 99.6% (486/488)<br>98.5% - 99.9% | 100% (56/56)<br>93.6% - 100% |
|--------------------|---------|-----|----|-----|----------------------------------|------------------------------|
| | EQ | 1 | 0 | 1 | | |
| | Neg (-) | 1 | 56 | 57 | | |
| | Total | 488 | 56 | 544 | | |

The comparison between the BioPlex RPR results and the predicate RPR results is shown below.

| | | | Predicate RPR Result | | | | |
|-------------------------------------------------|--------------------------------------|--------|----------------------|--------|-------|--------------------------------|---------------------------------|
| | Retrospective Samples | | R (+) | NR (-) | Total | % Pos Agreement (95% CI) | % Neg Agreement (95% CI) |
| | RPR/VDRL<br>Positive | R (+) | 390 | 11 | 401 | 98.5%(390/396)<br>96.7%-99.3% | 21.4% (3/14)<br>7.6%-47.6% |
| | | NR (-) | 6 | 3 | 9 | | |
| | | Total | 396 | 14 | 410 | | |
| | Pregnant<br>Positive<br>Women | R (+) | 25 | 1 | 26 | 100% (25/25)<br>86.7%-100% | 85.7% (6/7)<br>48.7%-97.4% |
| | | NR (-) | 0 | 6 | 6 | | |
| | | Total | 25 | 7 | 32 | | |
| | Pregnant<br>Women<br>with STD | R (+) | 0 | 1 | 1 | N/A | 97.8% (44/45)<br>88.4% - 99.6% |
| BioPlex 2200 RPR Assay<br>(Non-Treponemal Test) | | NR (-) | 0 | 44 | 44 | | |
| | | Total | 0 | 45 | 45 | | |
| | HIV/<br>Syphilis<br>Dual<br>Positive | R (+) | 7 | 9 | 16 | 77.8% (7/9)<br>45.3% - 93.7% | 81.3% (39/48)<br>68.1%- 89.8% |
| | | NR (-) | 2 | 39 | 41 | | |
| | | Total | 9 | 48 | 57 | | |
| | All known<br>positive | R (+) | 422 | 22 | 444 | 98.1%(422/430)<br>96.4% -99.1% | 80.7% (92/114)<br>72.5% - 86.9% |
| | | NR (-) | 8 | 92 | 100 | | |
| | | Total | 430 | 114 | 544 | | |

### c. Clinical Performance in Clinically Diagnosed Individuals

Samples were collected from 160 individuals diagnosed with primary, secondary or latent syphilis with treatment status including 43 females (18 – 78 years old) and 117 males (19 – 64 years old). Four samples with no results were excluded due to repeated instrument error flags observed. The comparison results between BioPlex Syphilis Total and RPR assays and predicate results are shown below.

Sensitivity of the BioPlex Syphilis Total and RPR assays and predicate assays in individuals clinically diagnosed with syphilis along with the treatment status is shown below.

| | Treatment<br>Status | N | Reactivity in Medically Diagnosed Syphilis Patients (95% CI) | | | |
|----------------|---------------------|----|--------------------------------------------------------------|----------------|---------------------|---------------|
| Syphilis Phase | | | BioPlex<br>Syphilis Total | BioPlex<br>RPR | Final<br>Comparator | Predicate RPR |
| Primary | Untreated | 26 | 96.2% | 92.3% | 100% | 88.5% |

{21}------------------------------------------------

| Syphilis Phase | Treatment<br>Status | N | Reactivity in Medically Diagnosed Syphilis Patients (95% CI) | | | |
|----------------|---------------------|-----|--------------------------------------------------------------|-----------------------------------|-----------------------------------|-----------------------------------|
| | | | BioPlex<br>Syphilis Total | BioPlex<br>RPR | Final<br>Comparator | Predicate RPR |
| | | | (25/26)<br>81.1% - 99.3% | (24/26)<br>75.9% - 97.9% | (26/26)<br>87.1% - 100% | (23/26)<br>71.05 - 96.0% |
| | Treated | 29 | 86.2%<br>(25/29)<br>69.4% - 94.5% | 65.5%<br>(19/29)<br>47.3% - 80.1% | 86.2%<br>(25/29)<br>69.4% - 94.5% | 75.9%<br>(22/29)<br>57.9% - 87.8% |
| Secondary | Untreated | 25 | 100%<br>(25/25)<br>86.7% - 100% | 100%<br>(25/25)<br>86.7% - 100% | 100%<br>(25/25)<br>86.7% - 100% | 100%<br>(25/25)<br>86.7% - 100% |
| | Treated | 26 | 100%<br>(26/26)<br>87.1% - 100% | 88.5%<br>(23/26)<br>71.05 – 96.0% | 100%<br>(26/26)<br>87.1% -100% | 80.8%<br>(21/26)<br>62.1% - 91.6% |
| | Untreated | 23 | 100%<br>(23/23)<br>85.7% - 100% | 95.7%<br>(22/23)<br>79.0% - 99.2% | 100%<br>(23/23)<br>85.7% - 100% | 95.7%<br>(22/23)<br>79.0% - 99.2% |
| Latent | Treated | 27 | 100%<br>(27/27)<br>85.1% -100% | 66.7%<br>(18/27)<br>47.8% - 81.4% | 100%<br>(27/27)<br>85.1% -100% | 66.7%<br>(18/27)<br>47.8% - 81.4% |
| All Phases | Untreated | 74 | 98.6%<br>(73/74)<br>92.7% - 99.8% | 95.9%<br>(71/74)<br>88.7% - 98.6% | 100%<br>(74/74)<br>95.1% - 100% | 95.0%<br>(70/74)<br>86.9% - 97.9% |
| | Treated | 82 | 95.1%<br>(78/82)<br>88.1% - 98.1% | 73.2%<br>(60/82)<br>62.7% - 81.6% | 95.1%<br>(78/82)<br>88.1% - 98.1% | 74.4%<br>(61/82)<br>64.0% - 82.6% |
| Total | | 156 | 96.8% (151/156)<br>92.7% - 98.6% | 84.0% (131/156)<br>77.4% - 88.9% | 97.4% (152/156)<br>93.6% - 99.3% | 84.0% (131/156)<br>77.4% - 88.9% |

# d. Clinical Performance in HIV Positive Individuals

Clinical performance in the HIV positive samples was evaluated by testing a total of 362 samples from 305 HIV positive individuals (97 females, 208 males, 17 -7…